WO1997006468A2 - Pattern formation, replication, fabrication and devices thereby - Google Patents

Pattern formation, replication, fabrication and devices thereby Download PDF

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Publication number
WO1997006468A2
WO1997006468A2 PCT/IB1996/000912 IB9600912W WO9706468A2 WO 1997006468 A2 WO1997006468 A2 WO 1997006468A2 IB 9600912 W IB9600912 W IB 9600912W WO 9706468 A2 WO9706468 A2 WO 9706468A2
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Prior art keywords
εaid
relief
molecular
array
patterned
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PCT/IB1996/000912
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French (fr)
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WO1997006468A3 (en
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Ely Michael Rabani
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Ely Michael Rabani
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Priority to AU67524/96A priority Critical patent/AU6752496A/en
Publication of WO1997006468A2 publication Critical patent/WO1997006468A2/en
Publication of WO1997006468A3 publication Critical patent/WO1997006468A3/en

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    • GPHYSICS
    • G03PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03FPHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/0035Multiple processes, e.g. applying a further resist layer on an already in a previously step, processed pattern or textured surface
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00Manufacture or treatment of nanostructures
    • GPHYSICS
    • G03PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03FPHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/0002Lithographic processes using patterning methods other than those involving the exposure to radiation, e.g. by stamping
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00382Stamping
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/00527Sheets
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00605Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00605Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00605Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614Delimitation of the attachment areas
    • B01J2219/00617Delimitation of the attachment areas by chemical means
    • B01J2219/00619Delimitation of the attachment areas by chemical means using hydrophilic or hydrophobic regions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00605Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623Immobilisation or binding
    • B01J2219/0063Other, e.g. van der Waals forces, hydrogen bonding
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00605Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632Introduction of reactive groups to the surface
    • B01J2219/00637Introduction of reactive groups to the surface by coating it with another layer
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00639Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00659Two-dimensional arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00725Peptides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/10Libraries containing peptides or polypeptides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to the fabrication of two- and three- dimensional patterns and the production of devices thereby; the present invention finds application in the areas of microelectronics, micromechanics, microfluidics, scanning probe microscopy, mass data storage, scientific instrumentation, clinical diagnostics, molecular assembly, and other area ⁇ .
  • metal layers may h ⁇ ched to form wiring patterns and regions of a semiconductint .bstrate may be doped to form electronic components in an integrated device.
  • Metal layers may also be patterned by lift-off processes involving the local dissolution of a pre-patterned resist underlayer-, which carries away with it the immediately overlying regions of the metal layer.
  • Fault Tolerant Circuit Designs One approach that has been applied in the field of microelectronics to increasing the tolerable physical defect rate arising from a particular fabrication process involves fault tolerant circuit design. This is a category of methods which includes redundancy of functions or components, self-testing or quality checking during the fabrication process, and rerouting of connections between functional blocks to serve functions lost to defect ⁇ . For example, such methods have been applied to dynamic RAM fabrication, where the memory bit array is divided into a number of blocks, the total capacity of which is larger than the device specification. All blocks are checked for defects or functionality, and where defects are found, the involved blocks are deactivated. Functional blocks are then reconfigured, as needed, to contiguously fill the device addre ⁇ s space. Analogous methods have been applied in the design of gate arrays, field programmable gate arrays, memory devices and other microelectronic devices.
  • J.L. Nistler has disclosed a method for the reduction of defects in lithographic phase shift masks.
  • the es ⁇ ence of this method is to form a resist pattern, use it to etch the underlying quartz surface only partially, remove said resist pattern and any defects it may include, form a substantially identical pattern of resi ⁇ t on said quartz surface again, partially etch again and remove said substantially identical pattern of resist again, in repetitive cycles.
  • etch resistant region ⁇ appearing in all resist patterns will be reproduced in the final patterned quartz article at the fully etched depth.
  • this method checks one pattern, which may contain random defects, against other patterns which may also contain random defects which are not likely to appear at the ⁇ ame location a ⁇ those random defect ⁇ in any other pattern.
  • the ca ⁇ e of the pha ⁇ e ⁇ hifting reticles produced by that invention only regions etched such that the tran ⁇ mitted light i ⁇ shifted by 180 degree ⁇ exposes the lithographic resi ⁇ t the ⁇ e reticle ⁇ are u ⁇ ed to expose.
  • correspondence between multiple source patterns is required for a feature to be represented in the final product mask produced therefrom.
  • Thi ⁇ method form ⁇ lithographic plate ⁇ by expo ⁇ ing materials similar or identical to those used as resists in conventional microfabrication, such that exposed regions have affinity or repel ink or other liquids, such that a pattern of differential retention of ink or ⁇ aid other liquid ⁇ may be u ⁇ ed to form a pattern of ⁇ aid ink or other liquids on said lithographic plates and then transfer this pattern to the sub ⁇ trate. While thi ⁇ method reduce ⁇ the extent to which photolithographic equipment i ⁇ needed for patterning, production of micropatterned articles by this method still requires routine acces ⁇ to ⁇ uch photolithographic equipment for lithographic plate production.
  • a relief is produced by selectively exposing regions of a photoresist coated onto a first surface and mechanically transferring either the exposed or non- exposed regions to a polymeric foil second surface by juxtaposing said second surface to the image-expo ⁇ ed polymeric re ⁇ ist and relying on the differential adhesion properties resulting from expo ⁇ ure. Thu ⁇ , a po ⁇ itive image i ⁇ produced on one ⁇ urface while the other ⁇ urface retains the corresponding negative pattern.
  • Such relief patterns may provide differential wetting or liquid-retention properties differing from tho ⁇ e of the underlying ⁇ urface ⁇ , and thus be used as offset lithographic plates.
  • G. Gal has, in U.S. Patent Number 5,310,623, taught a method whereby microlens array ⁇ are formed.
  • grey ⁇ cale expo ⁇ ure of a resist material may be used to cause the differential etching (in the preferred case by ion milling) of the underlying substrate, to produce a correspondingly curved surface.
  • Microelectromechanical Systems Microfabrication technique ⁇ have been applied to multilayered substrates comprising sacrificial layers which permit under-etching. Under-etching permit ⁇ the fabrication of ⁇ tructures with suspended or overhanging members and freely moving microscale mechanical part ⁇ . Such a proce ⁇ may be combined with conventional microfabrication ⁇ tep ⁇ or applied to ⁇ ub ⁇ trate ⁇ comprising microelectronic device ⁇ to integrate both electronic and mechanical components of micron and submicron dimen ⁇ ion. Such integrated devices have been termed microelectromechanical sy ⁇ tems (MEMS) 5 .
  • MEMS microelectromechanical sy ⁇ tems
  • Data is recorded in the form of pit ⁇ on ⁇ aid ⁇ urface.
  • a la ⁇ er is used to heat said polycarbonate above the glas ⁇ tran ⁇ ition temperature, and the AFM tip impre ⁇ e ⁇ a pit into the locally heated region.
  • the size of the resulting pit corresponds to the apical geometry of said AFM tip.
  • Readout is accomplished by conventional contact-mode AFM detection of said pits.
  • a relief pattern is formed which encodes the corresponding, stored data.
  • Scanning Probe Pattern Formation Scanning Resist Patterning: Scanning Tunneling Micro ⁇ cope ⁇ (STM) 11 ' 12 , Atomic Force Microscopes (AFM) and Near Field Scanning Optical Microscopes (NFSOM) 13 have been used to selectively form patterns in surfaces on the nanometer scale. These include patterns in materials or layers which mask the underlying sub ⁇ trate from the action of etchant ⁇ when not modified by the micro ⁇ copic probe u ⁇ ed.
  • STM Scanning Tunneling Micro ⁇ cope ⁇
  • AFM Atomic Force Microscopes
  • NFSOM Near Field Scanning Optical Microscopes
  • B.J. Mclntyre, M. Salmeron and G.A. Somorjai 15 have ⁇ i ilarly effected po ⁇ itional control of metallic ⁇ urface cataly ⁇ is using a platinum- rhodium STM tip in an atmo ⁇ pheric-pressure chemical reactor to catalyze the rehydrogenation of molecules comprising alkyne functionalities on (111) platinum surfaces.
  • M.A. Voelker 16 has proposed a method for the patterning of chemical moietie ⁇ on ⁇ urface ⁇ , and the formation of patterns in multicomponent Langmuir-Blodgett (L-B) film ⁇ according to the interaction of affinity groups of molecules in said L-B films with complementary affinity moieties on ⁇ aid surfaces.
  • Such a method would entail that the molecules of which said L-B film is composed have good mobility in the as-formed L-B film prior to any polymerization, and that transport permits good segregation of molecular species within ⁇ aid L-B film without any congestion problems or other hindrance to the proper assortment of specie ⁇ to match the pattern on the underlying ⁇ urface.
  • Integrated optical devices and other micro ⁇ cale optical components have been fabricated by the replication of relief patterns into polymeric material ⁇ 17 .
  • the optical propertie ⁇ of the ⁇ e materials and the structure ⁇ resulting from pattern replication determine device function and characteristics.
  • Replication methods used include injection molding and casting into polymeric, elastiomeric or metallic molds or mold inserts, and hot embos ⁇ ing with relief ⁇ , for example, metal relief ⁇ .
  • Multiple cycle ⁇ of replication may be performed to yield a large number of relief ⁇ for the rapid ma ⁇ production of devices, according to the fidelity of replication and acceptable device tolerances.
  • This first generation mold may then be as a mold for the casting or microinjection molding of a second polymeric relief nearly identical to the first relief.
  • the molding or casting proces ⁇ may be repeated with the fir ⁇ t generation mold, and the electroforming proce ⁇ may be repeated with ⁇ econd or subsequent generation polymeric relief structures produced with said molds by appropriate methods.
  • polymeric object ⁇ and metallic objects with sub-micron patterns may be economically reproduced.
  • Gale et al., 20 extend method ⁇ from the embo ⁇ ed diffractive foil and compact di ⁇ k (CD) industries by the replication of an original (e.g. microfabricated) microrelief on the surface of a replication shim through electroforming, followed by replication of this first shim by surface passivation followed again by electroforming.
  • the ⁇ e workers find that the shim replication proce ⁇ occur ⁇ with only slight ( ⁇ 2nm) increases in surface roughne ⁇ per generation, thus enabling the reproduction of nanoscale relief features, with corresponding cost advantages.
  • Related injection molding methods are described by A. Neyer et al., 21 and R. Klein and A. Neyer. 22
  • micropatterned reliefs may be used to micromold ceramic pattern ⁇ .
  • these workers used a polyimide relief, prepared by reactive ion etching through a Ti mask, was used as a "cookie cutter" which was impressed into a chemically softened ethyl-methacrylate tape comprising powdered ceramic particles (ceria-zirconia) of average size 0.3 micron ⁇ . After embos ⁇ ing, the tape was dried, removed from the micromold, heated at 600 degrees centigrade to remove the EMA binder and then sintered at 1500 degrees centigrade. Features as small as 4 micron ⁇ could be thu ⁇ produced. The ⁇ e workers have developed this method for the fabrication of piezoelectric ceramics.
  • Self-Assembling Monolayers and Resists Therefrom A paradigmatic example of self-a ⁇ sembling monolayers is provided by the self assembly into monolayer ⁇ of alkanethiols onto gold ⁇ urface ⁇ , according to the strong affinity of thiol groups for gold ⁇ urface ⁇ .
  • the ⁇ e ⁇ elf-assembling monolayer ⁇ y ⁇ tem ⁇ also include organosilanes and derivatives thereof (e.g. RSiX 3 , R2 ⁇ iX2 or R3SiX where R groups may be identical or distinct) , which form covalent linkages to surface hydroxyl groups (e.g.
  • monolayers thus formed may be modified such that the molecules of said monolayers thus formed bear terminal hydroxyl groups, which may then serve as donors for the succe ⁇ ive, covalent addition of monolayer ⁇ , forming cro ⁇ slinked multilayered structures on said surface ⁇ .
  • the organic chains of such monolayers may additionally comprise polymerizable or photopolymerizable groups such as alkynes. 25 Further, such monolayers may be used to render surfaces hydrophobic, such that they are protected from a solution with which said surfaces are contacted, ⁇ uch a ⁇ an aqueou ⁇ etchant solution.
  • SAMs homogenous ⁇ elf-a ⁇ sembling monolayers
  • metal films generally gold films
  • SAMs homogenous ⁇ elf-a ⁇ sembling monolayers
  • These methods include scraping with sharpened tools 26 to produce the desired pattern as well as applying increased force to an AFM tip during scanning operation ⁇ or increa ⁇ ing the ⁇ etpoint current to cause physical contact of an STM tip so said metal surface with increased local force ⁇ re ⁇ ulting.
  • Whitesides and co-workers 27 have also produced a rapidograph- like micropen for the application of organothiol compounds to specified surface regions, and used this writing instrument to produce organothiol patterns on gold surface ⁇ .
  • the underlying ⁇ urface i ⁇ exposed; where said underlying surface is, for example, cry ⁇ talline ⁇ ilicon, it may be wet etched to produce a relief pattern corre ⁇ ponding to the mirror image of the relief surface used in said printing.
  • the ⁇ e method ⁇ are found by these workers to yield a defect rate (of over 1/mm 2 per mask-etch cycle) 28 which they deem unacceptably high to make these methods competitive with conventional lithographic techniques for the production of microelectronic devices. They speculate that these defects primarily owe to microcrystallite ⁇ in ⁇ aid gold film ⁇ , ari ⁇ ing from incomplete coverage of said microcrystallites by said organothiol molecules.
  • Pattern ⁇ of organothiol SAM ⁇ by the ⁇ e method ⁇ have reproducible resolution of features smaller than lOOnm. Such patterns have also been formed on the surfaces of glas ⁇ cylinders and fibers, which have been used ⁇ ubsequently to replicate the corresponding pattern on flat surfaces by a rolling technique, analogous to that u ⁇ ed in the printing of hologram ⁇ in metallized polymeric film ⁇ but on a smaller scale.
  • said first organothiol may have a terminal methyl group such that regions with a SAM of said first organothiol composition are wetted well by organic liquid ⁇ and poorly by aqueous or polar solutions
  • said second organothiol may have a polar terminal group, such a ⁇ a hydroxyl group ⁇ uch that region ⁇ with a SAM of ⁇ aid second organothiol are wetted well by aqueous or polar liquids and poorly by organic liquid ⁇ .
  • ⁇ urface-ten ⁇ ion defined ⁇ tructure ⁇ such as a microlens array 29 from solutions comprising optically clear polymerizable compound ⁇ .
  • Wetting may be conducted, for example, by vapor condensation or dipping into solutions.
  • S.P.A. Fodor et al. 30 have disclosed methods for the spatially directed synthesis of copolymers including biopolymers such a ⁇ oligonucleotides and polypeptide ⁇ . These worker ⁇ bind initiator chemical group ⁇ protected with photolabile protecting groups to a ⁇ ub ⁇ trate. Spatially ma ⁇ ked exposure to appropriate wavelength actinic radiation removes said photolabile protecting groups from expo ⁇ ed region ⁇ , where the extent of ⁇ aid exposed regions is delimited by an exposure mask having a de ⁇ ired radiation ma ⁇ king pattern.
  • Said masked exposure step, said protected monomer contacting steps and said washing ⁇ tep ⁇ are repeated ⁇ o a ⁇ to synthesize desired oligomers at desired locations on said surface ⁇ uch that an array of diver ⁇ e oligomer ⁇ ⁇ ituated on said surface i ⁇ produced and the location of an array element comprising oligomers bears a predetermined relationship to the sequence composition of said oligomer copolymer located at said array element.
  • Such copolymer arrays may be used, depending on the compositions of said copolymer, to detect affinity binding to members of a population of molecules (comprised within the compo ⁇ ition of ⁇ aid array) of a ⁇ ample, which method ⁇ are u ⁇ eful to ⁇ creen antibody specificity, to detect the presence of polynucleotide sequence ⁇ in a ⁇ ample or mixture, and for ⁇ equencing by hybridization method ⁇ .
  • Array Fabrication by Masking for Materials Screening X.-D. Xiang et al. 31 have recently described a method for the ⁇ creening of material ⁇ compri ⁇ ing the repetitive evaporation of different material ⁇ onto a ⁇ urface occluded by a ma ⁇ k, ⁇ uch that an array is formed comprising elements of different compo ⁇ ition, which array i ⁇ then ⁇ intered. Array elements thus prepared may then be subjected to ⁇ creening for de ⁇ ired propertie ⁇ in analogy to the methods of the field of combinatorial chemistry.
  • Whitesides and co-workers 32 have used elastiomeric patterned relief ⁇ urfaces to produce surfaces with correspondingly patterned depo ⁇ it ⁇ of protein ⁇ .
  • thi ⁇ method a gold ⁇ urface i ⁇ patterned with an organothiol, and then expo ⁇ ed to and organothiol comprising terminal hydrophilic moieties are then adsorbed to unpattemed region ⁇ .
  • Such a surface is then contacted with a solution of the de ⁇ ired protein or protein ⁇ , which ad ⁇ orb to the hydrophobic ⁇ urface region ⁇ .
  • Such a method does not provide for the formation of pattern ⁇ of variation ⁇ of combinations of protein ⁇ from one region to the next.
  • Various workers 33 , 34 , 35 have de ⁇ cribed method ⁇ for the stepwise addition of monolayers or bilayers to a surface, such that a multilayer ⁇ tructure compri ⁇ ing a predetermined number of layer ⁇ of only a few nanometer ⁇ in thicknes ⁇ may be formed.
  • a monolayer or bilayer i ⁇ thu ⁇ deposited on a surface including surfaces comprising monolayers, bilayers or multilayers
  • said monolayer or bilayer remains situated upon said surface.
  • S.I. Stupp et al. 36 have ⁇ hown that ⁇ ub ⁇ tantially linear molecule ⁇ compri ⁇ ing extended linear region ⁇ along their length, a polar enantiomeric ordering center and two polymerizable reactive groups of distinct polymerization chemi ⁇ try at a ⁇ pecific positions along their length spontaneously assemble into layered meso ⁇ cale structures which may then be polymerized to yield two dimensional polymer ⁇ .
  • Thi ⁇ work continue ⁇ the teachings of S.I. Stupp disclosed in U.S. Patent Number 5,229,474.
  • M. Kotera, J.-M. Lenh, and J.-P. Vigneron 37 have disclo ⁇ ed compound ⁇ comprising rigid rod molecular segment ⁇ with ⁇ ingle terminal nucleoba ⁇ e moietie ⁇ . Said compounds self as ⁇ emble in organic ⁇ olvent ⁇ to form supramolecular rods according to well-known Watson-Crick pairing rules. The re ⁇ ulting rods self-organize into meso ⁇ cale ⁇ tructures.
  • oligonucleotides were ⁇ ynthesized to comprise a thiol group which was cros ⁇ linked to an amine functionalized organosilane derivatized silica AFM tip. Disruption of association of complementary oligonucleotides was shown to be reversible.
  • Musgrave ha ⁇ undertaken theoretical analyses of factors related to the assembly of diamondoid structures from reactive carbon specie ⁇ under mechanical or po ⁇ itional control and force ⁇ exerted on reactants thereby (referred to a ⁇ mechano ⁇ ynthe ⁇ i ⁇ ) .
  • Objects of the Invention It i ⁇ an object of the pre ⁇ ent invention to reduce the capital co ⁇ ts of microscale and submicron patterning in the fields of microelectronics, micromechanics and MEMS. It i ⁇ a further object to reduce the minimum feature ⁇ ize below that attainable with vi ⁇ ible light in certain embodiment ⁇ . It is an object of further embodiments to facilitate the inexpensive prototyping and low-volume production of such device ⁇ . It i ⁇ a yet further object of the present invention to provide for the inexpensive production of miniaturized ⁇ canned probe device ⁇ . It i ⁇ a further object of the pre ⁇ ent invention to provide for the used of said miniaturized scanned probe devices in the parallel positional synthe ⁇ i ⁇ of complex molecule ⁇ and ⁇ upramolecular assemblages.
  • an object of the pre ⁇ ent invention to provide an inexpen ⁇ ive method for the ⁇ ynthe ⁇ i ⁇ of complex copolymer array ⁇ without exten ⁇ ive u ⁇ e of lithography. It i ⁇ a further object to extend the methods of use of ⁇ uch complex copolymer array ⁇ to provide enhanced capabilitiesitie ⁇ and refinement of discrimination.
  • the present invention employs either contact printing , contact molding, or devices produced thereby, in combination with pattern replication, and applies these to the fabrication of numerous device ⁇ .
  • An initial or master relief pattern or pattern of surface composition is produced by prior art methods or by method ⁇ of the pre ⁇ ent invention.
  • Final generation templates are used in device production.
  • Macroscale to nanoscale features may be achieved by the ⁇ e methods, including within the same fabrication proces ⁇ and article of manufacture.
  • Preferred embodiment ⁇ include the generation of masking patterns for etch control and diffusion barrier formation in semiconductor fabrication, microfluidics fabrication, MEMS fabrication and the like, patterned chemical synthesis and copolymer synthe ⁇ i ⁇ , the formation of patterned molecular monolayer ⁇ and multilayer ⁇ , replication of chemical patterns, fabrication of microstructured and nano ⁇ tructured materials and combinations of any of the foregoing.
  • patterned relief ⁇ urface ⁇ are compo ⁇ ed of materials that are either unreactive to any chemical species which are applied to them or may be pretreated to render them similarly unreactive, or otherwise that any reactivity to said chemical species which are applied to said patterned relief surfaces occurs or may be caused to occur to only a negligible extent.
  • precaution ⁇ must be taken to avoid microscopic contaminants, which cause defects at micron and submicron scales; such precautions include ultrafiltration of solutions, and protection from atmospheric contaminants.
  • adhe ⁇ ion layer ⁇ , re ⁇ i ⁇ t layer ⁇ and ⁇ acrificial layer ⁇ may be used as needed without departing from the scope of the present invention.
  • a re ⁇ i ⁇ t or ma ⁇ king re ⁇ i ⁇ t pattern may be formed upon a ⁇ urface by molding an appropriate material in the de ⁇ ired pattern on ⁇ aid ⁇ urface or by analogou ⁇ technique ⁇ ⁇ uch a ⁇ casting, injection molding, or alternatively by a method comprising the step ⁇ of ⁇ heet stamping followed by a brief pre- etch to eliminate protecting material in reduced thicknes ⁇ region ⁇ formed by ⁇ aid ⁇ heet stamping step.
  • a fir ⁇ t relief pattern i ⁇ replicated in a convenient material, ⁇ uch a ⁇ by the polymerization of a prepolymer (preferably an ela ⁇ tiomer) on ⁇ aid surface, ca ⁇ ting a plastic, polymeric or other appropriate solidifiable liquid material with ⁇ aid fir ⁇ t relief ⁇ urface, plating with one or more metal ⁇ by known art technique ⁇ , or, ⁇ tamping into a melted material such as is practiced in hot-foil technology and CD replication, to define a mold.
  • a prepolymer preferably an ela ⁇ tiomer
  • a mold is thu ⁇ formed with a pattern defined by ⁇ aid fir ⁇ t relief pattern, and has surface features corresponding to the negative pattern formed by ⁇ aid first relief (defined a ⁇ the po ⁇ itive pattern) .
  • ⁇ aid fir ⁇ t relief pattern may be fabricated ⁇ o a ⁇ to it ⁇ elf be the negative pattern, from which further negative pattern ⁇ are generated in even numbered replication generation ⁇ .
  • Said fir ⁇ t relief pattern i ⁇ fabricated either by prior art mean ⁇ or by method ⁇ di ⁇ clo ⁇ ed within the pre ⁇ ent invention.
  • Relief feature height (or depth) is predefined so as to permit reliable pattern formation, reliable cast or molded material self-cohe ⁇ ion and substrate surface adhesion, and also reliable release of said cast or molded material from said from ⁇ aid negative replicated relief.
  • Optimal feature ⁇ izes will vary according to the materials and materials combination ⁇ cho ⁇ en, and ⁇ pecifics of any particular embodiment of this category of methods.
  • Said initial pattern may be used repetitively to form the opposite pattern on a replica surface such that high quality first generation replicas are formed in a quantity increasing arithmetically with each replication cycle.
  • First generation replicas formed by said first relief pattern may be used in turn to generate ⁇ ub ⁇ equent generation replica ⁇ , with multiplicative increa ⁇ e in replica quantity per replication generation.
  • Thu ⁇ tradeoff ⁇ will exist between the quantity of replicas formed in a particular length of time and the quality of replicas thu ⁇ formed.
  • High fidelity replication method ⁇ such a ⁇ are used in the fabrication of integrated optical devices and component ⁇ thu ⁇ permit the rapid production of replica ⁇ ; negative replica ⁇ are then u ⁇ ed to mold a re ⁇ i ⁇ t pattern onto a ⁇ urface.
  • a prototype ma ⁇ ter is used repetitively to create negative replicas neighboring each other on a first generation replica ⁇ urface, which i ⁇ then in turn u ⁇ ed repetitively to create replica ⁇ of the entire adjacent group of first generation replicas, again in an adjacent configuration.
  • a single pattern is repeated an increasing number of times on the surface generated by each generation of replication.
  • advantageous tradeoffs between relief life, rapidity of pattern production, and pattern fidelity may be attained according to the demands (e.g. time, volume, quality) of production. Calculations relating the ⁇ e will be obviou ⁇ from the nece ⁇ ary empirical data and production con ⁇ traints.
  • ⁇ e methods exclude masking material from regions contacted by the highest extent of ⁇ aid negative replicated relief, which, if of elastiomeric composition, will conform to the contours of the sub ⁇ trate ⁇ urface when juxtaposed to said surface under sufficient normal force.
  • molded resist layers may favorably be formed on the ⁇ urface ⁇ of adhesion layers which have first been deposited or coated onto said ⁇ ub ⁇ trate ⁇ urface without departing from the above a ⁇ pect of the present invention.
  • an ultra-thin, uniform adhesion layer which i ⁇ ⁇ ensitive to an etching solution or procedure comprising polymerizable chemical functional groups may first be ⁇ ituated upon a ⁇ ubstrate, which is then, for example, coated uniformly with a photopolymerizable polymeric re ⁇ ist, which i ⁇ then patterned under pre ⁇ ure with a clear relief ⁇ urface and then photopolymerized by expo ⁇ ure to appropriate wavelength light, with the re ⁇ ulting polymeric re ⁇ i ⁇ t material covalently linked to ⁇ aid polymerizable chemical functional groups of said adhesion layer.
  • Said adhe ⁇ ion layer is chosen to not protect the underlying sub ⁇ trate surface from etching treatments or procedures, and is formed at a thickness sufficiently small to prevent the significant under-etch of the overlying resi ⁇ t material (due to the ⁇ mall cro ⁇ - ⁇ ectional area and correspondingly small diffusion rate through such an area) .
  • an underlayer may, when po ⁇ ses ⁇ ed of sufficient elasticity, alternatively permit the u ⁇ e of a metal relief replica a ⁇ said negative replicated relief because ⁇ aid ela ⁇ ticity ⁇ erve ⁇ the ⁇ ame function a ⁇ the ela ⁇ ticity of an ela ⁇ tiomeric mold, i.e.
  • a yet further variation involves the u ⁇ e of metal or other rugged relief ⁇ a ⁇ "cookie-cutter ⁇ " which mechanically exclude a ⁇ oftened re ⁇ i ⁇ t precur ⁇ or such as a melt or sol-gel from surface regions underlying outermost exten ⁇ ion ⁇ of the applied relief ⁇ tructure.
  • a ⁇ oftened re ⁇ i ⁇ t precur ⁇ or such as a melt or sol-gel from surface regions underlying outermost exten ⁇ ion ⁇ of the applied relief ⁇ tructure.
  • Fabrication by Layered Molding of Patterned Article Material and Sacrificial Material Technique ⁇ ⁇ uch a ⁇ tho ⁇ e de ⁇ cribed above for molding of ⁇ tructure ⁇ u ⁇ ing relief masters may be used recur ⁇ ively where each repetition add ⁇ another patterned layer (i.e. a portion of a layer in a predetermined pattern which may additionally have ⁇ urface feature ⁇ or height contour ⁇ ) of material for the de ⁇ ired final article or another patterned layer of ⁇ acrificial material u ⁇ eful in the production of un ⁇ upported or overhanging ⁇ tructure ⁇ .
  • another patterned layer i.e. a portion of a layer in a predetermined pattern which may additionally have ⁇ urface feature ⁇ or height contour ⁇
  • ⁇ acrificial material ⁇ may include waxe ⁇ which are melted away at elevated temperature ⁇ or di ⁇ olved in solvents such as alcohols.
  • the ⁇ e would mo ⁇ t favorably be u ⁇ ed as sacrificial material with polymers that polymerize in aqueous solution or other solution ⁇ in which said wax i ⁇ ⁇ ubstantially insoluble.
  • Sacrificial material may also be used to flatten intermediate surface ⁇ of article material a ⁇ an article i ⁇ being built up by this method.
  • article material is patterned by molding, ca ⁇ ting or ⁇ tamping, and when hardened (if hardening i ⁇ required,) the article under fabrication is juxtaposingly contacted to a flat ⁇ urface which i ⁇ coated with ⁇ oftened or liquefied sacrificial material (where a ⁇ nece ⁇ ary a relea ⁇ e agent i ⁇ u ⁇ ed between said flat surface and ⁇ aid ⁇ acrificial material to facilitate removal of ⁇ aid flat ⁇ urface after ⁇ aid flattened material has set or hardened.)
  • This method is advantageous where an article is conveniently built up in layers with ⁇ ub ⁇ tantially flat border ⁇ .
  • article material i ⁇ preferably cho ⁇ en ⁇ uch that where article precur ⁇ or material ( ⁇ uch a ⁇ a prepolymer) i ⁇ overlaid onto polymerized or hardened article material, chemical cro ⁇ link ⁇ will form between the ⁇ e region ⁇ of two such juxtaposed layers.
  • This will be the ca ⁇ e, for example, for polymers where polymerization occur ⁇ via ⁇ ide-group ⁇ or where there i ⁇ an exce ⁇ of one of the two chemical functionalitie ⁇ which are involved in polymerization, such that a plurality of unreacted functional groups are available on the surface of ⁇ uch article material after polymerization of that portion of article material ha ⁇ proceeded to completion.
  • i. Metal Film Annealing The defects encountered by Whiteside ⁇ and co-worker ⁇ are attributed by the ⁇ e workers to crystallite ⁇ arising in the gold film depo ⁇ ition process. It has been determined 45 that an acetylene flame annealing step yields atomically flat terraces extending hundreds of nanometers. STM shows these surfaces to be better coated with an organothiol derivative (dimethylaminoethanethiol) than the unannealed film.
  • M.D. Ward and co-workers 46 have previously found that annealing a gold wire with a hydrogen flame yield ⁇ atomically flat terraces extending over hundreds of microns.
  • thermal annealing ⁇ tep ⁇ will thu ⁇ reduce the occurrence of defect ⁇ when added, before the formation of the patterned organothiol SAM, to the procedure of White ⁇ ide ⁇ and co-worker ⁇ . It i ⁇ , of cour ⁇ e, nece ⁇ ary that the annealed film be permitted to cool (i.e. thermally equilibrate with the temperature of the relief and applied ⁇ olution) before application of the SAM forming material, becau ⁇ e thermal contraction will otherwi ⁇ e di ⁇ tort the de ⁇ ired pattern.
  • Multilayer Vetoing Defect ⁇ which are ⁇ mall and occur approximately randomly, a ⁇ i ⁇ the ca ⁇ e as the gold-organothiol re ⁇ i ⁇ t ma ⁇ king method of Whitesides et al. , may be prevented from appearing in the etched final product by a vetoing method which bears analogy to the reticle voting method de ⁇ cribed in U.S. Patent Number 5,308,722 by J.L. Ni ⁇ tler.
  • An elastiomeric relief may be prepared by related art methods or as otherwise disclo ⁇ ed in the pre ⁇ ent invention, and then coated with an organothiol ⁇ olution.
  • a thin layer of gold is deposited by suitable means onto a substrate.
  • the pattern of ⁇ aid relief are thu ⁇ tran ⁇ ferred, in the form of a SAM of said organothiol, to the surface of ⁇ aid gold film, in a first printing step.
  • the patterned ⁇ urface thu ⁇ produced i ⁇ then coated again with gold, forming a ⁇ econd gold film layer, and again patterned in a ⁇ econd printing ⁇ tep with the same relief surface coated either with the same or a different organothiol compound ⁇ olution.
  • the uncoated regions of said second gold film layer are then etched by appropriate ⁇ olution ⁇ , ⁇ uch a ⁇ ⁇ trong acid ⁇ , such that the underlying sub ⁇ trate i ⁇ expo ⁇ ed in these unprotected regions and thu ⁇ ⁇ u ⁇ ceptible to etching and impurity diffu ⁇ ion.
  • ⁇ ub ⁇ trate i ⁇ compo ⁇ ed of ⁇ emiconductive material ⁇ electronic, microelectronic, MEMS and other device ⁇ may be produced through processe ⁇ compri ⁇ ing the above method.
  • a ⁇ uming a defect 1 ⁇ quare micron in ⁇ ize occur ⁇ on average once, randomly, in an area of 1mm 2 , the relative defect area of 10 ⁇ ° yields a defect rate per two-layer mask of 10 ⁇ 12 , which should be tolerably low for most applications.
  • Such a defect rate may be reduced further by three or more repetition of the above coating and printing step ⁇ .
  • reliefs may favorably incorporate features which facilitate alignment between succe ⁇ ively applied relief pattern ⁇ , such as features, favorably placed at borders which generate Moire patterns under appropriate illumination and imaging (which may include electron microscopy) due to the overlap of pattern ⁇ in ⁇ aid ⁇ ub ⁇ trate and said negative relief.
  • the methods of the present a ⁇ pect of the present invention very directly also permit accurate tactile alignment, where ⁇ aid feature ⁇ compri ⁇ e interlocking relief ⁇ which only fit together when ⁇ aid relief and ⁇ aid substrate are in precise registry with each other.
  • Such tactile alignment methods may thu ⁇ permit repeatable accuracy to within a few nanometer ⁇ with appropriately de ⁇ igned pattern ⁇ and sufficiently resilient materials.
  • Initiator Polymerization A further approach to defect reduction i ⁇ applicable to defect ⁇ arising from incomplete SAM formation in relief contacted regions.
  • the chemical composition of said SAM is chosen such that the exposed surface of said SAM comprises chemical functional groups which serve as initiators for ⁇ ome polymerizable material.
  • compounds which do not form SAMs may also be used, provided these are reliably deposited on said sub ⁇ trate and do not diffuse; compounds used need only suitably form patterned initiator compound region ⁇ .
  • a quantity of material of ⁇ aid chemical compo ⁇ ition (for example, dissolved in a first solution) i ⁇ applied in ⁇ ufficient but not exce ⁇ ive quantity to a de ⁇ ired patterned ⁇ urface, which i ⁇ then contacted with a ⁇ ub ⁇ trate to tran ⁇ fer ⁇ aid material of ⁇ aid chemical composition to ⁇ aid surface in a pattern determined by the pattern of said patterned ⁇ urface. Said ⁇ urface i ⁇ then washed to remove any exces ⁇ reactant ⁇ or reagent ⁇ .
  • Said material of said desired chemical compo ⁇ ition now ⁇ ituated in a patterned manner on ⁇ aid ⁇ urface, is then contacted with a second solution comprising monomer reactant species which are capable of reacting with said chemical functional groups which serve a ⁇ initiator ⁇ .
  • Said ⁇ econd solution preferably contain ⁇ ⁇ aid monomer reactant species at low concentrations such that molecules of said monomer reactant ⁇ pecies are more likely to react with said chemical functional groups which serve a ⁇ initiator ⁇ , or oligomers or polymers therefrom, rather than with other said molecule ⁇ of said monomer reactant species.
  • the limit on the concentration of said monomer reactant species are those po ⁇ ed by non-specific polymerization, i.e.
  • the term ⁇ ignificant here refer ⁇ to the quality of the overlayer ⁇ thu ⁇ produced and the non-coating of ⁇ ub ⁇ trate region ⁇ not compri ⁇ ing ⁇ aid chemical functional group which ⁇ erve ⁇ a ⁇ initiators, i.e. reduction of resolution in non-resi ⁇ t coated regions.
  • Said second solution may further comprise multimeric monomers or other cro ⁇ linking reagent ⁇ which serve to cause branching of polymer chains during a polymerization proces ⁇ .
  • the concentration of said monomer reactant specie ⁇ , and the proportion or ⁇ urface density of said chemical functional groups are chosen such that the polymer coating formed will be substantially limited to the regions of ⁇ aid ⁇ urface originally contacted by ⁇ aid patterned ⁇ urface, i.e. not extending far from any of ⁇ aid chemical functional groups which serve a ⁇ initiator ⁇ .
  • monomeric species may be added at a limiting quantity, or at a concentration sufficiently low that exces ⁇ ive polymerization does not occur within some conveniently short period of time.
  • Thi ⁇ method of ⁇ urface coating patterning followed by coating thickening (which may be termed overlayer formation) ⁇ erve ⁇ to enclose any small resi ⁇ t layer defect ⁇ without ⁇ ignificantly compromi ⁇ ing patterning re ⁇ olution.
  • Such enclo ⁇ ure reduce ⁇ or prevents transport of etching agents to defect site ⁇ and etching products from said defect sites.
  • this method requires that overlayer thicknes ⁇ be increa ⁇ ed corre ⁇ pondingly to ensure enclosure or encap ⁇ ulation of defect ⁇ of ⁇ aid defect ⁇ ize.
  • said chemical composition of said SAM may be chosen to comprise an alkanethiol moiety and a methacrylate moiety.
  • a solution compri ⁇ ing a quantity of ⁇ aid chemical composition is applied to a gold film situated on a substrate, such that a SAM is formed according to the pattern of said patterned ⁇ urface. Said gold film and substrate is then wa ⁇ hed to remove any exce ⁇ unbound material.
  • Resist Formation by Wettability Control The methods of G.M. Whiteside ⁇ and co-worker ⁇ involving the wetting of patterned surface regions, which these worker ⁇ have used to form microlen ⁇ array ⁇ from polymer ⁇ olution ⁇ , may al ⁇ o be applied to the elimination of small defects.
  • patterns of resist are formed by fir ⁇ t patterning a SAM which i ⁇ preferentially wet by a re ⁇ i ⁇ t monomer ⁇ olution or precursor solution on a surface which is poorly wet by said solution. Said patterning is accomplished by one or more of the methods described herein, such as microcontact printing, and the patterned said surface is then contacted with a resi ⁇ t precursor ⁇ olution, which i ⁇ then withdrawn.
  • Portions of said resist precursor solution retained on said surface are then caused or permitted to polymerize, such that a developed resi ⁇ t layer i ⁇ formed.
  • defect ⁇ related to incomplete ma ⁇ king may be eliminated by their enclosure by the overlying resi ⁇ t precur ⁇ or ⁇ olution and hence the re ⁇ ulting developed resi ⁇ t.
  • Etch ⁇ tep ⁇ are then performed a ⁇ in conventional microfabrication practice.
  • resi ⁇ t depth-dependent etching in analogy to that described by G. Gal in U.S. Patent Number 5,310,623, may be performed, for example by reactive ion etching, to replicate the self-a ⁇ embled resist structure in the underlying solid sub ⁇ trate material.
  • Patterned Metallization for example, for the formation of electrical contact ⁇ , may be accompli ⁇ hed according to the patterning method ⁇ of the present invention and corresponding exten ⁇ ion ⁇ of related art methods, as well as by the application of related art method ⁇ to article ⁇ produced by the method ⁇ of the present invention (e.g. formation of alkanethiol patterns by microcontact printing on gold films, employing, as neces ⁇ ary, the defect reduction method ⁇ disclosed herein) .
  • Molded materials such a ⁇ those used above for resi ⁇ t purposes may, with appropriate solvent ⁇ , be u ⁇ ed a ⁇ lift-off layers which eliminate any metallic film ⁇ ituated on the surface of ⁇ aid molded material ⁇ upon expo ⁇ ure of the ⁇ urface compri ⁇ ing the ⁇ e to said appropriate solvent. Patterns of said metallic film remain in regions which were not covered by said molded materials.
  • metal film ⁇ may be accomplished by vacuum evaporation of the corre ⁇ ponding metallic material onto the ⁇ urface of the article under production.
  • ⁇ uch lift-off patterning may be applied to thin electroformed (e.g. electrodepo ⁇ ited or electrole ⁇ plated) metallization layers.
  • metallization may be accomplished by the hot embos ⁇ ing of a first ⁇ urface with a metallized polymeric foil with the metallized ⁇ urface juxtapo ⁇ ed to ⁇ aid fir ⁇ t ⁇ urface, under ⁇ ufficient pre ⁇ ure and at ⁇ ufficient temperature to en ⁇ ure good electrical contact with expo ⁇ ed region ⁇ of said first ⁇ urface, followed by di ⁇ olution of ⁇ aid polymeric foil and underlying lift-off region ⁇ .
  • Such transferred film ⁇ may be ⁇ ubjected to brief electrodepo ⁇ ition steps to ensure good electrical contact, though care.mu ⁇ t be taken to avoid the formation of ⁇ hort circuits.
  • the foregoing fabrication methods compri ⁇ ing the u ⁇ e of relief pattern ⁇ u ⁇ ed to pattern re ⁇ ist or masking layers may be u ⁇ ed to control the etching and doping (impuritie ⁇ diffu ⁇ ion) of ⁇ emiconductor materials and overlying dielectric layer ⁇ , as well as the formation of metallization layers for electrical interconnection. These methods serve all of the patterning functions required in microelectronic ⁇ fabrication.
  • re ⁇ i ⁇ t molding and ca ⁇ ting methods may similarly be applied to conductive polymers compo ⁇ itions such a ⁇ tho ⁇ e comprising polyparaphenylene and derivatives thereof, polypyrrole and derivatives thereof, polythiophene and derivatives thereof, or other such conducting or electroactive polymer ⁇ , ⁇ o a ⁇ to directly form polymeric device ⁇ rather than re ⁇ i ⁇ t patterns.
  • the ⁇ e material ⁇ and devices based thereupon may further be combined with more conventional microelectronic device ⁇ fabricated by the method ⁇ of the pre ⁇ ent invention.
  • Initial relief pattern ⁇ may be formed in, for example, gla ⁇ or ⁇ ilicon ⁇ urface ⁇ by known art method ⁇ including photolithography, or may be formed by appropriate scanning probe based patterning method ⁇ of related art, or combination ⁇ thereof.
  • a gla ⁇ ⁇ urface may be coated with a gold film, upon which an organothiol SAM i ⁇ formed, which i ⁇ then patterned by STM or field emi ⁇ sion mode STM, followed by an acid etch to remove exposed gold regions and a base etch to remove thus exposed gla ⁇ region ⁇ .
  • a large patterned area may be formed by the replication of a ⁇ maller pattern from an initial relief acro ⁇ ⁇ aid large patterned area, a ⁇ , for example, a ⁇ ingle integrated electronic device pattern layer (e.g. corre ⁇ ponding to a ma ⁇ k layer) repeated many time ⁇ acro ⁇ the ⁇ urface area corre ⁇ ponding to a wafer diameter.
  • the replication methods of the present invention may be applied both to expand the number of occurrences of a pattern on a single surface a ⁇ well a ⁇ the number of ⁇ urface ⁇ thu ⁇ patterned.
  • a relief patterned surface preferably of elastiomeric compo ⁇ ition, may be used to spatially control the pattern of addition of monomer or macromonomer reactant ⁇ to a ⁇ ub ⁇ et of appropriate chemical reactive ⁇ ite ⁇ on a ⁇ ub ⁇ trate ⁇ urface.
  • reactant ⁇ are added to region ⁇ of the ⁇ urface of ⁇ aid ⁇ ub ⁇ trate according to the pattern of ⁇ aid patterned ⁇ urface, permitted or cau ⁇ ed to react, and unreacted or unbound ⁇ pecies are then washed away.
  • said sub ⁇ trate will comprise one or more sub ⁇ tantially flat surface region ⁇ and be prepared by the reaction or ad ⁇ orption of ⁇ ome chemical ⁇ pecie ⁇ to ⁇ aid ⁇ urface ⁇ uch that chemical ⁇ pecie ⁇ having desired reactivitie ⁇ are bound to ⁇ aid ⁇ ubstrate, which is thus capable of being reacted with desired reactant specie ⁇ .
  • ⁇ aid a ⁇ olution may further compri ⁇ e inert compounds which increase the visco ⁇ ity of ⁇ aid ⁇ olutions.
  • Said relief patterned surface is then contacted, in a contacting step, with said substrate to transfer ⁇ aid de ⁇ ired reactant to one or more predetermined regions of said ⁇ ub ⁇ trate.
  • said desired reactant is applied only to the desired region ⁇ , according to the predetermined pattern of said relief patterned surface.
  • the control of precise copolymer sequence thu ⁇ ⁇ ynthe ⁇ ized is favorably achieved, for example, through the u ⁇ e of protection/deprotection or activation chemi ⁇ trie ⁇ to effect ⁇ tep control over reactant addition to the monomer or copolymer ⁇ pecie ⁇ with which said desired reactants are reacted.
  • reactants added will generally comprise a protecting group or an activatable reactive group, which serve to prevent multiple monomer or macromonomer addition to the ⁇ ame molecule.
  • different patterned relief surface ⁇ are u ⁇ ed at different ⁇ tep ⁇ , and may optionally be ⁇ hifted in location from one ⁇ tep to the next; similarly, different desired reactant species may be used during different cycles.
  • This is in analogy to multiple masking step ⁇ in related art microfabrication, and directly related to the multiple masking method ⁇ of other a ⁇ pects of the present invention.
  • arrays of copolymer ⁇ of predetermined ⁇ equence, ⁇ uch a ⁇ oligonucleotide ⁇ , polypeptide ⁇ , biomimetic copolymer ⁇ and non- biological copolymer ⁇ may be produced at a resolution equal to that with which a relief master may be fabricated.
  • Pattern ⁇ having feature ⁇ many time ⁇ ⁇ maller than one micron are thu ⁇ achievable, and corre ⁇ pondingly high den ⁇ itie ⁇ (such as 10° to 10 10 array elements per cm 2 ) may be achieved without extensive use of costly lithographic equipment.
  • Such methods thus carry several advantages over the methods of S.P.A. Fodor et al. in that protection/deprotection chemi ⁇ trie ⁇ (and the corre ⁇ ponding reactant compounds or protected/deactivated) need not be limited to those respon ⁇ ive to actinic radiation or other energetic beam ⁇ .
  • reactant ⁇ useful for ordinary (e.g. not light controlled) stepwi ⁇ e ⁇ ynthe ⁇ es are u ⁇ eful with the ⁇ e methods. Further, these methods are ⁇ uitable for the synthesis of copolymer of compositions which might be or are desired to be sen ⁇ itive to photochemical or other energy induced degradation.
  • ⁇ uch a ⁇ aid patterned ⁇ urface may be u ⁇ ed to control the ⁇ patially controlled activation or deprotection of copolymer ⁇ or copolymer precur ⁇ or ⁇ , ⁇ uch that only the copolymer ⁇ or copolymer precursor ⁇ located at predetermined region ⁇ of ⁇ aid surface are prepared for subsequent reaction with a monomer or macromonomer, which is thereby added only to said copolymers or copolymer precursor ⁇ located at said predetermined region ⁇ .
  • a relief is for example coated with solution ⁇ comprising deprotection or activation reagents at sufficient concentrations (and preferably also inert constituents increasing the viscosity of said solutions) and contacted with said surface to effect the spatially controlled deprotection of the product precursor copolymer ⁇ ⁇ ituated thereupon.
  • a ⁇ olution compri ⁇ ing ⁇ aid monomer or macromonomer i ⁇ then contacted with said s.ub ⁇ trate ⁇ uch that the ⁇ e specie ⁇ may react with any available corresponding reactive groups.
  • the methods of this aspect of the present invention thus permit the synthe ⁇ i ⁇ of a potentially large array of diver ⁇ e copolymer ⁇ pecie ⁇ ⁇ ituated on a ⁇ urface according to a predetermined pattern, ⁇ uch that compo ⁇ ition of the copolymer ⁇ at a particular region of said surface correspond in a known fashion to the location of said region.
  • the copolymer sequence composition respon ⁇ ible for ⁇ aid activitie ⁇ may directly be deduced from information about the spatial location of said particular region ⁇ .
  • thi ⁇ method may be u ⁇ ed to synthesize or otherwise target copolymer ⁇ , affinity groups or other molecular functionalities or decorations to predefined region ⁇ of the surfaces of microreliefs, micromolded component ⁇ and component ⁇ of micromechanical or microelectromechai-ical systems.
  • the reliefs used for pattern formation are produced from master structures which are identical to the molded surfaces in regions to which said reliefs are to contact ⁇ aid microrelief ⁇ , ⁇ aid micromolded component ⁇ , etc., and thus situate reactant monomers or other reactive compounds, but which have surfaces or surface features of increased height at regions not to be targeted by ⁇ aid reactant monomers or other reactive compounds.
  • the reliefs used to target reactant ⁇ to predetermined region ⁇ of contoured ⁇ urface ⁇ conform to the geometry of tho ⁇ e ⁇ urface ⁇ in region ⁇ to be targeted or modified, but do not contact regions to not be affected due to clearance within said relief that prevent ⁇ contact of the ⁇ urface of said relief with regions of ⁇ aid contoured ⁇ urface ⁇ not targeted.
  • reliefs may be mounted on a roller apparatus, in analogy to arrangements used in holograph stamping processes, to facilitate rapid patterned deposition of reactants.
  • rin ⁇ e ⁇ teps may favorably be performed with reagent ⁇ that react with unreacted monomer reactants to inactivate said unreacted reactants such that these will not, upon washing, react with specie ⁇ bound to region ⁇ onto which ⁇ aid unreacted reactant ⁇ were not contacted by ⁇ aid relief.
  • alcoholic ⁇ olution ⁇ may be u ⁇ ed to inactivate and wash away unreacted phosphoramidite monomers, which will react with the hydroxyls of alcohol molecules.
  • a first surface such as a glass surface, is patterned with a first alkyl ⁇ ilane compound compri ⁇ ing an extended methylene or polyethylene glycol linker, or other substantially linear linker chosen to be well solvated or wetted by oligonucleotide synthesis solvents or chosen to be readily modified so as to be well ⁇ olvated or wetted by oligonucleotide synthesis solvent, and an esterified terminal hydroxyl group by use of an elastiomeric relief with a square grid pattern such that ⁇ uare regions of said fir ⁇ t ⁇ urface are modified by said first alkyl ⁇ ilane compound with di ⁇ tinct unmodified border ⁇ (which are hydroxylated) remaining.
  • a first alkyl ⁇ ilane compound compri ⁇ ing an extended methylene or polyethylene glycol linker, or other substantially linear linker chosen to be well solvated or wetted by oligonucleotide synthesis solvents or chosen to be readily modified so as to be well
  • Untreated region ⁇ of ⁇ aid ⁇ urface may then optionally be permitted to react with a ⁇ econd alkyl ⁇ ilane compound compri ⁇ ing a terminal functionality which will be poorly wetted by the ⁇ olvent ⁇ containing oligonucleotide ⁇ ynthe ⁇ i ⁇ reactant monomer ⁇ .
  • Said fir ⁇ t surface is then treated with appropriate reagents such as bases to hydrolyze the terminal ester group of said first alkylsilane and thu ⁇ deprotect the terminal hydroxyl group.
  • a ⁇ olution of 3' or 5' protected oligonucleotide synthesis reactant monomers of one nucleoba ⁇ e type is then coated onto a second ⁇ urface, onto which an ela ⁇ tiomeric relief i ⁇ impre ⁇ ed to tran ⁇ fer ⁇ ome of ⁇ aid reactant to the surface of high regions of said ela ⁇ tiomeric relief (with raised square regions corresponding to a subset of the raised ⁇ quare region ⁇ of the relief u ⁇ ed to form the regular array of said fir ⁇ t alkyl ⁇ ilane) , or the entire ⁇ urface of said elastiomeric relief is directly coated with a solution of ⁇ aid reactant monomer.
  • Such a proce ⁇ s is carried out with different relief pattern ⁇ and the remaining three nucleotide monomers, such that all array elements experience the positionally controlled addition of exactly one of the four nucleotide precur ⁇ or ⁇ pecie ⁇ .
  • ⁇ aid fir ⁇ t ⁇ urface region ⁇ which may have optionally been treated to be poorly wetted by said reactant ⁇ olutions will serve as borders impeding the spreading of these reactants beyond targeted array elements; said well solvated linker moieties are drawn into the solution ⁇ with which they are contacted, even where the ⁇ e are ⁇ ituated on surfaces which are poorly wetted by said solutions with which they are contacted (in which case undesirable spreading of reactants to inappropriate surface regions i ⁇ prevented while ⁇ ynthe ⁇ is is not frustrated) .
  • Said first surface is then thoroughly rinsed.
  • Capping reagents ( ⁇ uch a ⁇ acylating reagent ⁇ ) may optionally be applied to all of ⁇ aid first surface to prevent any further elongation of unreacted hydroxyl groups.
  • Other standard oligonucleotide ⁇ ynthe ⁇ i ⁇ ⁇ tep ⁇ , ⁇ uch a ⁇ oxidation, may then be performed as required by the particular chemistry u ⁇ ed.
  • the entire array i ⁇ then treated with reagents to deprotect the reactant monomers added during the previou ⁇ cycle (e.g.
  • Such cycle ⁇ are repeated until the array thu ⁇ ⁇ ynthesized sub ⁇ tantially consists of oligonucleotides of the de ⁇ ired length (i.e. n cycle ⁇ total to produce an array of n-mer ⁇ ) and de ⁇ ired element ⁇ equence according to po ⁇ ition within ⁇ aid array.
  • ⁇ tep ⁇ deprotecting nucleobase moieties and, where applicable, deprotecting the phosphate backbone are performed to yield the desired final molecular structure, a ⁇ in conventional oligonucleotide synthesis methods, excepting support removal step ⁇ .
  • Alternative copolymer array patterned synthesis methods may be used to produce a stencil capable of masking surface regions from exposure to reactants.
  • a substrate is first coated uniformly with a sacrificial layer, which is hardened or set.
  • an ela ⁇ tiomer precursor i ⁇ applied to the surface of said sacrificial layer and then molded by a negative relief.
  • analogous ca ⁇ ting or injection molding may be utilized, or embo ⁇ ing of a polymeric film with brief po ⁇ t-etching of the embossed film may be used.
  • Said sacrificial layer is then di ⁇ olved to free the patterned elastiomeric stencil.
  • Such a stencil is used in combination with an array of microfabricated columns, said columns being of uniform height, and with a pitch smaller than the smallest stencil feature.
  • Said stencil may either be combined with said array of microfabricated columns into one ⁇ tenciling body, or said stencil and said columns may merely be fu ⁇ ed together. In either case, said stencil is juxtaposed to the substrate which is to be subjected to patterned copolymer synthesis, and said array of microfabricated columns i ⁇ pre ⁇ ed onto ⁇ aid stencil, such that said columns force the ⁇ tencil ⁇ urface into good ma ⁇ king contact with ⁇ aid ⁇ ub ⁇ trate.
  • Solution ⁇ containing reactants e.g.
  • deprotection reagents or monomer reactants are then cau ⁇ ed to flow through said array of microfabricated columns, such that areas not masked by said stencil are exposed to said ⁇ olution ⁇ .
  • ⁇ tencil i ⁇ released from said sub ⁇ trate thorough wa ⁇ h ⁇ tep ⁇ are performed to remove re ⁇ idual reactant ⁇ or reagent ⁇ .
  • Differently patterned ⁇ tencil ⁇ are used in predetermines sequence with different monomer type ⁇ to produce the desired diverse copolymer array with sequence correlated to location in a predetermined manner.
  • said array microfabricated columns is to form one body with said stencil, these may be joined together, e.g. by coating the upper surface of said stencil with an adhesive or cro ⁇ linking agent which will react with both the material of ⁇ aid columns and the material of said stencil.
  • Compounds used for such joining mu ⁇ t be selected so as to be inert to the reagents and solvents which will be pas ⁇ ed over them.
  • said array of microfabricated columns may favorably be produced by LIGA methods or other replication methods including those of the present invention, and may favorably be composed of metals, e.g. formed by electrodeposition or electroless plating methods.
  • a ⁇ pect of the present invention may be used in a variety of ways including for sequencing by hybridization (where said copolymers are oligonucleotide ⁇ or oligonucleotide analogs) , sequence confirmation by hybridization, for the ⁇ creening of small peptides and the determination of affinity interactions (a ⁇ described by Fodor et al., referenced elsewhere herein), for the detection or determination of antibodies, in clinical medical as ⁇ ays, and in the screening of organic material for desired propertie ⁇ according to known method ⁇ in combinatorial chemi ⁇ try.
  • Sequencing by hybridization, a ⁇ well a ⁇ other technique ⁇ involving binding determination may favorably be performed by contacting ⁇ ample ⁇ with such an array, where sample molecule ⁇ binding to said array is monitored by any of se ral known art techniques, while physical or chemical condition ⁇ are changed and binding is recorded (e.g. by capture of signal ⁇ detected by ⁇ uch monitoring and recorded electronically) a ⁇ a function of condition ⁇ .
  • the condition dependent propertie ⁇ of set ⁇ of array elements (i.e. copolymer sequences) of interest are determined by observation of condition dependent binding of known samples, and information thus obtained i ⁇ used to further refine the discrimination of correct (i.e. most highly matched) or most specific binding from incorrect or relaxed specificity binding.
  • condition ⁇ which may be varied include temperature, ionic ⁇ trength, divalent cation concentration, pre ⁇ ence and/or concentration of tetramethylamonium chloride, pre ⁇ ence and/or concentration of denaturant compound ⁇ ⁇ uch a ⁇ formamide or dimethylformamide, pH, and other factors known to affect the hybridization specificity and stringency of oligonucleotides or polynucleotides, may be varied individually or in combination.
  • the binding, and also di ⁇ ociation of a particular ⁇ equence with the complementary n-mer oligonucleotide will occur at a characteri ⁇ tic temperature (i.e. having a characteri ⁇ tic T m melting temperature at which half of all homoduplex complexes dis ⁇ ociate) , and thi ⁇ property verifie ⁇ the correctne ⁇ of ba ⁇ e pairing.
  • Such array ⁇ may be used to detect very low quantities, approaching single molecules, of complementary molecules in samples by a few distinct methods.
  • an oligonucleotide array is produce by forming first square regions, subdivided into four square region ⁇ each containing one of adenosine, guanosine, cyto ⁇ ine or thymidine nucleotide ⁇ (e.g.
  • each ⁇ tep adding one nucleotide to one subdivision region) .
  • succe ⁇ ive synthesi ⁇ ⁇ tep ⁇ identical ⁇ quare ⁇ with ⁇ ides one half that of those of the preceding ⁇ tep and area one quarter that of the preceding ⁇ tep are formed, again ⁇ ubdivided into four square regions each containing one of the four nucleotide ⁇ , preferably in the same relative configuration as that of the preceding set of ⁇ tep ⁇ , are added, ⁇ uch that one subdivi ⁇ ion ⁇ quare of the previou ⁇ step is thus subdivided into four subdivisions.
  • a set of array ⁇ of oligonucleotide ⁇ of increa ⁇ ing length e.g. ⁇ (n-m) , (n-m+1) ... (n-l) , n ⁇ -mers in the case of single nucleotide increments, is produced as above.
  • This set of array ⁇ i ⁇ designed such that each successive ⁇ ive array in the series is in the mirror image of the preceding array of said series, with at least one further ⁇ ynthe ⁇ i ⁇ cycle (set of synthesi ⁇ steps) further subdividing the region ⁇ oppo ⁇ ed to the smallest array elements of the preceding array.
  • ⁇ aid sample is bound first to the array consisting of the smalle ⁇ t number of elements.
  • the sample is divided such that each oligonucleotide targets a region of some ⁇ ub ⁇ et of molecule ⁇ in ⁇ aid sample.
  • Hybridization to such a lower density array by rare species will be more accurate under appropriate conditions in that fewer array elements of larger area present more molecular target ⁇ and fewer degree ⁇ of freedom.
  • the kinetic limitation on binding depends mainly on the diffu ⁇ ional transit time from across said array.
  • Conditions are changed to denature the as ⁇ ociation of ⁇ ample molecules with said fir ⁇ t array, and then to binding conditions sufficiently stringent that ⁇ aid ⁇ ample molecule ⁇ will favorably bind to their target oligonucleotide ⁇ on said second array in preference to forming the lower energy association with their shorter targets on said first array.
  • This proce ⁇ s result ⁇ in ⁇ uccessive subdivision of said sample, such that each subdivision step ⁇ ucce ⁇ ively narrow ⁇ the volume which mu ⁇ t be traver ⁇ ed by a ⁇ ample molecule in order to find it ⁇ correct binding target, and further correspondingly reduces the chances that a molecule will find an incorrect target.
  • An analogous method within the scope of this fir ⁇ t method may be applied where an array is designed, as above, such that array elements are similar (but not identical) in sequence to most of their nearest neighbors, such that spatial proximity is related to ordered sequence ⁇ imilarity.
  • This similarity may be used to serve a ⁇ imilar "narrowing down" function accompli ⁇ hed above by juxtapo ⁇ ition of hierarchially related mirror image array ⁇ .
  • condition ⁇ affecting binding ⁇ tringency are varied ⁇ uch that after a sample is first applied, only a few contiguous base pairs are sufficient for binding.
  • the regions bound by a sample molecule comprising a specific sequence may either be within the contiguous region compri ⁇ ing many array element ⁇ having m of the total n monomer ⁇ in common ( ⁇ tarting from the fir ⁇ t variable nonomer added during the fir ⁇ t cycle of array synthe ⁇ is), or with m contiguous monomers off ⁇ et from the copolymer terminal from which ⁇ ynthesis began, in which ca ⁇ e, for each cla ⁇ s of array elements with said m monomers in common, contiguou ⁇ region ⁇ of element ⁇ compri ⁇ ing said m monomer ⁇ equence complementary to said specific sequence, where cla ⁇ ses are di ⁇ tingui ⁇ hed according to the number of monomer ⁇ by which ⁇ aid m monomer ⁇ equence i ⁇ off ⁇ et from ⁇ aid fir ⁇ t variable monomer, become increasingly small and increasingly di ⁇ tant from each other.
  • Stringency i.e. the condition ⁇ affecting ⁇ tringency
  • Stringency may be o ⁇ cillated in an increa ⁇ ing trend, such that sample molecules are mo ⁇ t likely to home in on their final target oligonucleotide ⁇ .
  • sample molecules which do not initially bind sufficiently near their targets will enjoy increased diffusional mobility as those sample molecules which have bound to said array are less likely to impede their diffusional transport through the sample ⁇ olution, e.g. after they no longer bind m-mer comprising elements further from their correct targets under more stringent condition ⁇ .
  • Generalizing thi ⁇ method to any copolymer with sequence dependent binding properties, phy ⁇ ical and chemical condition ⁇ may be varied to examine the characteri ⁇ tic ⁇ of, or detect the pre ⁇ ence of, binding activitie ⁇ corresponding to respective ⁇ et ⁇ of condition ⁇ , or ⁇ patial or temporal gradient ⁇ thereof.
  • a second method, applicable to polynucleotide ⁇ ample ⁇ involve ⁇ the amplification of rare specie ⁇ , or ⁇ equence fragments thereof.
  • e ⁇ tablished art methods such as the polymerase chain reaction (e.g. with PCR primers flanking the diverse target sequence) or the ligase chain reaction (e.g. for an 8-mer array, involving all 4-mer oligos under conditions ⁇ ufficiently ⁇ tringent to preclude any mi ⁇ match within said 4-mer ⁇ equence ⁇ for binding) are used to amplify the sample target sequence ⁇ , which may be a diverse sequence in a conserved sequence context.
  • Amplification reagent ⁇ e.g. primer ⁇ or nucleotide ⁇ for PCR or oligonucleotides for LCR
  • Amplification reagent ⁇ may be labeled, for example, with one or more affinity moieties (e.g. biotin, digoxigenin, etc.,) or one or more fluorescent dye moietie ⁇ (e.g. Texa ⁇ red, rhodamine, fluoroe ⁇ cien, etc.,) or one or more other reporter group ⁇ or portion ⁇ .
  • affinity moieties e.g. biotin, digoxigenin, etc.,
  • fluorescent dye moietie ⁇ e.g. Texa ⁇ red, rhodamine, fluoroe ⁇ cien, etc.
  • amplification will produce a geometrically increasing quantity of sequences matching the amplified sequences but also a related quantity (which may be biased by such known art methods as asymmetric PCR)
  • Similar operations as used to synthesize copolymers on a ⁇ urface in a ⁇ patially predetermined manner de ⁇ cribed above may similarly be used to collocalize multiple distinct macromolecule or macromolecule type ⁇ to predetermined ⁇ urface regions.
  • such methods may be u ⁇ ed to generate a very large number of po ⁇ itionally predetermined combination ⁇ , ⁇ hapes and/or patterns of large numbers of adhesion proteins which may be used , for example, to test the effect on cell adhe ⁇ ion, cell motility, cell ⁇ hape or cell phy ⁇ iology in biological and clinical a ⁇ ay ⁇ , as shown by Whiteside ⁇ and co-workers for patterns consisting of a single protein specie ⁇ , as well as in other biotechnological uses, including the interfacing of cells to microelectronic device ⁇ and sensors.
  • the pre ⁇ ent a ⁇ pect of thi ⁇ invention thus depends on the u ⁇ e of a relief pattern compri ⁇ ing raised surface elements to contact a solution containing one or more protein or polypeptide to portions of a fir ⁇ t ⁇ urface to which ⁇ aid protein or polypeptide will adsorb, wherein contact only occurs between said rai ⁇ ed surface elements of said relief pattern and said fir ⁇ t surface, such that a pattern of said protein or polypeptide is depo ⁇ ited in the pattern corre ⁇ ponding to ⁇ aid rai ⁇ ed surface elements onto said first surface, wherein such patterning step ⁇ are repeated to yield depo ⁇ it ⁇ of plural different proteins or polypeptide ⁇ at different region ⁇ of ⁇ aid fir ⁇ t ⁇ urface, ⁇ uch that different combination ⁇ result in a spatially predetermined manner.
  • Patterned Combinatorial Materials Deposition :
  • X.-D. Xiang et al. 49 have recently de ⁇ cribed a method for the production of array ⁇ of combination ⁇ of material, which are then ⁇ creened to di ⁇ cover combinations with useful properties.
  • the patterning methods de ⁇ cribed herein may be extended to the production of such arrays but further permitting the rapid, selective deposition of materials which are not conveniently evaporated or without the use of evaporation equipment.
  • a su ⁇ pen ⁇ ion of very fine particle ⁇ of a material is applied to the surface of a wettable relief, which i ⁇ preferably of ela ⁇ tiomeric compo ⁇ ition.
  • said flat ⁇ urface mu ⁇ t be that of a porous filter, so that negative pressure may be used to draw the suspen ⁇ ion solution through said filter, depositing suspended material onto said filter surface.
  • Template Patterned Synthesis of Sheet-like Complexes and Copolymers permits the use of ⁇ uch chemically patterned ⁇ urfaces as templates for the production of patterned molecular complexes and copolymers, which themselves may be used as templates for co plementarily patterned molecular complexes and copolymers.
  • This aspect of the present invention i ⁇ analogous with other aspects of the present invention in that spatially patterned surface ⁇ may be replicated from an initial pattern and thereafter from each other.
  • patterned compositions of sheet-like molecules serve as molecular template ⁇ for the definition of patterns in thus complementarily patterned ⁇ heet- like molecules in a molecular replication process analogous to the polymerase chain reaction invented by K. Mulli ⁇ for the amplification of polynucleotide molecule ⁇ .
  • a ⁇ well as scanning probe based patterning methods 50 ' 51 ' 52 may be used to perform the patterned chemical derivatization of surfaces.
  • modifications may cause the preferential binding of molecular or macromolecular species to predetermined regions of said surface ⁇ , which are demarcated by said patterned chemical derivatization.
  • Such surface modifications may be used, for example with the methods of G.M. Whitesides et. al. or those of W.T. Muller, P.G. Schultz, et al., to bind specific fir ⁇ t affinity group ⁇ or species to predetermined regions of said surface.
  • Said affinity group ⁇ or ⁇ pecie ⁇ are chosen so as to bind affinity derivatized layer forming molecules comprising a second set of affinity groups or species which are in communication with a layer forming precursor moiety (such as a polymethylene chain, or rigid rod oligomer or polymer) .
  • the affinity moiety of ⁇ aid second ⁇ et of affinity group ⁇ i ⁇ preferably located at one terminu ⁇ of ⁇ aid affinity derivatized layer forming molecule ⁇ , which are preferably ⁇ ub ⁇ tantially linear in ⁇ tructure.
  • Said affinity derivatized layer forming molecule ⁇ preferably but not nece ⁇ arily further compri ⁇ e a moiety of third affinity group or ⁇ pecie ⁇ , which i ⁇ preferably located at the terminus opposite that at which said affinity moiety of said ⁇ econd ⁇ et of affinity group ⁇ i ⁇ located;
  • ⁇ aid affinity derivatized layer forming molecules may also comprise one or more (preferably two or more) distinct reactive, cross-linkable or photopolymerizable chemical functional groups, situated along said layer forming molecules such that each of said distinct reactive, cro ⁇ -linkable or photopolymerizable chemical functional group ⁇ occur ⁇ at a particular po ⁇ ition along the length of said layer forming molecules, and may, after a layered structure ha ⁇ been formed by a ⁇ ociation of moieties ⁇ elected from said second set of affinity groups with said fir ⁇ t affinity group ⁇ pattered on ⁇ aid ⁇ urface, be cau ⁇ ed to react to form a covalently linked network joining said layer forming
  • Said first affinity groups and said second affinity groups are preferably chosen such that the interaction between them i ⁇ ⁇ ufficiently ⁇ trong and ⁇ pecific under a first set of condition ⁇ but i ⁇ substantially weakened or neutralized under a second set of conditions; thus, under said first said of conditions, layer forming molecules assemble at surface ⁇ comprising regions of said fir ⁇ t affinity group in pattern ⁇ corre ⁇ ponding to the pattern of ⁇ aid region ⁇ , while ⁇ aid ⁇ econd ⁇ et of condition ⁇ serve ⁇ to ⁇ eparate layer ⁇ compri ⁇ ing said affinity group ⁇ chosen from among ⁇ aid second set of affinity groups from surfaces (including surfaces of layers) comprising said first affinity groups.
  • Said third affinity group or species is chosen such that it does not bind to either of ⁇ aid fir ⁇ t affinity group ⁇ or ⁇ aid second set of affinity groups, but will bind to affinity groups selected from a fourth set of affinity groups, which in turn will not bind to said first affinity groups, other affinity groups selected from said fourth set of affinity groups, nor to said second affinity group ⁇ (i.e. ⁇ aid fir ⁇ t affinity group ⁇ and affinity groups of said second ⁇ et of affinity groups bind only to each other and not to identical affinity group ⁇ ; said affinity groups cho ⁇ en from ⁇ aid third ⁇ et of affinity groups and said affinity groups chosen from said fourth set of affinity groups bind only to each other and not to them ⁇ elves; e.g.
  • sets of affinity groups may each consist of one of adenine, thymine, cytosine or guanosine nucleobases) .
  • the composition of said surface in unpattemed (or differently derivatized region ⁇ ) i ⁇ cho ⁇ en to not bind ⁇ aid affinity derivatized layer forming molecule ⁇ .
  • a solution containing ⁇ aid affinity derivatized layer forming molecules is contacted with said surface under appropriate conditions and for sufficient time to permit the binding of ⁇ aid affinity derivatized layer forming molecules to all available ⁇ ite ⁇ on ⁇ aid ⁇ urface. Unbound species are washed from said surface.
  • said layer forming molecules comprising affinity groups from said ⁇ econd set of affinity group ⁇ are bound to ⁇ aid ⁇ urface in a pattern replicating the pattern of said fir ⁇ t affinity group ⁇ on said surface. These may then be cros ⁇ -linked via said reactive group ⁇ , for example by the addition of suitable cros ⁇ -linking agent ⁇ , photopolymerized, or cau ⁇ ed to react together by e ⁇ tabli ⁇ hing appropriate phy ⁇ ical or chemical conditions such that a covalent network is formed.
  • said affinity group chosen from said third set of affinity group ⁇ may be initially caged or protected to ⁇ uppress the formation of association ⁇ until a deprotection step is performed.
  • affinity group ⁇ chosen from said fourth set of affinity group may be reacted with regions of said surface not containing said fir ⁇ t affinity group, in direct analogy to the complementarily patterned two component monolayers disclo ⁇ ed by G.M. White ⁇ ide ⁇ and co-workers, formed by patterning a first component monolayer and then permitting a second component compound to self-assemble into a monolayer on remaining uncoated regions.
  • pattern ⁇ composed of domains of multiple distinct affinity moietie ⁇ may be formed by multiple relief contacting ⁇ tep ⁇ , or by multiple spatially selective activation ⁇ tep ⁇ (example ⁇ include: light directed, for photodeprotectable or photoactivatable compound derivated surface ⁇ ; ⁇ canning probe controlled surface cataly ⁇ i ⁇ such a ⁇ the method ⁇ of W.T. Muller, P.G. Schultz et al., or of B.J. Mclntyre, M. Salmeron and G.A.
  • Somorjai, or other ⁇ canning probe based patterning methods capable of directing the adsorption or reaction of molecules comprising the desired affinity moieties to the selected regions of patterned surfaces; and, the replication methods of related art or of the present invention, e.g. involving microcontact printing) .
  • Multiple distinct molecular ⁇ pecie ⁇ comprising multiple distinct affinity moiety types or clas ⁇ e ⁇ and type ⁇ may be patterned onto the ⁇ ame ⁇ urface by repetitive cycle ⁇ of patterned activation or patterned masking followed by deposition of the respective compound (e.g. by contacting a solution of said respective compound to said patterned activated or patterned ma ⁇ ked ⁇ urface) ⁇ uch that deposition occurs at all available site ⁇ .
  • a fir ⁇ t pattern may be formed, and the region ⁇ thu ⁇ ⁇ pecified cau ⁇ ed to react with a first chemical compound by contacting ⁇ aid surface with ⁇ aid fir ⁇ t chemical compound or ⁇ olution ⁇ thereof, a ⁇ econd pattern may be formed and the regions thus specified cau ⁇ ed to react with a second chemical compound by contacting said surface with said second chemical compound or solution ⁇ thereof, and so on, until patterns of all de ⁇ ired pattern ⁇ of all de ⁇ ired chemical compound ⁇ (comprising the corresponding di ⁇ tinct affinity moietie ⁇ ) are formed.
  • affinity groups on each side of said sheet-like polymer may then be used, by similar steps as those used to produce said sheet-like polymer, as a template for the formation of complementarily patterned ⁇ heet-like polymer ⁇ by expo ⁇ ure to appropriate affinity group terminally functionalized layer forming monomer ⁇ .
  • Repetition of ⁇ uch layer forming ⁇ tep ⁇ thus provides geometric amplification (here for fabrication purpose ⁇ ) of the patterned ⁇ heet-like polymer, in analogy to PCR, provided that the sheet-like polymer produced at each step is freed, as above, from the template surface which directed its formation.
  • Pattern ⁇ thu ⁇ generated may be designed to facilitate orderly binding together of such layers under appropriate conditions (e.g. thermal annealing) into bi-layer or multilayer ⁇ tructure ⁇ , in analogy to the hybridization of DNA molecule ⁇ .
  • Such design will favorably include pattern elements that ensure proper sheet-like alignment with proper registry before bulk or random associations between surface regions of said layers are permitted to occur.
  • one corner of each of two sheet-like structures to be juxtaposed may comprise thiol groups, which thus target the ⁇ e corners to each other before affinity interactions are permitted to occur.
  • a second set of corner ⁇ may compri ⁇ e primary hydroxyl ⁇ , which are then cro ⁇ linked by an appropriate crosslinking agent, such as a bifunctional acid anhydride compound.
  • an appropriate crosslinking agent such as a bifunctional acid anhydride compound.
  • Dissolving such a substrate permits the overlying layer to be situated at a phase boundary, and i ⁇ favorably accomplished by slowly lowering said ⁇ ub ⁇ trate into ⁇ aid ⁇ olution which di ⁇ olve ⁇ it, such that once it is dis ⁇ olved, said overlying layer is situated at ⁇ aid pha ⁇ e boundary. After the preceding ⁇ tep ⁇ , there would be two stacked monolayer ⁇ at thi ⁇ point. Condition ⁇ (e.g. temperature, compo ⁇ ition, etc.,) are adjusted such that a dissoluble solid may again be pas ⁇ ed through said ⁇ olution without di ⁇ solution, to capture said overlying layer previously situated at said phase boundary.
  • Condition ⁇ e.g. temperature, compo ⁇ ition, etc.
  • ⁇ uch a method comprises the steps of: forming an interfacial layer, e.g.
  • a ⁇ ub ⁇ trate used with this embodiment may be of a polymeric composition comprising photolabile linkages.
  • Said polymeric composition comprising photolabile linkages is preferably chosen such that the photodegradation products will be soluble in the ⁇ olution u ⁇ ed to form interfacial film ⁇ or to support thus liberated films, and to not bind with or otherwise interfere with these films.
  • compo ⁇ ition ⁇ are suitable for substrates.
  • sub ⁇ trate ⁇ are of polymeric compo ⁇ ition
  • the ⁇ e are mo ⁇ t preferably ca ⁇ t with a highly flat surface, simple examples of which include glass and freshly cleaved mica.
  • substrate ⁇ are floated or otherwise held at the surface of the solution to support the overlying film supported by said substrate after dissolution of said substrate, and the substrate is exposed to a sufficient intensity of light of a frequency chosen to cau ⁇ e the degradation of said photolabile linkages for a sufficient length of time to effect complete di ⁇ olution of ⁇ aid ⁇ ub ⁇ trate, with further time being allowed a ⁇ nece ⁇ sary for degradation products to diffuse away in thi ⁇ solution from the interfacial layer.
  • a proce ⁇ may alternatively avail a release layer, which may be produced a ⁇ a ⁇ elf-as ⁇ embling monolayer, for example compri ⁇ ing photolabile or thermolabile linkage ⁇ situated on the surface of an insoluble ⁇ ub ⁇ trate.
  • composition of said relea ⁇ e layer is preferably chosen ⁇ o that the di ⁇ rupted layer sub ⁇ equently produced is well wetted by the solution ⁇ u ⁇ ed, and such that the free degradation product ⁇ (tho ⁇ e not bound to the sub ⁇ trate ⁇ urface) are well solvated by the solution ⁇ u ⁇ ed.
  • This embodiment describes first device ⁇ for the parallel po ⁇ itional ⁇ ynthe ⁇ i ⁇ of molecular and ⁇ upramolecular object ⁇ or article ⁇ (termed workpiece ⁇ or workpiece precursors) which may also be devices, method ⁇ for the fabrication of ⁇ aid fir ⁇ t device ⁇ according to the general microfabrication method ⁇ of the pre ⁇ ent invention, and method ⁇ for the use of said fir ⁇ t devices to effect positionally controlled molecular and supramolecular synthe ⁇ i ⁇ .
  • Terminology u ⁇ ed will include terminology from the field of art of ⁇ canning probe micro ⁇ copy.
  • a capacitance based actuator is produced by forming electrodes on a surface which serves as a first plate of a parallel plate capacitor. Two categories of capacitive actuators are possible: unfilled (i.e.
  • capacitors where the coulombic force between capacitor plates ari ⁇ ing from the charge separation due to applied voltage difference is opposed by force ⁇ associated with mechanical flexure of a second capacitor plate partially or completely ⁇ uspended over said first plate ⁇ uch that a cantilever arrangement results; and filled capacitors wherein ⁇ aid fir ⁇ t plate i ⁇ coated with an ela ⁇ tiomeric or other compre ⁇ sible substance, according to the molding or casting methods of the present invention, upon which a second plate of a parallel plate capacitor is formed, where compre ⁇ ion of ⁇ aid ela ⁇ tiomeric or other compressible substance oppose ⁇ the coulombic force ⁇ resulting from charge separation acros ⁇ the gap of ⁇ aid parallel plate capacitor.
  • compo ⁇ ition of the ⁇ ubstance between capacitor plates may further be chosen according to dielectric properties in addition to mechanical propertie ⁇ .
  • the functional characteri ⁇ tic ⁇ of both leg ⁇ of actuator ⁇ will be highly dependent on geometrical factor ⁇ and on material ⁇ properties, providing a large range of realizable device characteri ⁇ tics according to tradeoffs elected in design and fabrication.
  • Each such capacitive actuator may be placed under control of a different voltage signal, under electronic control, favorably with the electronic circuitry effecting thi ⁇ control integrated into the same array device, according to conventional electronic device integration de ⁇ ign practice ⁇ and, for example, the microfabrication method ⁇ of the pre ⁇ ent invention, such that each capacitive actuator may be tran ⁇ lated to a position independently of other capacitive actuators in the same array.
  • the direction normal or perpendicular to the ⁇ urface upon which said array of actuator ⁇ i ⁇ formed shall be denoted as the Z-axi ⁇ , which is also the axis ⁇ ub ⁇ tantially perpendicular to the plane of the plate ⁇ of ⁇ aid parallel plate capacitor actuator ⁇ .
  • the portions of said Z-actuators furthe ⁇ t from the subatrate may further compri ⁇ e or be in communication with a small cantilever, the purpose of which i ⁇ to facilitate the mea ⁇ urement of force ⁇ acting upon any tips associated with said cantilever.
  • Said forces may conveniently be measured by reflecting a laser beam from the bottom surface (i.e. the surface nearer to said substrate, which in this instance is chosen to be transparent) and monitoring and changes in the reflected angle of said la ⁇ er beam (e.g. with a split photodiode arrangement as is commonly used as detection means in AFM ⁇ ), or by other mean ⁇ which have been employed in the design of AFM cantilever deflection monitoring means (e.g. capacitive detection, tunneling detection, piezore ⁇ i ⁇ tivity change detection, etc.,.)
  • the extent (proximity) of said tunneling contact to said conductive layer on said cantilever may be self-aligned by electroplating at said tunneling contact until a desired current is observed between ⁇ aid conductive layer and ⁇ aid tunneling contact, while ⁇ aid cantilever i ⁇ at it ⁇ equilibrium (undeflected) po ⁇ ition, optionally followed by a brief rever ⁇ ed potential to di ⁇ olve a very ⁇ mall portion of the depo ⁇ ited metal from the ⁇ urface of said tunneling contact to establi ⁇ h a predetermined gap ⁇ ize.
  • Detection methods relying on ⁇ uch ⁇ tructure ⁇ may additionally rely on detection of variation in field emitted current between ⁇ aid conductive layer and the conductive ⁇ tructure termed a tunneling contact above in the ca ⁇ e of tunneling current ba ⁇ ed deflection detection.
  • the signals corresponding to the deflections thus detected may, as in conventional AFM sy ⁇ tem ⁇ , be u ⁇ ed to adjust the position of said Z- actuators to restore a desired set point value.
  • other regimes of AFM and SPM will be pos ⁇ ible with the appropriate modification ⁇ or exten ⁇ ions of the present array format.
  • the externally exposed surface of the topmost plate of said parallel plate capacitor, or layers formed upon ⁇ aid externally expo ⁇ ed ⁇ urface may be further patterned ⁇ uch that a confined ⁇ urface region, of predetermined ⁇ ize and ⁇ urface elemental or chemical compo ⁇ ition i ⁇ produced in communication with the moving portion of ⁇ aid capacitor actuator.
  • a ma ⁇ king pattern may be formed on the exposed surface of a gold said topmost plate ⁇ uch that only a ⁇ mall region, for example ⁇ maller than lOOnm by lOOnm ⁇ quare of bare gold remain ⁇ exposed.
  • ⁇ aid square of bare gold may be u ⁇ ed a ⁇ a target for the random ad ⁇ orption of organothiol molecule ⁇ or molecular complexes.
  • adsorption i ⁇ preferably carried out ⁇ uch that depo ⁇ ition of ⁇ aid organothiol molecule ⁇ or molecular complexes occurs at a rate of one per pad or region, i.e. preferably exactly one per actuator, but at least on average one per actuator in the case of random deposition.
  • small objects may be ⁇ ubstituted for said organothiol molecules or molecular complexe ⁇ , in which case the ⁇ aid externally expo ⁇ ed ⁇ urface or coating ⁇ thereupon are cho ⁇ en so as to ⁇ tably bind the corre ⁇ ponding ⁇ aid ⁇ mall objects selected.
  • Said small object ⁇ may be ⁇ elected from among: macromolecules, enzymes and conjugates thereof, biological receptors, immunoglobulins or fragment ⁇ thereof or conjugate ⁇ thereof, colloids, nanocrystallite ⁇ , polymeric bead ⁇ , dendrimers, fullerene derivatives, nanotube derivatives, mesoscopically structured single objects or other small objects of predetermined geometries or geometrical place ⁇ .
  • the ⁇ e molecule ⁇ , molecular complexe ⁇ or other small objects serve as an extremities or projections in communication with actuator pad ⁇ ; the ⁇ e will be generically referred to herein a ⁇ tip ⁇ .
  • tips include any of the above small objects in communication via a flexible molecular linker to a chemical functional group which binds stably to said pads, such that ⁇ aid small object i ⁇ po ⁇ itionally con ⁇ trained according to the flexibility and tethering of ⁇ aid linker, but not precisely po ⁇ itioned.
  • another type of tip may be formed by electron beam depo ⁇ ition of vaporized ⁇ ub ⁇ tance ⁇ onto ⁇ aid pad ⁇ .
  • actuator array ⁇ may homogeneously consi ⁇ t of a ⁇ ingle pad type decorated by a ⁇ ingle type of tip, multiple pad type ⁇ (i.e. multiple pads of each type where each of said types has a different ⁇ urface chemical composition) decorated on a one-to-one composition basis by corresponding multiple small object types, or may comprise different pads decorated, according to foregoing spatially predetermined copolymer ⁇ ynthesis method ⁇ , with di ⁇ tinct oligonucleotides, polypeptides or other copolymers with di ⁇ tinct binding propertie .
  • Array ⁇ of workpiece pad ⁇ are produced by forming small exposed regions of a ⁇ urface of a convenient composition on a substrate surface, such that said workpiece pads may line up with actuator pads and/or tips of the above actuator arrays.
  • Workpiece pad ⁇ may al ⁇ o comprise or overlie actuators, which may for example serve to tilt ⁇ aid workpiece pad ⁇ relative to ⁇ aid tip ⁇ , or may merely compri ⁇ e pad array ⁇ which facilitate the colocalization of tip and workpiece, depending on the de ⁇ ign of the article of fabrication and the tip or tip ⁇ u ⁇ ed.
  • molecular tips within tip array ⁇ (tip flat ⁇ ) ⁇ uch a ⁇ tho ⁇ e de ⁇ cribed by Drexler 53 may be ⁇ elected according to the tilt of ⁇ aid workpiece pad effected by one or more actuator ⁇ which adju ⁇ t the angle of the plane of ⁇ aid workpiece pad relative to the normal defined by the Z-actuator of the tip pad.
  • the tip arrays described by Drexler pertain to conventional atomic force microscopes and cantilevers u ⁇ ed therewith, ⁇ aid cantilever ⁇ modified to further comprise ⁇ aid tip flat ⁇ .
  • the pre ⁇ ent invention provide ⁇ that an array of actuator ⁇ po ⁇ ition ⁇ molecular tip ⁇ or array ⁇ thereof along the Z-axi ⁇ , while one or more actuators associated with each workpiece pad orients said workpiece pad to orient said workpiece pad such that one molecular tip of the opposed array is selected according to the orientation dependent proximity.
  • Such molecular tip array ⁇ may, following Drexler, be ⁇ ituated on a curved ⁇ urface, here each in communication with one of ⁇ aid Z- actuators, where the approach of a particular molecular tip in one of said arrays is selected according to the tilt of the workpiece relative to said one of said arrays which selects a distinct normal to said curved surface, thus selecting said molecular tip situated on said curved surface at the locus of the line normal to both said curved surface and said workpiece pad (i.e.
  • provi ⁇ ion of workpiece pads comprising one or more actuator ⁇ effecting tilted orientation of said workpiece pads relative to the Z-axis translation of said Z-actuators of said tip pad arrays permits the use of array ⁇ of tip arrays in the parallel modification of arrays of workpieces.
  • the particular advantage of being able to ⁇ elect individual tip ⁇ from an array of tip ⁇ in the modification of a workpiece accrues from the ability thereby to selectively interact ⁇ aid workpiece with a different, predetermined chemical functionality or compo ⁇ ition of ⁇ aid individual tip ⁇ , while maintaining a determini ⁇ tic relation ⁇ hip between the spatial location of said tip array with said workpiece (i.e. eliminating the need to locate a workpiece upon changeover of tip ⁇ .)
  • An array of tilt actuator ⁇ permit ⁇ ⁇ uch advantage to be availed in coinbination with the advantage ⁇ of paralleli ⁇ m accruing from the simultaneous positionally controlled modification of workpieces situated upon said workpiece pad array comprising said array of tilt actuator ⁇ .
  • ⁇ aid X-Y positioning means is a piezoelectric tube ⁇ canner
  • additional care mu ⁇ t be taken in calibration and scanning voltage waveform generation to en ⁇ ure that deviation ⁇ from planar orientation of the tube opening do not cause a rocking motion of the array mounted thereupon (i.e.
  • Placement of ⁇ aid molecular precur ⁇ or ⁇ of workpieces or said workpiece ⁇ on ⁇ aid workpiece pad ⁇ of ⁇ aid workpiece pad array by said molecular tip ⁇ or other molecular po ⁇ itioning member ⁇ en ⁇ ure ⁇ correct relative po ⁇ itioning of said workpiece precursors or workpieces relative to said molecular tips or other molecular positioning members and the reactants later carried thereupon.
  • a molecular workpiece precursor comprising a thiol group
  • a molecular workpiece precursor may be deposited onto a workpiece pad comprising an exposed gold surface by a molecular tip, such that the resulting position of as ⁇ ociation of ⁇ aid thiol with said gold surface corresponds to the position on said tip pad or cantilever of said molecular tip.
  • said workpiece precursor may additionally comprise a cleavable linkage susceptible to a particular predetermined physical or chemical treatment, and a first reactive group, for example a hydroxyl.
  • a ⁇ econd molecular workpiece precursor molecule for example of identical composition, may be depo ⁇ ited by the ⁇ ame proce ⁇ at a second location on said workpiece pad.
  • two predetermined reactive groups are placed in a predetermined arrangement on ⁇ aid workpiece pad.
  • a third molecular precur ⁇ or molecule may ⁇ imilarly be positioned at a third point, in which ca ⁇ e the three positioned reactive groups thu ⁇ fix a coordinate system admitting no gros ⁇ rotations (i.e.
  • molecular component ⁇ may be reacted with the reactive groups of said workpiece precursor ⁇ according to the method ⁇ de ⁇ cribed below.
  • neces ⁇ ity of ⁇ uch ⁇ teps in practice will depend on the ⁇ ize of such surface ⁇ and the ability to obtain or fabricate ideally flat sub ⁇ trate ⁇ urface ⁇ as materials for the production of tip pad arrays and workpiece pad arrays.
  • Constraint-Based Simplification of Positional Synthesis A more immediately practical approach to the positional synthesi ⁇ propo ⁇ ed by Drexler fabricate ⁇ structure ⁇ from structure ⁇ larger than single atoms, and avails conventional solution and solid-phase chemistry where positional control is not essential. These methods permit the synthetic fabrication of extended, complex heterogeneous structure ⁇ which would be difficult or impo ⁇ sible to produce by known art chemical methods.
  • the size of molecular components or reactant ⁇ and the linker ⁇ which hold the ⁇ e in communication with said molecular tips or molecular positioning means are chosen to be of a sufficiently great size that angstrom positioning accuracy is not ⁇ trictly required to exert po ⁇ itional control over ⁇ ynthe ⁇ i ⁇ .
  • ⁇ aid linkers which may for example be composed of a polymethylene (polyethylene) polymer chain or other preferably straight chain flexible polymer chain, which are attached via ⁇ ome cleavable linkage to said molecular components or reactant ⁇ , con ⁇ train ⁇ ⁇ aid molecular component ⁇ or reaction ⁇ to a volume defined by the linear length of ⁇ aid linker ⁇ (and the ⁇ ize of ⁇ aid molecular component ⁇ or reactant ⁇ ) with a probability di ⁇ tribution (or effective concentration a ⁇ a function of position within said volume) which may be predicted u ⁇ ing Flory theory.
  • Said volume to which ⁇ aid molecular components or reactant may be termed a con ⁇ traint volume.
  • control over translation of said constraint volumes i ⁇ u ⁇ ed to ⁇ elect which reactive sites of said workpieces are reacted.
  • the re ⁇ olution of ⁇ uch a method is limited by the extent of said constraint volume, and workpiece ⁇ are preferably de ⁇ igned according to ⁇ aid re ⁇ olution (otherwi ⁇ e re ⁇ ultant workpieces will obey the corresponding probability distribution related to the number of pos ⁇ ible ⁇ ite ⁇ within an approached con ⁇ traint volume and the effective concentration di ⁇ tribution at each of said pos ⁇ ible sites.)
  • such a linker may alternatively have a large central portion of their length composed of a rigid polymeric member (with terminal flexible polymeric linkages), in which case the attached molecular components or reactants are confined rather to a shell, which may be termed a constraint shell.
  • the configuration of said constraint shell is determined by the structure of the partially rigid said linker, i.e. defining a volume beyond which the terminally situated said molecular components or reactant ⁇ may not extend and also a closest point of approach to the point on said tip pad at which said partially rigid said linker is attached beyond which said molecular components or reactants may not approach.
  • Thi ⁇ may be accompli ⁇ hed by monitoring any forces exerted by said communication as said tip pad array i ⁇ withdrawn from ⁇ aid workpiece pad array or, alternatively, a ⁇ ⁇ aid Z-actuator ⁇ of ⁇ aid tip pad ⁇ are tran ⁇ lated away from ⁇ aid workpiece ⁇ .
  • Said force ⁇ may be monitored conveniently if each of ⁇ aid molecular tip (or said linker) is in communication with a cantilever, the position of which i ⁇ tracked by any known art method ⁇ uch as optical beam deflection, monitoring of a tunneling current between said cantilever (coated by a metal) and a tunneling tip, change ⁇ in piezore ⁇ i ⁇ tivity, changes is capacitance where an electrode situated on said cantilever forms one plate of a capacitor, etc.,.
  • a linker is preferably of elastiomeric compo ⁇ ition such that tension i ⁇ gradually applied to ⁇ aid workpiece a ⁇ deflection of ⁇ aid cantilever occur ⁇ during ⁇ aid withdrawal.
  • tension i ⁇ gradually applied to ⁇ aid workpiece a ⁇ deflection of ⁇ aid cantilever occur ⁇ during ⁇ aid withdrawal.
  • gross severing of linkage ⁇ which would be expected with applied ten ⁇ ion ⁇ of less than 10 nN for single bonded linkages, are avoided. Otherwise care must be taken to ensure that applied tensions remain under a tolerable maximum to prevent such severing.
  • the tip once a reaction between said molecular component and said workpiece has occurred, the tip remain ⁇ in communication with ⁇ aid workpiece, until chemical or phy ⁇ ical treatment severs the resulting linkage.
  • Linkages i.e.
  • linkage composition is chosen according to the de ⁇ ired method ⁇ of linker cleavage.
  • linker ⁇ may compri ⁇ e bond ⁇ ⁇ u ⁇ ceptible to cleavage by particular chemical reagents, may be heat labile or may be photocleavable, e.g. comprising esters to compounds such as nitroveratryloxycarbonyl compounds, such that said ester linkages are cleaved by exposure to appropriate wavelength actinic radiation.
  • reactant molecular components are situated on ⁇ aid molecular tip ⁇ or tip pads by contacting said tip pad array with a solution comprising the said reactant molecular components to be added to said workpieces during the subsequent reaction step and permitted to bind to the available ⁇ ites on said tip pad array, i.e. to molecular tips or to the affinity groups thereupon.
  • a molecular component will generally be bound to a workpiece at more than one location, i.e. will generally have greater than one point of connectivity with ⁇ aid workpiece.
  • a positionally controlled reaction links ⁇ uch a reactant molecular component to a fir ⁇ t reactive ⁇ ite on a workpiece, and the ⁇ tructure of said reactant molecular component and said workpiece are such that the reactive groups on ⁇ aid reactant molecular component and ⁇ aid workpiece will react together for only one relative topological orientation of ⁇ aid reactant molecular component and ⁇ aid workpiece.
  • rigid ⁇ tructure i.e.
  • those comprising rigid polymeric ⁇ tructure ⁇ ) ⁇ imple de ⁇ ign rules may yield such a result, namely, that the pairwise distances of all similar reactive group ⁇ are different and that once a fir ⁇ t bond i ⁇ formed, under po ⁇ itional constraint imposed by linkage to said tip, no incorrect second bonds to reactive group ⁇ on ⁇ aid workpiece may form after release of said reactant molecular component from said tip.
  • no rotation of said reactant molecular component about the first bond formed under positional con ⁇ traint by ⁇ aid tip will yield proximity of reactive group ⁇ situated on said reactant molecular component to correspondingly reactive groups of said workpiece except that rotation which permits the de ⁇ ired reaction and hence the desired connectivity.
  • flexible components may al ⁇ o be employed according to ⁇ uch constraint-ba ⁇ ed de ⁇ ign method ⁇ , but here flexibility entail ⁇ that the positional constraints on reactions of these components obtaining after a first bond or linkage is formed between such flexible component ⁇ and a workpiece will concern the maximum possible reach of other reactive groups on ⁇ aid flexible component ⁇ .
  • the selection of rigid or flexible components for different parts of a workpiece structure will be according to considerations concerning both the de ⁇ ired structure and the preferred molecular as ⁇ emble strategy.
  • component ⁇ et ⁇ may be synthesized comprising different topologie ⁇ ⁇ uch as different degrees of branching, different length ⁇ of branch arms, different chemical functionalization of regions or location of said branch arms, etc.,.
  • workpiece ⁇ may be treated with other reactant ⁇ or chemical modifying reagent ⁇ from bulk ⁇ olution.
  • reactive group ⁇ on molecular components may all be primary hydroxyl group ⁇ .
  • each such cross-linking agent molecule will react with each hydroxyl on said workpiece, while the second such functional group ⁇ of each such cros ⁇ -linking agent molecule, now a reactive group on said workpiece, will only react with approaching hydroxyls of new molecular components.
  • thi ⁇ entails that no two hydroxyls available on a workpiece may be clo ⁇ er together than the length ⁇ panned by the reactive functionalitie ⁇ of ⁇ uch cross-linking groups.
  • molecular components may carry protected functional groups, which, once ⁇ aid molecular component ⁇ are reacted onto said workpieces and relea ⁇ ed from said tips, are deprotected by contacting ⁇ aid workpiece pad array, a ⁇ in the ca ⁇ e of activation with cross-linking agents, in this case with deprotection reagents.
  • function groups which were held inert with respect to each other to prevent unwanted reactions between molecular component ⁇ a ⁇ the ⁇ e are prepared and bound to said tip ⁇ , are activated once on ⁇ aid workpiece.
  • inert ⁇ olvent media mu ⁇ t be chosen.
  • control over the step ⁇ at which functional group ⁇ on ⁇ aid workpiece ⁇ are activated or deprotected provides another method of control over synthesis, i.e. a method by which a sub ⁇ et of functional group ⁇ are held inert until a de ⁇ ired a ⁇ embly step, whereupon they are deprotected or activated by treatment with appropriate reagents.
  • a method by which a sub ⁇ et of functional group ⁇ are held inert until a de ⁇ ired a ⁇ embly step, whereupon they are deprotected or activated by treatment with appropriate reagents It will be obviou ⁇ to those ⁇ killed in organic ⁇ ynthe ⁇ i ⁇ that other reactive chemi ⁇ trie ⁇ , as will a ⁇ multiple, concurrently u ⁇ ed chemi ⁇ trie ⁇ will be po ⁇ ible and often preferably advantageou ⁇ over the ⁇ imple example pre ⁇ ented here merely for purpo ⁇ e ⁇ of illustration.
  • molecular tips and any affinity group ⁇ thereupon may thu ⁇ be ⁇ pared from expo ⁇ ure to highly reactive group ⁇ (e.g. oligonucleotide moietie ⁇ of molecular tip ⁇ need never be expo ⁇ ed to acid chloride containing compounds, which would inactivate said oligonucleotides for proper binding) .
  • reactive groups may be primary hydroxyls on alkyl chain linkers
  • rigid structural members may compri ⁇ e polyparaphenylene derivative ⁇ 57 or the phenylacetylene polymer ⁇ di ⁇ clo ⁇ ed by J. Zhang, J.S. Moore et al. 58 , in either ca ⁇ e comprising component ⁇ with ⁇ tructures synthetically acces ⁇ ible, for example, by the methods of J. Zhang, J.S. Moore et al.,.
  • Affinity members for reversibly binding these components to molecular tips may comprise oligonucleotide ⁇ , oligopeptide ⁇ , peptide nucleic acid oligomer ⁇ or ⁇ mall molecule hapten ⁇ joined to ⁇ aid component ⁇ (e.g. via methylene linker ⁇ ), according to the complementary affinity members on ⁇ aid molecular tip ⁇ .
  • Other molecular component structures of interest in the construction of molecular devices by such as ⁇ embly method ⁇ include rotaxane ⁇ comprising macrocycles which themselves comprise reactive group ⁇ , and catenane ⁇ .
  • Molecular component ⁇ ba ⁇ ed on the ⁇ e topological compounds provide structure ⁇ u ⁇ eful a ⁇ moving part ⁇ in object ⁇ and device ⁇ fabricated by the ⁇ e synthesis method ⁇ .
  • Affinity groups such as oligonucleotides may be included within the structure ⁇ of ⁇ aid molecular component ⁇ or may be po ⁇ itioned by tip ⁇ into proximity with proximity ⁇ elected reactive group ⁇ on said workpieces which will react with linkers (e.g. at terminal hydroxyls) attached to said affinity group ⁇ .
  • linkers e.g. at terminal hydroxyls
  • Said affinity groups used to bind reactant molecular component ⁇ to said molecular tips or molecular positioning means may be in communication with the remainder of the structure of the reactant molecular component which they serve to target to said molecular tip ⁇ or molecular po ⁇ itioning mean ⁇ via linker ⁇ cleavable by predetermined phy ⁇ ical and/or chemical treatment ⁇ (e.g.
  • hydroly ⁇ is of an ester linkage
  • chemical functional groups may be primary amine ⁇ , which may be protected by esterification.
  • a molecular tip may comprise or may be linked (e.g. by known art conjugate chemistry) to a protease enzyme molecule, such as Proteinase K. Only tho ⁇ e primary amine ⁇ within the reach of such enzyme molecules tethered to a molecular tip or tip pad are su ⁇ ceptible to enzymatic deprotection, and hence only the ⁇ ub ⁇ et of such amines on said workpieces will be deprotected.
  • Other catalysts such a ⁇ ribozymes, metal tip surface ⁇ , or metallic colloid ⁇ partially embedded in polymer matrices may similarly be used, as may tethered molecule ⁇ which effect deprotection by non-catalytic reaction ⁇ .
  • the large data capacitie ⁇ realizable with the variou ⁇ data storage methods comprising the use of scanning probe technology based data readout are not accompanied by a commensurate increase in the rate of data recording except where large numbers of scanned probes are used to record a data pattern onto a storage medium surface. Because one particular advantage of such storage technologies is in application ⁇ related to data publishing, a rapid method for the duplication of predefined bit patters readable with such readout technologies would be de ⁇ irable, a ⁇ ha ⁇ been the ca ⁇ e with read-only optical data ⁇ torage technologie ⁇ .
  • the method ⁇ of the pre ⁇ ent invention are well ⁇ uited to the fabrication of ⁇ urface ⁇ with in ⁇ cribed nano ⁇ cale data pattern ⁇ readable with variou ⁇ scanning probe readout mean ⁇ .
  • the ⁇ e include near-field photon transfer (corresponding to NFSOM), electron tunneling and electron field emis ⁇ ion (corre ⁇ ponding to STM and field emis ⁇ ion mode STM) , and surface profile detection (corresponding to AFM and variation ⁇ thereon) .
  • a transparent surface such as glas ⁇ or polycarbonate i ⁇ coated with a metal film, which i ⁇ patterned by the foregoing method ⁇ of the pre ⁇ ent invention for the ⁇ patially predetermined protection or ma ⁇ king of metallic film ⁇ urface ⁇ from etchant ⁇ such that the desired bit pattern is formed compri ⁇ ing apertures in said metallic film and intact regions in ⁇ aid metallic film which reduce or eliminate the local tran ⁇ mi ⁇ ion of light.
  • Such a bit pattern may be read out by conventional NFSOM apparatu ⁇ or variations thereupon, or the near-field detection means described below.
  • the patterned said metal layer may mask the local transmi ⁇ ion of photon ⁇ to a probe aperture which conduct ⁇ received photon ⁇ to a detector, ⁇ aid photon ⁇ originating from a source on the oppo ⁇ ite ⁇ ide of the tran ⁇ parent material comprising said transparent surface, in which case said metal layer serves as a photon tran ⁇ mi ⁇ ion ma ⁇ k.
  • ⁇ aid pattern in said metal layer may ma ⁇ k the tran ⁇ mi ⁇ sion of photons emitted due to the fluore ⁇ cence of compound ⁇ in said transparent material excited by photons either emanating from an NFSOM tip aperture which may be the same aperture used for the detection of fluorescentiy emitted photons or emanating from a source on the other side of ⁇ aid tran ⁇ parent ⁇ urface.
  • a metallization pattern may similarly be formed, here on an insulator, or alternatively a relief pattern may be formed in one or more metal layer ⁇ with all expo ⁇ ed ⁇ urface ⁇ having conductive propertie ⁇ .
  • a bit pattern which may be replicated by the methods of the pre ⁇ ent invention i ⁇ encoded in ⁇ uch a ⁇ urface relief pattern.
  • Such a bit pattern may be read out with instruments such as STMs, microfabricated STM ⁇ or the array device ⁇ di ⁇ clo ⁇ ed herein comprising plural Z-actuators each a ⁇ ociated with a tip, which entire array i ⁇ ⁇ canned acro ⁇ a juxtaposed surface by an X-Y po ⁇ itioning mean ⁇ and wherein data pattern ⁇ are detected by monitoring, with electronic mean ⁇ , voltage, current or height of each tip either ⁇ ingly or in the various po ⁇ ible combination ⁇ and where ⁇ aid current may be either a tunneling current or a field emi ⁇ ion current according to particular implementation .
  • relief surfaces comprising surface patterns are replicated by casting, injection molding or embos ⁇ ing method ⁇ .
  • an original relief comprising the desired bit patterns may be formed by scanning probe technology based patterning methods such as those of related art reviewed above.
  • a CCD array fabricated by methods of the present invention or by conventional methods may be modified to serve as a highly parallel detection means for optical data readout relying on near-field photon transmission through a small aperture.
  • An array of waveguide elements for example compo ⁇ ed of PMMA, i ⁇ molded onto the ⁇ urface of said CCD array such that each photodiode of said CCD array will capture photons conducted by each of ⁇ aid waveguide element ⁇ .
  • the array comprising said waveguide elements must therefore be aligned with the photodiode array of ⁇ aid CCD.
  • Gap ⁇ between ⁇ aid waveguide ⁇ are filled in with, for example, an opaque polymeric material, or may be electroplated provided thi ⁇ will not cau ⁇ e short circuits in said CCD, which may be designed to prevent such problems.
  • Said waveguide element array and any material filling said gaps are formed such that a substantially flat surface result ⁇ .
  • an array of pinhole ⁇ of predetermined size and location is fabricated with as ⁇ ociated waveguide ⁇ , which conduct any photon ⁇ received at the aperture formed by ⁇ aid pinhole ⁇ to the photodiode ⁇ of ⁇ aid CCD in a one-to-one manner.
  • Such an array of pinholes with associated waveguides may al ⁇ o be fabricated by LIGA method ⁇ and variation ⁇ thereupon, and then ⁇ ituated in an appropriately aligned manner onto a CCD array ⁇ urface.
  • the ⁇ ame type of structure comprising an array of pinhole ⁇ with associated waveguides may be fabricated by other molding and electroforming method ⁇ de ⁇ cribed herein and then, ⁇ imilarly, ⁇ ituated in an aligned manner onto a CCD array.
  • Such an array may be termed an aperture limited CCD array (ALCCD) .
  • the CCD array device ⁇ u ⁇ ed in thi ⁇ a ⁇ pect of the present invention are preferably of high sensitivity, ⁇ uch that a reduced number of photon ⁇ must be received by each photodiode to register a signal.
  • Such an ALCCD may be used for data readout by juxtaposing the ⁇ urface of said ALCCD comprising ⁇ aid pinhole array to a patterned metal film surface of a transparent material such glas ⁇ , such a ⁇ that described above which pattern may be replicated as described above.
  • Said pattern metal film surface is held parallel to the ⁇ urface of ⁇ aid ALCCD compri ⁇ ing ⁇ aid pinhole array, at a di ⁇ tance of le ⁇ than one half the wavelength of the light employed and preferably at a distance of le ⁇ than one-quarter of thi ⁇ wavelength. Smaller ⁇ eparation ⁇ are further preferred.
  • the region of ⁇ aid patterned metal film oppo ⁇ ite each pinhole on the ⁇ urface of said ALCCD is either be coated with ⁇ aid metal film or may expose the underlying said transparent material, which distinction provides for the detection of the corresponding data pattern.
  • the den ⁇ ity achievable with thi ⁇ data readout apparatu ⁇ and method is primarily determined by the dimension ⁇ of ⁇ aid pinhole, which limit ⁇ the photons thus received to tho ⁇ e originating from narrow expo ⁇ ed regions.
  • Said patterned metal film surface is ⁇ canned, in a manner maintaining parallel alignment with the pinhole array surface of ⁇ aid ALCCD, ⁇ uch that an area corre ⁇ ponding to the ⁇ ize of each CCD array element pa ⁇ e ⁇ opposite each pinhole, by repeatable positioning mean ⁇ ⁇ uch a ⁇ piezoelectric or capacitor actuator ⁇ .
  • said patterned metal film ⁇ urface i ⁇ ⁇ canned in the X and Y while maintaining a ⁇ ub ⁇ tantially con ⁇ tant Z ⁇ eparation, such that the ⁇ ignal received by each photodiode of each CCD element correlate ⁇ , according to ⁇ can timing, with a particular po ⁇ ition on ⁇ aid patterned metal film surface.
  • Scanning is conducted slowly, such that said CCD captures the bit image corresponding to a particular position of said patterned metal film surface, which is then read out from said CCD according to conventional methods associated with the use of such electronic imaging or memory devices.
  • data rates will primarily depend on the device characteristics of the CCD used.
  • the X-Y location of said patterned metal film surface relative to said ALCCD are then translated by incremental ⁇ canning motion of said actuator, and the bit pattern as ⁇ ociated with this sub ⁇ equent po ⁇ ition i ⁇ then ⁇ imilarly collected.
  • photons originate from a source on the opposite side of said patterned metal film than said ALCCD, with transmis ⁇ ion occurring through exposed regions of said transparent surface as it is masked by said patterned metal film.
  • the number of photons received by each CCD element are limited by the relative size of said pinhole to the area of each CCD element.
  • ⁇ aid patterned metal film surface is situated between said microlen ⁇ array and ⁇ aid ALCCD, and remain ⁇ in a fixed po ⁇ ition relative to ⁇ aid ALCCD a ⁇ ⁇ aid patterned metal film ⁇ urface i ⁇ ⁇ canned between ⁇ aid microlen ⁇ array and ⁇ aid ALCCD surface.
  • a relief is formed by replication methods at the ⁇ urface of an elastiomer polymer composition, comprising a pattern of parallel lines comprising narrow raised regions generally of le ⁇ than 200nm in width and wider ⁇ unken regions preferably of greater than 1 micron in width.
  • the aspect ratio of said narrow raised regions may be reduced by sloping the surface of said wider sunken regions in proximity to ⁇ aid narrow rai ⁇ ed region ⁇ .
  • Said polymer compo ⁇ ition is chosen such that the re ⁇ ulting molecular network compri ⁇ e ⁇ chemical functional groups which may be reacted with cross-linking reagents to attach molecule ⁇ to the ⁇ urface of ⁇ aid relief.
  • An appropriate cro ⁇ -linking agent compri ⁇ ing a central linker of predetermined length, preferably shorter than 50nm is coupled to a nuclease enzyme (such as DNase I or a type II re ⁇ triction endonuclease, preferably recognizing a 4 base restriction site) according to known art conjugate chemistry.
  • Said cross-linking agent is chosen so as to link said nuclease to ⁇ aid elastiomer polymer compri ⁇ ing ⁇ aid chemical functional groups.
  • a small volume of a solution of ⁇ aid cro ⁇ s-linking agent conjugated to said nuclease is spread on a surface as a thin layer in the pattern of a single stripe, preferably of les ⁇ than a few millimeters in width.
  • Said relief is contacted with said stripe of said conjugate on said surface, oriented with said parallel lines perpendicular to said stripe, such that nuclease molecules are linked to the ⁇ urfaces of said narrow raised regions along the region juxtaposed with ⁇ aid ⁇ tripe.
  • the solution used to form said strip may favorably contain a dye or pigment to facilitate identification of the enzyme modified region, or other marking methods may be used to accomplish ⁇ uch identification.
  • a relief comprising parallel lines and trenches of predefined size and location is produced with a predefined region of rai ⁇ ed feature ⁇ upon which nuclea ⁇ e molecules are ⁇ ituated.
  • Linear or linearized DNA molecule ⁇ or chromatin are treated ⁇ o a ⁇ to be immobilized at ⁇ pecific region ⁇ along their length, preferably near to one distinct terminus, and optionally derivatized with bead ⁇ at the opposite terminus.
  • these may be hybridized with an oligonucleotide compri ⁇ ing a fir ⁇ t affinity moiety ⁇ uch a ⁇ biotin moiety and one or more p ⁇ oralen group ⁇ (where plural group ⁇ are preferred to favor the formation of covalent linkage ⁇ with both ⁇ trand ⁇ of the DNA double helix) .
  • ⁇ aid p ⁇ oralen group ⁇ are cau ⁇ ed to react with ⁇ aid DNA to form a covalent linkage ⁇ .
  • Streptavidin is bound to a glass surface in a narrow stripe ( ⁇ 1 micron in width) ⁇ horter in length than the width of ⁇ aid ⁇ trip of ⁇ aid conjugate above.
  • Said DNA terminally linked to said biotin is then contacted with said narrow stripe of streptavidin and affinity binding i ⁇ permitted to occur.
  • the immobilized DNA sample is optionally then briefly subjected to degradation by a 5' to 3' exonuclease, followed by treatment with a polymerase and second affinity group modified nucleotide triphosphate ⁇ to terminally decorate ⁇ aid linearized DNA molecule ⁇ with said second affinity group.
  • said immobilized DNA sample is.then contacted with a ⁇ olution comprising microscopic or sub-micron diameter beads, which may be fluore ⁇ cently labeled, ⁇ urface modified with receptor ⁇ (e.g. immunoglobulins, etc.) according to known art conjugate chemistry. Alignment of the immobilized DNA molecules is then performed by known art technique ⁇ .
  • Straightening of terminally immobilized molecule ⁇ may alternatively be accompli ⁇ hed, where said molecules underwent the optional addition of terminal beads as above, by methods such as those of T.T. Perkins et al. 62 whereby bead ⁇ ⁇ ituated on DNA molecule ⁇ are manipulated u ⁇ ing an optical trap (also known as la ⁇ er tweezer ⁇ ) , or, where ⁇ aid beads are of paramagnetic compo ⁇ ition, by the application of an appropriately directed magnetic field of sufficient strength to straighten, but not so large as to disrupt, the sample molecular and supramolecular ⁇ tructure.
  • an optical trap also known as la ⁇ er tweezer ⁇
  • ⁇ ample molecule ⁇ are end-immobilized to a preci ⁇ e, predetermined region, oriented and extended by mean ⁇ ⁇ uch as those above, the relief prepared above with rai ⁇ ed narrow line ⁇ modified with nuclea ⁇ e molecules i ⁇ then contacted with ⁇ aid gla ⁇ s surface on which said sample molecules have been straightened and aligned, in an orientation such that the parallel lines of said relief are perpendicular to ⁇ aid sample molecules, where said contacting i ⁇ performed under ⁇ light pre ⁇ ure to en ⁇ ure ⁇ ealing of the channel ⁇ thu ⁇ temporarily formed.
  • a gentle fluid flow is immediately established in said channel ⁇ , such that once a molecular region is freed at both ends by cleavage by said nuclease molecule ⁇ , it is carried down said channel away from said nuclease molecules, which might otherwise further degrade it.
  • Said flow then carrie ⁇ the cleaved molecule fragment ⁇ to collection volumes formed in said relief structure ⁇ at the ends of ⁇ aid channels, which are arranged such that the location of the channel in which a fragment was cleaved may be determined or inferred from the location of said collection volume.
  • the collection volumes forms a linearly ordered set, which set comprise ⁇ an ordered arrangement of fragment ⁇ of a well defined physical length and precise ordering with respect to their original location in the in-tact sample molecules.
  • Such a method may be repeated, as desired, with a slightly different offset of said relief with respect to said narrow stripe of streptavidin, such that different regions are occluded from the collected sample ⁇ et, to the extent that retention under ⁇ aid narrow rai ⁇ ed line ⁇ po ⁇ e ⁇ any difficultie ⁇ . Note that in all ⁇ teps, care is taken to avoid shearing sample molecules.
  • Nonethele ⁇ , thi ⁇ ⁇ imple proce ⁇ yield ⁇ ordered fragment populations u ⁇ eful for e ⁇ tabli ⁇ hing phy ⁇ ical map ⁇ of genetic material and producing ordered ⁇ ample ⁇ therefrom for purpo ⁇ e ⁇ such as library cloning or subcloning or other analysi ⁇ techniques, and particularly useful in genome sequencing projects.
  • Two alternative genetic material di ⁇ ection method ⁇ availing ..-lcropatterned relief ⁇ are al ⁇ o po ⁇ ible.
  • a relief compri ⁇ ing parallel rai ⁇ ed narrow line ⁇ and trenche ⁇ such as that described above, is formed.
  • This relief is then contacted with a first surface on which are coated particles ⁇ uch a ⁇ metallic or glass colloids or hard microcrystallite ⁇ , which are coated with chemical ⁇ pecies capable of cros ⁇ linking ⁇ aid particle ⁇ to ⁇ aid relief. Thu ⁇ , said particles are bound to the rai ⁇ ed narrow region ⁇ of said relief.
  • Genetic material is immobilized on a second ⁇ urface and aligned a ⁇ above, and the relief of the pre ⁇ ent alternative i ⁇ oriented a ⁇ above with said parallel lines perpendicular to the direction of alignment of said genetic material on said second surface and contacted with said second surface under an applied normal force. Liquid is caused to flow through the channel ⁇ formed by said trenches and said second surface as a result of the preceding contacting ⁇ tep. The relief i ⁇ then translated in the direction of said parallel lines as said normal force i ⁇ maintained and said liquid flow is maintained.
  • the genetic material i ⁇ abraded and therefore cut by said particles, and once cut, fragments are freed from communication with said second surface and said relief, such that said fragment ⁇ are carried by ⁇ aid liquid flow down said channels to collection volumes.
  • the cutting action availed in the present alternative of this embodiment is in direct analogy to the cutting of DNA molecules by an atomic force microscope tip.
  • Said second surface may be hard, such a ⁇ gla ⁇ , may be ela ⁇ tiomeric; ⁇ aid second surface may comprise bonded particles similar or identical to those used on said raised regions of said relief, in which case the cutting action may be compared to a ⁇ ci ⁇ oring action.
  • a relief compo ⁇ ed of opaque material ⁇ compri ⁇ ing parallel lines of narrow raised regions and trenche ⁇ i ⁇ u ⁇ ed, a ⁇ above, to define flow channel ⁇ when contacted under normal force with a ⁇ econd ⁇ urface, here of a ⁇ heet-like or flat material.
  • said second surface i ⁇ tran ⁇ parent to radiation of the appropriate frequency range but i ⁇ coated (preferably on said second surface, or instead on the surface not contacted with said relief) with material ⁇ which mask ⁇ aid radiation.
  • ⁇ aid flat material may be quartz coated with metallic mask materials and said radiation may be X-radiation, or said flat material may be glas ⁇ coated with any material opaque to vi ⁇ ible or ultraviolet light, which i ⁇ u ⁇ ed for irradiation.
  • X-irradiation genetic material expo ⁇ ed will be cleave directly due to double stranded breaks.
  • the masking pattern used is de ⁇ igned such that when said relief is contacted to ⁇ aid ⁇ econd ⁇ urface, the line ⁇ of narrow rai ⁇ ed region ⁇ are juxtapo ⁇ ed directly to unma ⁇ ked region ⁇ of said second surface.
  • said irradiation cause ⁇ break ⁇ of ⁇ aid genetic material at the loci of said narrow raised region ⁇ , which are in contact with ⁇ aid second surface, ⁇ uch that the fragment ⁇ thu ⁇ produced will be freed within the channel ⁇ formed by said second ⁇ urface and said trenches and carried away, by a fluid flow applied in said channels, to a collection volume corre ⁇ ponding to the re ⁇ pective channel.
  • Articles produced by the methods of the present invention may be used to facilitate the analysis of polynucleotide librarie ⁇ such as plasmid libraries, bacteriophage librarie ⁇ , co ⁇ mid librarie ⁇ , yea ⁇ t autonomou ⁇ chromosome (YAC) based libraries and the like.
  • polynucleotide librarie ⁇ such as plasmid libraries, bacteriophage librarie ⁇ , co ⁇ mid librarie ⁇ , yea ⁇ t autonomou ⁇ chromosome (YAC) based libraries and the like.
  • YAC yea ⁇ t autonomou ⁇ chromosome
  • transcripts are transcribed with radiolabeled ribonucleotides, and the RNA thus obtained is used to probe said arrays or replicas; those array elements or colonies or plaque ⁇ which hybridize the radiolabeled RNA tran ⁇ cript ⁇ thu ⁇ share common sequence ⁇ with the fragments from which said transcripts were obtained.
  • these methods determine which clonal isolates contain common sequence ⁇ , and by repetition identify ⁇ erie ⁇ of clonal i ⁇ olate ⁇ which are of contiguou ⁇ origin. Because such librarie ⁇ may contain well over 10 4 di ⁇ tinct chimera, ⁇ uch information is rarely if ever exhaustively determined for any given library, and i ⁇ u ⁇ ually only applied to small fraction ⁇ of such librarie ⁇ .
  • Copolymer array ⁇ such as those disclosed in related art or produced by the methods of the present invention may gainfully be applied to the ta ⁇ k of determining the linkage relation ⁇ hip between the individual chimera of a library and al ⁇ o to chromosomal mapping thereby.
  • each replicon must be rendered uniquely distinguishable, for example by the incorporation of a sufficiently long random nucleotide sequence (e.g.
  • variable base pair ⁇ (which need not all be contiguou ⁇ ] in a replicon de ⁇ igned for u ⁇ e in preparing librarie ⁇ of which ⁇ 10 ⁇ clones will be used.)
  • tag sequence Such a sequence will be referred to as a tag sequence, but it will be understood by those skilled in the relevant arts that different tagging and discrimination methods (e.g. the use of epitope library fused terminal-protein replicated viral or bacteriophage [e.g. PRD1, phi29, adenovirus], probed with peptide or antibody arrays) are po ⁇ sible, although nucleotide sequence based tagging with oligo array discrimination i ⁇ favorably convenient, and will therefore be empha ⁇ ized here.
  • the tag sequence is favorably placed ⁇ uch that it is transcribed under the influence of a promoter in the replicon sequence and the appropriate purified RNA polymera ⁇ e.
  • each cla ⁇ ifying oligo with a ⁇ ample molecule may be regarded as a positive result from a clas ⁇ ification test; each fragment is te ⁇ ted by the full ⁇ et of cla ⁇ ifying oligo ⁇ (i.e. all line ⁇ ), ⁇ uch that for n such lines such an array will be capable of discriminating up to 2 n different classification results.
  • Clas ⁇ ifying ⁇ equence ⁇ are therefore optimally cho ⁇ en to have about a 50% chance of probing any given ⁇ ample fragment or tran ⁇ cript, however large number ⁇ of cla ⁇ ifying ⁇ equence ⁇ (line ⁇ ) may obviate thi ⁇ effort.
  • Corre ⁇ pondence between tag identity and the regions of said lines of said cla ⁇ sifying oligos is enforced at the appropriate step with a relief (preferably of elastiomeric composition) of parallel raised lines and trenche ⁇ , of ⁇ ufficient spacing to accommodate both the tag probing oligo array element ⁇ and the length of the polynucleotide fragment ⁇ , juxtapo ⁇ ed to the detection oligo array compri ⁇ ing ⁇ aid one dimensional array compri ⁇ ing all sequences complementary to all pos ⁇ ible tag ⁇ equence ⁇ and ⁇ aid line ⁇ of ⁇ aid cla ⁇ sifying oligos, oriented such that said parallel raised line ⁇ and trenche ⁇ are perpendicular to ⁇ aid line ⁇ of said clas ⁇ ifying oligo ⁇ .
  • a relief preferably of elastiomeric composition
  • a library of said chimera, in mixture form, is either transcribed or fragmented, such that for each chimera, a first fragment type comprising a tag ⁇ equence and a ⁇ ufficient length of ⁇ ample fragment derived ⁇ equence i ⁇ obtained.
  • Such fragment ⁇ are either produced in labeled form (affinity, fluore ⁇ cent, radioactive or other label) or otherwi ⁇ e labeled before application to said array.
  • Said lines of said clas ⁇ ifying oligo ⁇ are covered to prevent contact with ⁇ aid first fragment type as a ⁇ olution containing ⁇ aid first fragment type i ⁇ contacted with a region of ⁇ aid array compri ⁇ ing ⁇ aid one dimensional array compri ⁇ ing all ⁇ equence ⁇ complementary to all po ⁇ ible tag ⁇ equences, to which the molecule ⁇ of ⁇ aid first fragment type are permitted to hybridize under sufficiently ⁇ tringent conditions to en ⁇ ure high ⁇ pecificity and accuracy of tag identification. Unbound ⁇ ample molecule ⁇ are washed away. Said relief is then juxtaposed to said array in said perpendicular orientation.
  • the hybrids formed comprising said sequences complementary to all possible tag sequences are then denatured and a flow of buffer solution is applied along the channels formed by the trenches of said relief and the surface of the substrate of said two dimensional array, and conditions permitting ⁇ tringent hybridization are then quickly re-e ⁇ tablished.
  • the molecules of said first fragment type which have been confined to particular channels according to the tag sequence which they comprise are swept by said flow acros ⁇ said lines of said cla ⁇ ifying oligos and which they thus have the opportunity to hybridize with if and only if they comprise sequence ⁇ complementary to the sequence ⁇ of any of said clas ⁇ ifying oligo ⁇ . Unbound molecules are then washed away.
  • Hybridization of sample molecules to ⁇ aid array is then detected by means which correspond to the labeling method used, and the corresponding data favorably recorded by digital computer.
  • Combination of data derived from en ⁇ emble ⁇ of ⁇ uch overlap information permit the reconstruction of maps of larger contiguou ⁇ region ⁇ ( ⁇ uch a ⁇ chromosomal material) from which sample fragments derive, and simultaneously yield information about which chimeras (as identified by tag sequences) are derived from particular regions of said maps.
  • Molecules of said first fragment type preferably consi ⁇ t of material from one extremity of the ⁇ ample fragment incorporated into the re ⁇ pective chimera.
  • Thi ⁇ minimize ⁇ the degree of overlap between two chimera ⁇ classified as contiguou ⁇ and thu ⁇ minimize ⁇ the number of chimera ⁇ nece ⁇ sary to represent the original linear ⁇ ample, reducing the work nece ⁇ ary for analy ⁇ i ⁇ . Note, however, that more redundant information may be obtained by permitting a greater degree of overlap, for example, by producing and then analyzing longer molecule ⁇ of ⁇ aid first fragment type.
  • two extreme case ⁇ are po ⁇ ible: said first fragment type comprising most all of a chimera; and said fir ⁇ t fragment type con ⁇ isting of a minimal length of ⁇ equence to permit useful clas ⁇ ification.
  • tradeoff ⁇ exi ⁇ t between these extremes, and intermediate cases are probably preferable according to the specific library and sample.
  • each of ⁇ aid molecule ⁇ of said fir ⁇ t fragment type mu ⁇ t compri ⁇ e ⁇ aid tag ⁇ equence and ⁇ equence from ⁇ aid original sample from which ⁇ aid library wa ⁇ derived.
  • ⁇ aid molecules of said fir ⁇ t fragment type are limited to one terminu ⁇ of the ⁇ ample fragment ⁇ equence (i.e. one ⁇ ample fragment sequence region adjacent to one arm of the replicon vector used to construct the library)
  • the method of preparation of said first fragment type must ensure that the tag sequence associated with one terminus may be correlated with the tag ⁇ equence a ⁇ sociated with the other terminus. At lea ⁇ t two instances conforming to this requirement are pos ⁇ ible.
  • ⁇ aid molecule ⁇ of ⁇ aid fir ⁇ t fragment type may be prepared by re ⁇ tricting aliquot ⁇ of the library of said chimera differently.
  • a the vector from which ⁇ aid library wa ⁇ prepared comprises a first unique restriction enzyme ⁇ ite, which may be cho ⁇ en according to infrequent occurrence in the ⁇ ubject genome or original ⁇ ample, on one ⁇ ide of a tag ⁇ equence, and a ⁇ econd unique re ⁇ triction enzyme site, which may be chosen according to infrequent occurrence in the subject genome or original sample, on the other side of a tag sequence.
  • fragments may be shortened by any convenient method ⁇ uch a ⁇ ⁇ hearing, random nuclea ⁇ e treatment, etc., to re ⁇ trict the length of polynucleotide fragment ⁇ captured by tag probe oligo ⁇ and ⁇ ubsequently analyzed to convenient length ⁇ .
  • each aliquot i ⁇ analyzed ⁇ eparately according to thi ⁇ aspect of the present invention, and result ⁇ are u ⁇ ed to correlate similarly cla ⁇ ed fragment ⁇ equence ⁇ found in said first aliquot with tho ⁇ e found in ⁇ aid ⁇ econd aliquot.
  • each molecule of ⁇ aid vector from which ⁇ aid library wa ⁇ prepared compri ⁇ e ⁇ two different, unique tag ⁇ equence ⁇ each located near a terminu ⁇ of said molecule, i.e. near the locus to which a ⁇ ample fragment i ⁇ ligated.
  • each tag sequence corre ⁇ pond ⁇ to a terminus of a sample fragment in the library constructed with said vector.
  • Molecules of said first fragment type having exactly one of said two different, unique tag sequences are produced by known art methods which will be related in consideration to the design of said vector (e.g. restriction digestion, transcription from an artificial promoter, etc., according to corresponding vector feature ⁇ in relation to said two different, unique tags and the in ⁇ erted ⁇ aid sample fragment.) Said molecules of said first fragment type having exactly one of said two different, unique tag sequences are analyzed according to the above library analysi ⁇ method ⁇ , probing in each ca ⁇ e with the member ⁇ of only one tag probe ⁇ et. Becau ⁇ e each terminus is as ⁇ ociated with a different tag in each clone, the tag from one terminu ⁇ mu ⁇ t be correlated with the tag of the other terminu ⁇ .
  • Thi ⁇ ⁇ econd correlation ⁇ tep is accomplished by the ⁇ ame method, binding chimera or fragment ⁇ compri ⁇ ing the vector termini with a one dimen ⁇ ional tag probing oligo array complementary to the first tag ⁇ et, where line ⁇ of different cla ⁇ ifying oligo ⁇ in the array are replaced with line ⁇ of different tag probing oligo ⁇ , corre ⁇ ponding to tho ⁇ e which bind tag ⁇ equence ⁇ of the second tag set.

Abstract

Methods for the formation and replication of patterns are disclosed, with objectives including economical microfabrication and nanofabrication, suitable for both low and high volume production. A patterned relief is formed on a surface by various techniques, which is then replicated on daughter surfaces. Methods for the reduction or elimination of defects are included. Various compositions may be patterned to address a wide range of applications. Spatial control over copolymer synthesis, synthesis of patterned molecular monolayers and multilayers, fabrication of microelectronic, microelectromechanical and microfluidic devices may be effected by the methods of the present invention. Various articles of manufacture and uses thereof, including improved methods and means for genome analysis, are further disclosed.

Description

PATTERN FORMATION . REPLICATION . FABRICATION AND
DEVICES THEREBY
Field of the Invention: The invention relates to the fabrication of two- and three- dimensional patterns and the production of devices thereby; the present invention finds application in the areas of microelectronics, micromechanics, microfluidics, scanning probe microscopy, mass data storage, scientific instrumentation, clinical diagnostics, molecular assembly, and other areaε.
Related Art:
Microfabrication and Microelectronics:
The field of integrated microelectronics depends critically upon the generation of predetermined two dimensional patterns. Generally, some lithographic process (e.g. involving actinic radiation) is employed to transfer a pattern from a mask to a resist, which is developed to expose portions of the surface of the underlying substrate. Such exposure permits the patterned etching of the exposed surface, or the patterned diffusion of impurities into regions near said surface. Thus, metal layers may h< ched to form wiring patterns and regions of a semiconductint .bstrate may be doped to form electronic components in an integrated device. Metal layers may also be patterned by lift-off processes involving the local dissolution of a pre-patterned resist underlayer-, which carries away with it the immediately overlying regions of the metal layer. Such resist masking and modification steps are repeated with different mask patterns and different modifications are performed which spatially vary according to the pattern formed in εaid resist. The alignment of mask patterns during each step with respect to all other steps is a critical limit on fabrication resolution. A summary of the basic methods is given is described by Millman . Microfabrication of integrated optics devices relies on similar techniques, as well as further additional techniques, a discussion of which is given by H. Nishihara, M. Haruna and T. Suhara.3
Fault Tolerant Circuit Designs One approach that has been applied in the field of microelectronics to increasing the tolerable physical defect rate arising from a particular fabrication process involves fault tolerant circuit design. This is a category of methods which includes redundancy of functions or components, self-testing or quality checking during the fabrication process, and rerouting of connections between functional blocks to serve functions lost to defectε. For example, such methods have been applied to dynamic RAM fabrication, where the memory bit array is divided into a number of blocks, the total capacity of which is larger than the device specification. All blocks are checked for defects or functionality, and where defects are found, the involved blocks are deactivated. Functional blocks are then reconfigured, as needed, to contiguously fill the device addreεs space. Analogous methods have been applied in the design of gate arrays, field programmable gate arrays, memory devices and other microelectronic devices.
Microscale and Nanoscale Lithographic Methods: Defect Reduction bv Resist "Voting":
In U.S. Patent Number 5,308,722, J.L. Nistler has disclosed a method for the reduction of defects in lithographic phase shift masks. The esεence of this method is to form a resist pattern, use it to etch the underlying quartz surface only partially, remove said resist pattern and any defects it may include, form a substantially identical pattern of resiεt on said quartz surface again, partially etch again and remove said substantially identical pattern of resist again, in repetitive cycles. Thus, only etch resistant regionε appearing in all resist patterns will be reproduced in the final patterned quartz article at the fully etched depth. Conceptually, this method checks one pattern, which may contain random defects, against other patterns which may also contain random defects which are not likely to appear at the εame location aε those random defectε in any other pattern. In the caεe of the phaεe εhifting reticles produced by that invention, only regions etched such that the tranεmitted light iε shifted by 180 degreeε exposes the lithographic resiεt theεe reticleε are uεed to expose. Thus, correspondence between multiple source patterns is required for a feature to be represented in the final product mask produced therefrom.
Contact Printing of Resist bv Lithographic Plate:
In U.S. Patent Number 5,380,620, T. Suzuki and F. Shinozaki teach a method whereby a lithographic plate comprising regions of ink binding and ink repelling regions is contacted with an ink or resiεt sheet and then contacted with a substrate to which said ink or reεist which bound to said ink binding regions of said ink. Thiε method formε lithographic plateε by expoεing materials similar or identical to those used as resists in conventional microfabrication, such that exposed regions have affinity or repel ink or other liquids, such that a pattern of differential retention of ink or εaid other liquidε may be uεed to form a pattern of εaid ink or other liquids on said lithographic plates and then transfer this pattern to the subεtrate. While thiε method reduceε the extent to which photolithographic equipment iε needed for patterning, production of micropatterned articles by this method still requires routine accesε to εuch photolithographic equipment for lithographic plate production.
Further, primary reliance on differenceε in affinity of liquidε to those regions of said lithographic plate treated to have such affinity compared to the affinity of εaid liquids to the subεtrate to be thus imprinted entailε that thiε method will require considerable effort for optimization when applied to new inks or resistε to different εurf ceε.
Photolithography Generated Relief Patterns, Mechanical Transfer Thereof, and Use as Lithographic Plate:
Methodε for the production and transfer of relief patterns in polymeric resist materials or variants thereof are reviewed by B. Bednar, J. Kralicek and J. Zachoval.4 A relief is produced by selectively exposing regions of a photoresist coated onto a first surface and mechanically transferring either the exposed or non- exposed regions to a polymeric foil second surface by juxtaposing said second surface to the image-expoεed polymeric reεist and relying on the differential adhesion properties resulting from expoεure. Thuε, a poεitive image iε produced on one εurface while the other εurface retains the corresponding negative pattern. Such relief patterns may provide differential wetting or liquid-retention properties differing from thoεe of the underlying εurfaceε, and thus be used as offset lithographic plates.
Differential Resist and Differential Ion Milling Thereby: G. Gal has, in U.S. Patent Number 5,310,623, taught a method whereby microlens arrayε are formed. In this method, greyεcale expoεure of a resist material may be used to cause the differential etching (in the preferred case by ion milling) of the underlying substrate, to produce a correspondingly curved surface. (Note that this inventor useε the term replica in a different senεe than that uεed herein.) Greyscale exposure resultε in a different effective etch protection by the resist, according to the degree of exposure. This may be termed etch-resiεtance denεity or depth dependent etching.
Microelectromechanical Systems: Microfabrication techniqueε have been applied to multilayered substrates comprising sacrificial layers which permit under-etching. Under-etching permitε the fabrication of εtructures with suspended or overhanging members and freely moving microscale mechanical partε. Such a proceεε may be combined with conventional microfabrication εtepε or applied to εubεtrateε comprising microelectronic deviceε to integrate both electronic and mechanical components of micron and submicron dimenεion. Such integrated devices have been termed microelectromechanical syεtems (MEMS)5.
Microfabricated Scanning Probe Microscopes and Actuators:
J.J. Yao, N.C. MacDonald and S.C. Arney have described microfabricated actuators, springε and STM instruments6, and have taught methodε for producing componentε of theεe in U.S. Patent Numbers 5,179,499 and 5,235,187. These capacitor actuators have nanometer εcale accuracy and high resonant frequencies.
S. Akamine, C. Quate and co-workers7 have described a different microfabricated STM design relying on bimorph piezoelectric actuators.
Data Storage with Scanning Probe Devices:
The high detection resolution achieved by the various regimeε of scanning probe microscopy aε well aε the ability to modify εurfaceε and structures thereupon with these instruments has motivated much interest in the use of scanning probe technology for data εtorage and retrieval. It iε anticipated that meanε εuch aε theεe will be neceεεary to surpasε the phyεical limitε on recording density in magnetic and far-field optical εurface recording technologieε. Recently, T.C. Reiley, L.-S. Fan and H.J. Mamin8 have demonεtrated the recording and readout of data uεing and AFM based instrument and the polycarbonate coated surface of a transparent disk as a storage medium. Data is recorded in the form of pitε on εaid εurface. A laεer is used to heat said polycarbonate above the glasε tranεition temperature, and the AFM tip impreεεeε a pit into the locally heated region. The size of the resulting pit corresponds to the apical geometry of said AFM tip. Readout is accomplished by conventional contact-mode AFM detection of said pits. Thus, upon recording of a bit pattern onto such a polymer coated surface, a relief pattern is formed which encodes the corresponding, stored data. These workers have formed pitε aε small aε lOOnm across and lOnm deep, yielding an areal storage density of 25 Gb/in^.
R. Imura, S. Hosaka, et al.9 have also recently demonstrated a scanning probe technology based data storage method, also using an AFM-like instrument with a gold-coated AFM tip. These workers rely on filed evaporation of metal from said gold-coated AFM tip to form dots (representing data bits) on a silicon εurface, with readout again accompliεhed by conventional contact-mode AFM.
In a εomewhat different approach, R.E. Betzig et al. , in U.S. Patent Number 5,286,971 and elsewhere10, teach a method by which data may be stored by employing a medium which undergoeε phyεical transformation upon exposure to light and expoεing εaid medium to light according to a near-field tranεfer procesε, εuch that the region of said medium exposed is limited by the physical dimensions of the aperture transmitting said light rather than the wavelength of said light, where said physical transformation includes a local change in optical properties and where said local change in optical propertieε iε detectable by a method similar to Near Field Scanning Optical Microscopy (NFSOM) . In thiε method, denεity advantage iε gained compared to the wavelength related denεity limitations of far-field optical recording methods, with densitieε as high aε 45 Gb/in2 having been demonεtrated .
Scanning Probe Pattern Formation: Scanning Resist Patterning: Scanning Tunneling Microεcopeε (STM)11'12, Atomic Force Microscopes (AFM) and Near Field Scanning Optical Microscopes (NFSOM)13 have been used to selectively form patterns in surfaces on the nanometer scale. These include patterns in materials or layers which mask the underlying subεtrate from the action of etchantε when not modified by the microεcopic probe uεed.
Chemical Manipulation and Svnthesis with Scanning Probe Microscopes: W.T. Muller, P.G. Schultz et al.14 describe a patterning procesε whereby a surface iε adεorbed with moleculeε compriεing a chemical functional group which may be tranεformed to a εecond chemical functionality by contact with a metal catalyεt, an AFM iε uεed to scan a tip coated with said metal catalyst over areas of said εurface which are intended to be tranεformed, and εaid chemical functional groupε tranεformed by the reεulting, εpatially limited catalytic proceεε are then εubjected to further chemical reactions which are selective for the catalytic reaction product second chemical functional groups.
B.J. Mclntyre, M. Salmeron and G.A. Somorjai15 have εi ilarly effected poεitional control of metallic εurface catalyεis using a platinum- rhodium STM tip in an atmoεpheric-pressure chemical reactor to catalyze the rehydrogenation of molecules comprising alkyne functionalities on (111) platinum surfaces.
M.A. Voelker16 has proposed a method for the patterning of chemical moietieε on εurfaceε, and the formation of patterns in multicomponent Langmuir-Blodgett (L-B) filmε according to the interaction of affinity groups of molecules in said L-B films with complementary affinity moieties on εaid surfaces. Such a method would entail that the molecules of which said L-B film is composed have good mobility in the as-formed L-B film prior to any polymerization, and that transport permits good segregation of molecular species within εaid L-B film without any congestion problems or other hindrance to the proper assortment of specieε to match the pattern on the underlying εurface. Because said L-B film iε only expoεed to εaid pattern after depoεition onto εaid εurface, the mobility of molecular εpecieε within εaid film will be reduced as compared to mobility in the undeposited film, and this will reduce the efficacy of the proposed method. Further, though the proposed method provideε for multiple uses of the initial patterned surface, it doeε not provide for the replication of the L-B layer patternε thuε formed except where these comprise multilayers which remain aεsociated together. In other words, this method only provides for the additive accumulation of patterned L-B layers, whether these are then isolated from an initial surface or formed succeεεively in a multilayer molecular baεed structure.
Microfabrication with Replication:
Integrated optical devices and other microεcale optical components have been fabricated by the replication of relief patterns into polymeric materialε17. The optical propertieε of theεe materials and the structureε resulting from pattern replication determine device function and characteristics. Replication methods used include injection molding and casting into polymeric, elastiomeric or metallic molds or mold inserts, and hot embosεing with reliefε, for example, metal reliefε. Multiple cycleε of replication may be performed to yield a large number of reliefε for the rapid maεε production of devices, according to the fidelity of replication and acceptable device tolerances.
Over the last decade, methodε have been developed to form three dimenεional patternε on the micron and εub-micron εcaleε, combining lithography with electroforming, micromolding and mold replication.
An original microrelief iε formed by the lithographic depth patterning of a relief material, which iε then developed to form the deεired predetermined structure (first relief) . Electroforming iε then performed to yield a metal negative mold inεert conforming to the polymer resist structure, and then separated from the original resiεt. This first generation mold may then be as a mold for the casting or microinjection molding of a second polymeric relief nearly identical to the first relief. The molding or casting procesε may be repeated with the firεt generation mold, and the electroforming proceεε may be repeated with εecond or subsequent generation polymeric relief structures produced with said molds by appropriate methods. Thus, polymeric objectε and metallic objects with sub-micron patterns may be economically reproduced. This εet of methods, known as LIGA, was developed by E.W. Becker et al.,.18 These workerε reported reproduction of lateral featureε εmaller than 0.1 micron. Theεe methods are particularly uεeful in the production of devices with microscale featureε and moving partε. Where the lithographic method employed iε synchrotron X-Ray lithography, high aεpect ratios may be achieved with resiεts of up to millimeter depth. More recently, M. Abraham et al.19 have reported the fabrication of micro-optical εyεtems with LIGA, and further extend theεe methodε by uεing mold inserts thus produced for hot emboεεing. Similarly, M.T. Gale et al.,20 extend methodε from the emboεεed diffractive foil and compact diεk (CD) industries by the replication of an original (e.g. microfabricated) microrelief on the surface of a replication shim through electroforming, followed by replication of this first shim by surface passivation followed again by electroforming. Theεe workers find that the shim replication proceεε occurε with only slight (<2nm) increases in surface roughneεε per generation, thus enabling the reproduction of nanoscale relief features, with corresponding cost advantages. Related injection molding methods are described by A. Neyer et al.,21 and R. Klein and A. Neyer.22
Micromolding of Ceramics and Resonators Therefrom:
J.A. Bride et al.23 have shown that micropatterned reliefs may be used to micromold ceramic patternε. Specifically, these workers used a polyimide relief, prepared by reactive ion etching through a Ti mask, was used as a "cookie cutter" which was impressed into a chemically softened ethyl-methacrylate tape comprising powdered ceramic particles (ceria-zirconia) of average size 0.3 micronε. After embosεing, the tape was dried, removed from the micromold, heated at 600 degrees centigrade to remove the EMA binder and then sintered at 1500 degrees centigrade. Features as small as 4 micronε could be thuε produced. Theεe workers have developed this method for the fabrication of piezoelectric ceramics.
Self-Assembling Monolayers and Resists Therefrom: A paradigmatic example of self-aεsembling monolayers is provided by the self assembly into monolayerε of alkanethiols onto gold εurfaceε, according to the strong affinity of thiol groups for gold εurfaceε.
Aε reviewed by A. Ulman,24 theεe εelf-assembling monolayer εyεtemε also include organosilanes and derivatives thereof (e.g. RSiX3, R2≤iX2 or R3SiX where R groups may be identical or distinct) , which form covalent linkages to surface hydroxyl groups (e.g. formed on surfaceε with expoεed Siθ2 including native εilicon oxideε, Snθ2, Tiθ2) • Note that according to appropriate chemistries, monolayers thus formed may be modified such that the molecules of said monolayers thus formed bear terminal hydroxyl groups, which may then serve as donors for the succeεεive, covalent addition of monolayerε, forming croεslinked multilayered structures on said surfaceε. Note alεo that the organic chains of such monolayers may additionally comprise polymerizable or photopolymerizable groups such as alkynes.25 Further, such monolayers may be used to render surfaces hydrophobic, such that they are protected from a solution with which said surfaces are contacted, εuch aε an aqueouε etchant solution.
Simple Methods for Initial Patterning of Monolaver Resists:
Several groups have formed patterns in homogenous εelf-aεsembling monolayers (SAMs) formed on meta" surfaces or surfaceε of metal films, generally gold films, by methods related to milling or grinding. These methods include scraping with sharpened tools26 to produce the desired pattern as well as applying increased force to an AFM tip during scanning operationε or increaεing the εetpoint current to cause physical contact of an STM tip so said metal surface with increased local forceε reεulting. G.M. Whitesides and co-workers27 have also produced a rapidograph- like micropen for the application of organothiol compounds to specified surface regions, and used this writing instrument to produce organothiol patterns on gold surfaceε.
Microcontact Printing of Self-Assembling Monolayer Resists; G.M. Whitesides and co-workers have discloεed methodε for the microcontact printing of compoundε which form SAMs onto surfaces coated with evaporated metal films, with a strong emphasiε on organothiol compoundε used in conjunction with evaporated gold film coated surfaceε. These workers have demonstrated that metal regions coated by said monolayers are resiεtant to wet chemical etch εolutionε. After εuch a chemical etch εtep, the underlying εurface iε exposed; where said underlying surface is, for example, cryεtalline εilicon, it may be wet etched to produce a relief pattern correεponding to the mirror image of the relief surface used in said printing. Microcontact printing methods uεed by these workers haε been based upon relief patterns replicated in elastiomeric polymer surfaces, generally using (PDMS) elastiomerε and reliefε replicated from microfabricated εurfaceε. Theεe methodε are found by these workers to yield a defect rate (of over 1/mm2 per mask-etch cycle)28 which they deem unacceptably high to make these methods competitive with conventional lithographic techniques for the production of microelectronic devices. They speculate that these defects primarily owe to microcrystalliteε in εaid gold filmε, ariεing from incomplete coverage of said microcrystallites by said organothiol molecules.
Patternε of organothiol SAMε by theεe methodε have reproducible resolution of features smaller than lOOnm. Such patterns have also been formed on the surfaces of glasε cylinders and fibers, which have been used εubsequently to replicate the corresponding pattern on flat surfaces by a rolling technique, analogous to that uεed in the printing of hologramε in metallized polymeric filmε but on a smaller scale.
yj(?i- >-ability control by Self-Assembling Monolayer Composition and Structures Thereby: G.M. Whitesides and co-workers have further demonstrated that patterns of such organothiols affect the wettability of the εurface regionε they coat, and that the preεence of a pattern of a first organothiol can mask the adsorption of a second organothiol, εuch that when a pattern of a firεt organothiol iε disposed upon a gold surface and said gold surface is then contacted with a solution of a second organothiol, εaid εecond organothiol formε a SAM only on expoεed areaε of εaid gold surface; the resulting surface iε thus completely coated with two complementarily patterned organothiol SAMs. Thus, said first organothiol may have a terminal methyl group such that regions with a SAM of said first organothiol composition are wetted well by organic liquidε and poorly by aqueous or polar solutions, and said second organothiol may have a polar terminal group, such aε a hydroxyl group εuch that regionε with a SAM of εaid second organothiol are wetted well by aqueous or polar liquids and poorly by organic liquidε.
With appropriate patternε thus formed, the patterned wettability of such surfaces haε been uεed to fabricate εurface-tenεion defined εtructureε, such as a microlens array29 from solutions comprising optically clear polymerizable compoundε. Wetting may be conducted, for example, by vapor condensation or dipping into solutions.
Lithographically Patterned Copolymer Synthesis:
S.P.A. Fodor et al.30 have disclosed methods for the spatially directed synthesis of copolymers including biopolymers such aε oligonucleotides and polypeptideε. These workerε bind initiator chemical groupε protected with photolabile protecting groups to a εubεtrate. Spatially maεked exposure to appropriate wavelength actinic radiation removes said photolabile protecting groups from expoεed regionε, where the extent of εaid exposed regions is delimited by an exposure mask having a deεired radiation maεking pattern. Said εurface iε then contacted with a εolution of photolabile protecting group protected monomerε of deεired compoεition, which react only with the deprotected (photodeprotected) initiator chemical groups located at expoεed εurface regionε. Unbound monomers are then washed. Said masked exposure step, said protected monomer contacting steps and said washing εtepε are repeated εo aε to synthesize desired oligomers at desired locations on said surface εuch that an array of diverεe oligomerε εituated on said surface iε produced and the location of an array element comprising oligomers bears a predetermined relationship to the sequence composition of said oligomer copolymer located at said array element. Such copolymer arrays may be used, depending on the compositions of said copolymer, to detect affinity binding to members of a population of molecules (comprised within the compoεition of εaid array) of a εample, which methodε are uεeful to εcreen antibody specificity, to detect the presence of polynucleotide sequenceε in a εample or mixture, and for εequencing by hybridization methodε.
Other methodε for the production of εimilar copolymer arrayε have been demonstrated by others, albeit generally at lesser array element densities.
Array Fabrication by Masking for Materials Screening: X.-D. Xiang et al.31 have recently described a method for the εcreening of materialε compriεing the repetitive evaporation of different materialε onto a εurface occluded by a maεk, εuch that an array is formed comprising elements of different compoεition, which array iε then εintered. Array elements thus prepared may then be subjected to εcreening for deεired propertieε in analogy to the methods of the field of combinatorial chemistry.
Microcontact Patterning of Proteinaceous Arrays on Surfaces: Whitesides and co-workers32 have used elastiomeric patterned relief εurfaces to produce surfaces with correspondingly patterned depoεitε of proteinε. In thiε method, a gold εurface iε patterned with an organothiol, and then expoεed to and organothiol comprising terminal hydrophilic moieties are then adsorbed to unpattemed regionε. Such a surface is then contacted with a solution of the deεired protein or proteinε, which adεorb to the hydrophobic εurface regionε. Such a method does not provide for the formation of patternε of variationε of combinations of proteinε from one region to the next.
Production of Molecular Multilayers with
Monolayer Precision:
Various workers33 , 34 , 35 have deεcribed methodε for the stepwise addition of monolayers or bilayers to a surface, such that a multilayer εtructure compriεing a predetermined number of layerε of only a few nanometerε in thicknesε may be formed. According to theεe methodε, once a monolayer or bilayer iε thuε deposited on a surface (including surfaces comprising monolayers, bilayers or multilayers), said monolayer or bilayer remains situated upon said surface.
Synthesis of Sheet-like Polymers:
S.I. Stupp et al.36 have εhown that εubεtantially linear moleculeε compriεing extended linear regionε along their length, a polar enantiomeric ordering center and two polymerizable reactive groups of distinct polymerization chemiεtry at a εpecific positions along their length spontaneously assemble into layered mesoεcale structures which may then be polymerized to yield two dimensional polymerε. Thiε work continueε the teachings of S.I. Stupp disclosed in U.S. Patent Number 5,229,474.
Terminally Functionalized Rod-like Molecules:
M. Kotera, J.-M. Lenh, and J.-P. Vigneron37 have discloεed compoundε comprising rigid rod molecular segmentε with εingle terminal nucleobaεe moietieε. Said compounds self asεemble in organic εolventε to form supramolecular rods according to well-known Watson-Crick pairing rules. The reεulting rods self-organize into mesoεcale εtructures.
Immobilization of Molecules to Scanned Probes:
There have been some succeεεful effortε to derivatize the εurface of a scanning probe microεcope probe (or tip) with moleculeε. One εet of methodε coatε the tip εurface with gold and then incubateε with organic thiol compound to coat the tip to form εome functionalized surface.38'39'40
Another set of experiments has relied on the strong non-εpecific aεεociation of bovine εerum albumin (derivatized with biotin) and the εilicon nitride commonly uεed to produce εuch microfabricated tips.41'42 Similarly, G. Lee et al.43 have immobilized oligonucleotideε to the tipε of cantilevers and asεociated these to complementary oligonucleotideε bound to juxtaposed εurfaces, and have measured the forces developed as such asεociations are ruptured by withdrawal of εaid cantilever. Here, oligonucleotides were εynthesized to comprise a thiol group which was crosεlinked to an amine functionalized organosilane derivatized silica AFM tip. Disruption of association of complementary oligonucleotides was shown to be reversible.
Positional Synthesis of Molecules: D. Eigler has demonεtrate the direct manipulation of atomε adεorbed to metal εurfaceε with a Scanning Tunneling Microscope, and the direct asεembly of molecules from said atomε. Thiε work involved nonεpecific interactionε between εaid atomε and εaid surfaceε, and εaid atomε and the STM tip. Theεe experimentε were carried out at liquid helium temperatureε under ultra-high vacuum conditions, because theεe conditions were necessary for the stability of the interactions used and the molecules (multimerε of CsCl2) thus produced. These atom-by- atom manipulations provide useful insight into scientifically interesting processeε and the propertieε of otherwiεe inacceεεible compoundε, but becauεe of the extremity of the conditionε required, do not suggest practical methods for the asεembly of useful molecular structureε from atoms. K.E. Drexler44, after R. Feynman, has made elaborate proposalε concerning the poεitional εyntheεiε of nanoscale, three-dimensional structures, generally on an atom-by-atom basiε. Practical methodε for εuch εynthesiε have yet to be presented, though C. Musgrave haε undertaken theoretical analyses of factors related to the assembly of diamondoid structures from reactive carbon specieε under mechanical or poεitional control and forceε exerted on reactants thereby (referred to aε mechanoεyntheεiε) .
Objects of the Invention: It iε an object of the preεent invention to reduce the capital coεts of microscale and submicron patterning in the fields of microelectronics, micromechanics and MEMS. It iε a further object to reduce the minimum feature εize below that attainable with viεible light in certain embodimentε. It is an object of further embodiments to facilitate the inexpensive prototyping and low-volume production of such deviceε. It iε a yet further object of the present invention to provide for the inexpensive production of miniaturized εcanned probe deviceε. It iε a further object of the preεent invention to provide for the used of said miniaturized scanned probe devices in the parallel positional syntheεiε of complex moleculeε and εupramolecular assemblages.
It iε an object of the preεent invention to extend the applicability of patterning methods to molecular monolayers, multilayers and polymerε therefrom.
It iε an object of the preεent invention to provide an inexpenεive method for the εyntheεiε of complex copolymer arrayε without extenεive uεe of lithography. It iε a further object to extend the methods of use of εuch complex copolymer arrayε to provide enhanced capabilitieε and refinement of discrimination.
Summary of the Invention:
The present invention employs either contact printing , contact molding, or devices produced thereby, in combination with pattern replication, and applies these to the fabrication of numerous deviceε. An initial or master relief pattern or pattern of surface composition is produced by prior art methods or by methodε of the preεent invention. Thiε maεter iε replicated one or more timeε by methodε provided within the present invention, permitting rapid expansion of template size and number of templates . Final generation templates are used in device production. Macroscale to nanoscale features may be achieved by theεe methods, including within the same fabrication procesε and article of manufacture. Preferred embodimentε include the generation of masking patterns for etch control and diffusion barrier formation in semiconductor fabrication, microfluidics fabrication, MEMS fabrication and the like, patterned chemical synthesis and copolymer syntheεiε, the formation of patterned molecular monolayerε and multilayerε, replication of chemical patterns, fabrication of microstructured and nanoεtructured materials and combinations of any of the foregoing.
Description of the Invention:
Throughout this discloεure, it will be aεεumed that patterned relief εurfaceε are compoεed of materials that are either unreactive to any chemical species which are applied to them or may be pretreated to render them similarly unreactive, or otherwise that any reactivity to said chemical species which are applied to said patterned relief surfaces occurs or may be caused to occur to only a negligible extent. It is also asεumed, aε iε obviouε to thoεe εkilled in the relevant artε, that precautionε must be taken to avoid microscopic contaminants, which cause defects at micron and submicron scales; such precautions include ultrafiltration of solutions, and protection from atmospheric contaminants. It is further noted that it will be obvious to those skilled in the relevant arts that adheεion layerε, reεiεt layerε and εacrificial layerε may be used as needed without departing from the scope of the present invention.
All references are incorporated by citation.
Resist Molding:
Aε an alternative to the patterning methodε deεcribed by Whiteεideε et al. whereby patternε are defined by the tranεfer an organothiol to a gold εurface and formation of a SAM thereby, a reεiεt or maεking reεiεt pattern may be formed upon a εurface by molding an appropriate material in the deεired pattern on εaid εurface or by analogouε techniqueε εuch aε casting, injection molding, or alternatively by a method comprising the stepε of εheet stamping followed by a brief pre- etch to eliminate protecting material in reduced thicknesε regionε formed by εaid εheet stamping step.
In this procesε, a firεt relief pattern iε replicated in a convenient material, εuch aε by the polymerization of a prepolymer (preferably an elaεtiomer) on εaid surface, caεting a plastic, polymeric or other appropriate solidifiable liquid material with εaid firεt relief εurface, plating with one or more metalε by known art techniqueε, or, εtamping into a melted material such as is practiced in hot-foil technology and CD replication, to define a mold. A mold is thuε formed with a pattern defined by εaid firεt relief pattern, and has surface features corresponding to the negative pattern formed by εaid first relief (defined aε the poεitive pattern) . (Note that alternatively, εaid firεt relief pattern may be fabricated εo aε to itεelf be the negative pattern, from which further negative patternε are generated in even numbered replication generationε.) Said firεt relief pattern iε fabricated either by prior art meanε or by methodε diεcloεed within the preεent invention. Relief feature height (or depth) is predefined so as to permit reliable pattern formation, reliable cast or molded material self-coheεion and substrate surface adhesion, and also reliable release of said cast or molded material from said from εaid negative replicated relief. Optimal feature εizes will vary according to the materials and materials combinationε choεen, and εpecifics of any particular embodiment of this category of methods.
Said initial pattern may be used repetitively to form the opposite pattern on a replica surface such that high quality first generation replicas are formed in a quantity increasing arithmetically with each replication cycle. First generation replicas formed by said first relief pattern may be used in turn to generate εubεequent generation replicaε, with multiplicative increaεe in replica quantity per replication generation. Thuε tradeoffε will exist between the quantity of replicas formed in a particular length of time and the quality of replicas thuε formed. High fidelity replication methodε, such aε are used in the fabrication of integrated optical devices and componentε thuε permit the rapid production of replicaε; negative replicaε are then uεed to mold a reεiεt pattern onto a εurface.
In a preferred caεe, a prototype maεter is used repetitively to create negative replicas neighboring each other on a first generation replica εurface, which iε then in turn uεed repetitively to create replicaε of the entire adjacent group of first generation replicas, again in an adjacent configuration. Thus, a single pattern is repeated an increasing number of times on the surface generated by each generation of replication. Thuε, advantageous tradeoffs between relief life, rapidity of pattern production, and pattern fidelity may be attained according to the demands (e.g. time, volume, quality) of production. Calculations relating theεe will be obviouε from the neceεεary empirical data and production conεtraints. Several different procedures are available for the formation of a resist pattern on a surface with such a mold. Theεe methods exclude masking material from regions contacted by the highest extent of εaid negative replicated relief, which, if of elastiomeric composition, will conform to the contours of the subεtrate εurface when juxtaposed to said surface under sufficient normal force. Resist material iε not excluded from recessed regions of said negative replicated relief, and allowed to harden, polymerize, cure or is expoεed to appropriate illumination to photo-cure, aε appropriate, after which εaid negative replicated relief is evenly removed in a manner sufficiently gentle to ensure that the resulting resist pattern is not damaged.
Note that molded resist layers may favorably be formed on the εurfaceε of adhesion layers which have first been deposited or coated onto said εubεtrate εurface without departing from the above aεpect of the present invention. Here, for example, an ultra-thin, uniform adhesion layer which iε εensitive to an etching solution or procedure comprising polymerizable chemical functional groups may first be εituated upon a εubstrate, which is then, for example, coated uniformly with a photopolymerizable polymeric reεist, which iε then patterned under preεεure with a clear relief εurface and then photopolymerized by expoεure to appropriate wavelength light, with the reεulting polymeric reεiεt material covalently linked to εaid polymerizable chemical functional groups of said adhesion layer. Said adheεion layer is chosen to not protect the underlying subεtrate surface from etching treatments or procedures, and is formed at a thickness sufficiently small to prevent the significant under-etch of the overlying resiεt material (due to the εmall croεε-εectional area and correspondingly small diffusion rate through such an area) . Note that such an underlayer may, when poεsesεed of sufficient elasticity, alternatively permit the uεe of a metal relief replica aε said negative replicated relief because εaid elaεticity εerveε the εame function aε the elaεticity of an elaεtiomeric mold, i.e. enεureε that juxtapoεed surfaces subjected to normal forces conform to each other such that liquidε do not εignificantly interpenetrate between the contacting areaε or εaid juxtaposed surfaces; as above, reεiεt precurεor material is excluded from areas of εaid adheεion layer contacted by εaid negative. In thiε caεe, the article being patterned conforms to the relief rather than the reverεe εituation with elastiomeric negative replicated relief molds.
A yet further variation involves the uεe of metal or other rugged reliefε aε "cookie-cutterε" which mechanically exclude a εoftened reεiεt precurεor such as a melt or sol-gel from surface regions underlying outermost extenεionε of the applied relief εtructure.
Fabrication by Layered Molding of Patterned Article Material and Sacrificial Material: Techniqueε εuch aε thoεe deεcribed above for molding of εtructureε uεing relief masters may be used recurεively where each repetition addε another patterned layer (i.e. a portion of a layer in a predetermined pattern which may additionally have εurface featureε or height contourε) of material for the deεired final article or another patterned layer of εacrificial material uεeful in the production of unεupported or overhanging εtructureε.
Here, εacrificial materialε may include waxeε which are melted away at elevated temperatureε or diεεolved in solvents such as alcohols. Theεe would moεt favorably be uεed as sacrificial material with polymers that polymerize in aqueous solution or other solutionε in which said wax iε εubstantially insoluble.
Sacrificial material may also be used to flatten intermediate surfaceε of article material aε an article iε being built up by this method. Here, article material is patterned by molding, caεting or εtamping, and when hardened (if hardening iε required,) the article under fabrication is juxtaposingly contacted to a flat εurface which iε coated with εoftened or liquefied sacrificial material (where aε neceεεary a releaεe agent iε uεed between said flat surface and εaid εacrificial material to facilitate removal of εaid flat εurface after εaid flattened material has set or hardened.) This method is advantageous where an article is conveniently built up in layers with εubεtantially flat borderε.
Note that article material iε preferably choεen εuch that where article precurεor material (εuch aε a prepolymer) iε overlaid onto polymerized or hardened article material, chemical croεεlinkε will form between theεe regionε of two such juxtaposed layers. This will be the caεe, for example, for polymers where polymerization occurε via εide-groupε or where there iε an exceεε of one of the two chemical functionalitieε which are involved in polymerization, such that a plurality of unreacted functional groups are available on the surface of εuch article material after polymerization of that portion of article material haε proceeded to completion.
Complex mechanical and micromechanical syεtemε with conεiderable depth and complexity along the depth axiε may thuε be produced by repetitions of such a procedure, with removal of the sacrificial material used being performed by appropriate treatment after the final layer of article material haε been added. With this method, prototyping requires the production of initial reliefs corresponding to each pattern of article material, which may be uεed in turn to pattern εuch material.
Resist Defect Reduction Methods:
For microfabrication embodimentε of the present invention, variations of the methods of Whitesides et al. are used. The unacceptably high defect densitieε which have dissuaded these workerε from continuing effortε in applying this methods to commercial microfabrication and microelectronics fabrication may be prevented in a number of ways such that an acceptably low defect denεity may be achieved. By such methods, the simplicity, economy and high resolution capabilities of microcontact printing may favorably compete with deep-UV lithography and other more conventional high resolution patterning techniqueε.
i. Metal Film Annealing: The defects encountered by Whitesideε and co-workerε are attributed by theεe workers to crystalliteε arising in the gold film depoεition process. It has been determined45 that an acetylene flame annealing step yields atomically flat terraces extending hundreds of nanometers. STM shows these surfaces to be better coated with an organothiol derivative (dimethylaminoethanethiol) than the unannealed film. M.D. Ward and co-workers46 have previously found that annealing a gold wire with a hydrogen flame yieldε atomically flat terraces extending over hundreds of microns. Because atomically flat surfaceε are better subεtrateε for the formation of self-asεembling monolayerε, and because crystalliteε are reduced or eliminated by such thermal annealing, thermal annealing εtepε will thuε reduce the occurrence of defectε when added, before the formation of the patterned organothiol SAM, to the procedure of Whiteεideε and co-workerε. It iε, of courεe, neceεεary that the annealed film be permitted to cool (i.e. thermally equilibrate with the temperature of the relief and applied εolution) before application of the SAM forming material, becauεe thermal contraction will otherwiεe diεtort the deεired pattern.
ii. Multilayer Vetoing: Defectε which are εmall and occur approximately randomly, aε iε the caεe as the gold-organothiol reεiεt maεking method of Whitesides et al. , may be prevented from appearing in the etched final product by a vetoing method which bears analogy to the reticle voting method deεcribed in U.S. Patent Number 5,308,722 by J.L. Niεtler. An elastiomeric relief may be prepared by related art methods or as otherwise discloεed in the preεent invention, and then coated with an organothiol εolution. In the preεent caεe, a thin layer of gold is deposited by suitable means onto a substrate. This is then thoroughly cleaned and contacted with said patterned relief surface which has been coated with an organothiol εolution. The pattern of εaid relief are thuε tranεferred, in the form of a SAM of said organothiol, to the surface of εaid gold film, in a first printing step. The patterned εurface thuε produced iε then coated again with gold, forming a εecond gold film layer, and again patterned in a εecond printing εtep with the same relief surface coated either with the same or a different organothiol compound εolution. The uncoated regions of said second gold film layer are then etched by appropriate εolutionε, εuch aε εtrong acidε, such that the underlying subεtrate iε expoεed in these unprotected regions and thuε εuεceptible to etching and impurity diffuεion. Where εaid εubεtrate iε compoεed of εemiconductive materialε, electronic, microelectronic, MEMS and other deviceε may be produced through processeε compriεing the above method. For defects that occur due to incomplete SAM formation in the contacted regions, whether due to defects arising during the formation of said gold film layers or in the monolayer formation process (i.e. incomplete coverage of a satiεfactory gold εurface region) which are not due to large εurface protrusions, such a multilayer patterning method will prevent defects from leading to the improper exposure of subεtrate εurfaceε after εaid etch εtep becauεe only defects in the topmost layer organothiol or topmost layer gold film will permit exposure of the next underlying organothiol layer on the next gold layer. Where defect occurrences are independent events from layer to layer, defects are reduced in εuch a proceεε aε the power of the number of layerε uεed; for the above two layer proceεε, the probability of a defect occurring in a unit of area iε εquared. Aεεuming a defect 1 εquare micron in εize occurε on average once, randomly, in an area of 1mm2, the relative defect area of 10~° yields a defect rate per two-layer mask of 10~12, which should be tolerably low for most applications. Such a defect rate may be reduced further by three or more repetition of the above coating and printing stepε.
Of course, such methods will require correspondingly refined alignment between multiple masking steps, which may be ensured by more accurately machined alignment means which are designed to fasten to the reference pointε (e.g. drilled holeε of preciεe location and diameter, three or more per different reεiεt used in a pattern generation procedure) on relief negatives with high positional repeatability. Such considerations and methods for accomplishing the required alignment will be obvious to those skilled in the arts of microfabrication. Note that for purposeε of the present invention, reliefs may favorably incorporate features which facilitate alignment between succeεεively applied relief patternε, such as features, favorably placed at borders which generate Moire patterns under appropriate illumination and imaging (which may include electron microscopy) due to the overlap of patternε in εaid εubεtrate and said negative relief. Note further that the methods of the present aεpect of the present invention very directly also permit accurate tactile alignment, where εaid featureε compriεe interlocking reliefε which only fit together when εaid relief and εaid substrate are in precise registry with each other. Such tactile alignment methods may thuε permit repeatable accuracy to within a few nanometerε with appropriately deεigned patternε and sufficiently resilient materials. Higher tolerances will require increasingly careful choice of materials and control and uniformity of temperature to prevent significant diεtortions due to thermal expansion. As will be obvious, εuch alignment methodε may be uεed with other aεpectε and embodimentε of the present invention.
iii. Initiator Polymerization: A further approach to defect reduction iε applicable to defectε arising from incomplete SAM formation in relief contacted regions. Here, the chemical composition of said SAM is chosen such that the exposed surface of said SAM comprises chemical functional groups which serve as initiators for εome polymerizable material. Note that compounds which do not form SAMs may also be used, provided these are reliably deposited on said subεtrate and do not diffuse; compounds used need only suitably form patterned initiator compound regionε. A quantity of material of εaid chemical compoεition (for example, dissolved in a first solution) iε applied in εufficient but not exceεεive quantity to a deεired patterned εurface, which iε then contacted with a εubεtrate to tranεfer εaid material of εaid chemical composition to εaid surface in a pattern determined by the pattern of said patterned εurface. Said εurface iε then washed to remove any excesε reactantε or reagentε. Said material of said desired chemical compoεition, now εituated in a patterned manner on εaid εurface, is then contacted with a second solution comprising monomer reactant species which are capable of reacting with said chemical functional groups which serve aε initiatorε. Said εecond solution preferably containε εaid monomer reactant species at low concentrations such that molecules of said monomer reactant εpecies are more likely to react with said chemical functional groups which serve aε initiatorε, or oligomers or polymers therefrom, rather than with other said moleculeε of said monomer reactant species. The limit on the concentration of said monomer reactant species are those poεed by non-specific polymerization, i.e. εignificant polymerization of said monomer reactant specieε into moleculeε not in communication with εaid chemical functional groups which serve as initiators. The term εignificant here referε to the quality of the overlayerε thuε produced and the non-coating of εubεtrate regionε not compriεing εaid chemical functional group which εerveε aε initiators, i.e. reduction of resolution in non-resiεt coated regions. These polymerizations are thus preferably conducted so as to primarily yield solid phase immobilized polymer products. Said second solution may further comprise multimeric monomers or other croεεlinking reagentε which serve to cause branching of polymer chains during a polymerization procesε. Conditionε of reaction, the concentration of said monomer reactant specieε, and the proportion or εurface density of said chemical functional groups are chosen such that the polymer coating formed will be substantially limited to the regions of εaid εurface originally contacted by εaid patterned εurface, i.e. not extending far from any of εaid chemical functional groups which serve aε initiatorε. For example, monomeric species may be added at a limiting quantity, or at a concentration sufficiently low that excesεive polymerization does not occur within some conveniently short period of time. Thiε method of εurface coating patterning followed by coating thickening (which may be termed overlayer formation) εerveε to enclose any small resiεt layer defectε without εignificantly compromiεing patterning reεolution. Such encloεure reduceε or prevents transport of etching agents to defect siteε and etching products from said defect sites. As defect size increaseε, this method requires that overlayer thicknesε be increaεed correεpondingly to ensure enclosure or encapεulation of defectε of εaid defect εize.
For example, said chemical composition of said SAM may be chosen to comprise an alkanethiol moiety and a methacrylate moiety. A solution compriεing a quantity of εaid chemical composition is applied to a gold film situated on a substrate, such that a SAM is formed according to the pattern of said patterned εurface. Said gold film and substrate is then waεhed to remove any exceεε unbound material. Limiting quantitieε of methacrylate and any deεired polymerization acceleratorε, aε well aε a deεired ration of polyfunctional or branched methacrylate εpecieε are added, and overlayer polymerization is permitted to occur to a desired extent, correεponding to the defect εize to be thuε eliminated. Unreacted materialε are then waεhed away with appropriate solvents that do not degrade said overlayer. The surface thus treated is thus patterned with a resiεt layer of corresponding thicknesε which doeε not have pinhole defectε or other εmall defectε smaller than some critical size.
Note that the methods of this aspect of the preεent invention are readily applied to patterned thin film coatingε which are not SAM baεed, provided that appropriate chemical functional groupε which εerve aε initiatorε for εome polymerizable material may be incorporated. Note further that any chemical functionality which may be modified (e.g. deprotected, activated, or reacted with εome other initiator bearing chemical species) such that it may later serve aε an initiator for polymerization reactions is comprehended within the εpirit of this aspect of the present invention.
Resist Formation by Wettability Control: The methods of G.M. Whitesideε and co-workerε involving the wetting of patterned surface regions, which these workerε have used to form microlenε arrayε from polymer εolutionε, may alεo be applied to the elimination of small defects. In this approach, patterns of resist are formed by firεt patterning a SAM which iε preferentially wet by a reεiεt monomer εolution or precursor solution on a surface which is poorly wet by said solution. Said patterning is accomplished by one or more of the methods described herein, such as microcontact printing, and the patterned said surface is then contacted with a resiεt precursor εolution, which iε then withdrawn. Portions of said resist precursor solution retained on said surface are then caused or permitted to polymerize, such that a developed resiεt layer iε formed. Thus, defectε related to incomplete maεking may be eliminated by their enclosure by the overlying resiεt precurεor εolution and hence the reεulting developed resiεt. Etch εtepε are then performed aε in conventional microfabrication practice.
In the caεe of pattern which cauεe εelf-organization of εaid reεiεt precursor solution, and hence predetermined desired geometries of εaid developed reεiεt layer three dimenεional structure, resiεt depth- dependent etching, in analogy to that described by G. Gal in U.S. Patent Number 5,310,623, may be performed, for example by reactive ion etching, to replicate the self-aεεembled resist structure in the underlying solid subεtrate material.
Patterned Metallization: Metallization, for example, for the formation of electrical contactε, may be accompliεhed according to the patterning methodε of the present invention and corresponding extenεionε of related art methods, as well as by the application of related art methodε to articleε produced by the methodε of the present invention (e.g. formation of alkanethiol patterns by microcontact printing on gold films, employing, as necesεary, the defect reduction methodε disclosed herein) . Molded materials such aε those used above for resiεt purposes may, with appropriate solventε, be uεed aε lift-off layers which eliminate any metallic film εituated on the surface of εaid molded materialε upon expoεure of the εurface compriεing theεe to said appropriate solvent. Patterns of said metallic film remain in regions which were not covered by said molded materials.
As will be obvious, formation of metal filmε may be accomplished by vacuum evaporation of the correεponding metallic material onto the εurface of the article under production. Alternatively, εuch lift-off patterning may be applied to thin electroformed (e.g. electrodepoεited or electroleεε plated) metallization layers.
Alternatively, metallization may be accomplished by the hot embosεing of a first εurface with a metallized polymeric foil with the metallized εurface juxtapoεed to εaid firεt εurface, under εufficient preεεure and at εufficient temperature to enεure good electrical contact with expoεed regionε of said first εurface, followed by diεεolution of εaid polymeric foil and underlying lift-off regionε. Such transferred filmε may be εubjected to brief electrodepoεition steps to ensure good electrical contact, though care.muεt be taken to avoid the formation of εhort circuits.
Microelectronics Device Fabrication:
The foregoing fabrication methods compriεing the uεe of relief patternε uεed to pattern reεist or masking layers may be uεed to control the etching and doping (impuritieε diffuεion) of εemiconductor materials and overlying dielectric layerε, as well as the formation of metallization layers for electrical interconnection. These methods serve all of the patterning functions required in microelectronicε fabrication.
Note further that the above deεcribed reεiεt molding and caεting methods may similarly be applied to conductive polymers compoεitions such aε thoεe comprising polyparaphenylene and derivatives thereof, polypyrrole and derivatives thereof, polythiophene and derivatives thereof, or other such conducting or electroactive polymerε, εo aε to directly form polymeric deviceε rather than reεiεt patterns. Theεe materialε and devices based thereupon may further be combined with more conventional microelectronic deviceε fabricated by the methodε of the preεent invention. Initial relief patternε may be formed in, for example, glaεε or εilicon εurfaceε by known art methodε including photolithography, or may be formed by appropriate scanning probe based patterning methodε of related art, or combinationε thereof. For example, a glaεε εurface may be coated with a gold film, upon which an organothiol SAM iε formed, which iε then patterned by STM or field emiεsion mode STM, followed by an acid etch to remove exposed gold regions and a base etch to remove thus exposed glaεε regionε. It iε important to note that a large patterned area may be formed by the replication of a εmaller pattern from an initial relief acroεε εaid large patterned area, aε, for example, a εingle integrated electronic device pattern layer (e.g. correεponding to a maεk layer) repeated many timeε acroεε the εurface area correεponding to a wafer diameter. Thuε, the replication methods of the present invention may be applied both to expand the number of occurrences of a pattern on a single surface aε well aε the number of εurfaceε thuε patterned.
Spatially Controlled Copolymer Synthesis:
A relief patterned surface, preferably of elastiomeric compoεition, may be used to spatially control the pattern of addition of monomer or macromonomer reactantε to a εubεet of appropriate chemical reactive εiteε on a εubεtrate εurface. Here, instead of the addition of single species to surface regionε according to the pattern of an elaεtiomer εurface, aε done by Whiteεideε et al. , reactantε are added to regionε of the εurface of εaid εubεtrate according to the pattern of εaid patterned εurface, permitted or cauεed to react, and unreacted or unbound εpecies are then washed away. Generally, said subεtrate will comprise one or more subεtantially flat surface regionε and be prepared by the reaction or adεorption of εome chemical εpecieε to εaid εurface εuch that chemical εpecieε having desired reactivitieε are bound to εaid εubstrate, which is thus capable of being reacted with desired reactant specieε. Said relief patterned εurface iε partially or completely coated with a εolution of a deεired reactant, or εaid deεired reactant iε depoεited from vapor phaεe onto εaid relief patterned εurface. In the former caεe of reactantε diεεolved in εaid a εolution, εaid a εolution may further compriεe inert compounds which increase the viscoεity of εaid εolutions. Said relief patterned surface is then contacted, in a contacting step, with said substrate to transfer εaid deεired reactant to one or more predetermined regions of said εubεtrate. Thus, said desired reactant is applied only to the desired regionε, according to the predetermined pattern of said relief patterned surface. The control of precise copolymer sequence thuε εyntheεized is favorably achieved, for example, through the uεe of protection/deprotection or activation chemiεtrieε to effect εtep control over reactant addition to the monomer or copolymer εpecieε with which said desired reactants are reacted. Thus, reactants added will generally comprise a protecting group or an activatable reactive group, which serve to prevent multiple monomer or macromonomer addition to the εame molecule. Through successive cycleε compriεing the εtepε of contacting εaid εubstrate with deεired patterned εurfaceε coated with desired reactant specieε, to permit εpatial control over reactant addition, followed by εubεtrate waεhing steps, and steps preparing product copolymer species for subεequent reactions (e.g. deprotection stepε or activation εtepε by chemical or phyεical treatments of said substrate surface according to the polymerization chemistries and step control chemistrieε employed) , complete spatial control over synthesized copolymer sequence may be achieved. During successive cycles, different patterned relief surfaceε are uεed at different εtepε, and may optionally be εhifted in location from one εtep to the next; similarly, different desired reactant species may be used during different cycles. This is in analogy to multiple masking stepε in related art microfabrication, and directly related to the multiple masking methodε of other aεpects of the present invention. By such methods, arrays of copolymerε of predetermined εequence, εuch aε oligonucleotideε, polypeptideε, biomimetic copolymerε and non- biological copolymerε may be produced at a resolution equal to that with which a relief master may be fabricated. Patternε having featureε many timeε εmaller than one micron are thuε achievable, and correεpondingly high denεitieε (such as 10° to 1010 array elements per cm2) may be achieved without extensive use of costly lithographic equipment. Such methods thus carry several advantages over the methods of S.P.A. Fodor et al. in that protection/deprotection chemiεtrieε (and the correεponding reactant compounds or protected/deactivated) need not be limited to those responεive to actinic radiation or other energetic beamε. In other wordε, reactantε useful for ordinary (e.g. not light controlled) stepwiεe εyntheεes are uεeful with theεe methods. Further, these methods are εuitable for the synthesis of copolymer of compositions which might be or are desired to be senεitive to photochemical or other energy induced degradation.
In a εecond variation, εuch a εaid patterned εurface may be uεed to control the εpatially controlled activation or deprotection of copolymerε or copolymer precurεorε, εuch that only the copolymerε or copolymer precursorε located at predetermined regionε of εaid surface are prepared for subsequent reaction with a monomer or macromonomer, which is thereby added only to said copolymers or copolymer precursorε located at said predetermined regionε. Here, a relief is for example coated with solutionε comprising deprotection or activation reagents at sufficient concentrations (and preferably also inert constituents increasing the viscosity of said solutions) and contacted with said surface to effect the spatially controlled deprotection of the product precursor copolymerε εituated thereupon. A εolution compriεing εaid monomer or macromonomer iε then contacted with said s.ubεtrate εuch that theεe specieε may react with any available corresponding reactive groups.
The methods of this aspect of the present invention thus permit the syntheεiε of a potentially large array of diverεe copolymer εpecieε εituated on a εurface according to a predetermined pattern, εuch that compoεition of the copolymerε at a particular region of said surface correspond in a known fashion to the location of said region. Thus, where activitieε are uniquely diεplayed by particular regionε of εaid εurface, the copolymer sequence composition responεible for εaid activitieε may directly be deduced from information about the spatial location of said particular regionε.
Note that thiε method may be uεed to synthesize or otherwise target copolymerε, affinity groups or other molecular functionalities or decorations to predefined regionε of the surfaces of microreliefs, micromolded componentε and componentε of micromechanical or microelectromechai-ical systems. In these inεtanceε, the reliefs used for pattern formation are produced from master structures which are identical to the molded surfaces in regions to which said reliefs are to contact εaid microreliefε, εaid micromolded componentε, etc., and thus situate reactant monomers or other reactive compounds, but which have surfaces or surface features of increased height at regions not to be targeted by εaid reactant monomers or other reactive compounds. In other words, the reliefs used to target reactantε to predetermined regionε of contoured εurfaceε conform to the geometry of thoεe εurfaceε in regionε to be targeted or modified, but do not contact regions to not be affected due to clearance within said relief that preventε contact of the εurface of said relief with regions of εaid contoured εurfaceε not targeted. In the case of copolymer synthesis on preferably flat surfaces, reliefs may be mounted on a roller apparatus, in analogy to arrangements used in holograph stamping processes, to facilitate rapid patterned deposition of reactants. Note that for variations relying on selective contacting of εaid substrate with reactants rather than with deprotection reagentε, rinεe εteps may favorably be performed with reagentε that react with unreacted monomer reactants to inactivate said unreacted reactants such that these will not, upon washing, react with specieε bound to regionε onto which εaid unreacted reactantε were not contacted by εaid relief. For example, in the case of phosphoramidite oligonucleotide εyntheεiε chemiεtrieε, alcoholic εolutionε may be uεed to inactivate and wash away unreacted phosphoramidite monomers, which will react with the hydroxyls of alcohol molecules.
Example Synthesis Procedure:
A first surface, such as a glass surface, is patterned with a first alkylεilane compound compriεing an extended methylene or polyethylene glycol linker, or other substantially linear linker chosen to be well solvated or wetted by oligonucleotide synthesis solvents or chosen to be readily modified so as to be well εolvated or wetted by oligonucleotide synthesis solvent, and an esterified terminal hydroxyl group by use of an elastiomeric relief with a square grid pattern such that ε uare regions of said firεt εurface are modified by said first alkylεilane compound with diεtinct unmodified borderε (which are hydroxylated) remaining. Untreated regionε of εaid εurface may then optionally be permitted to react with a εecond alkylεilane compound compriεing a terminal functionality which will be poorly wetted by the εolventε containing oligonucleotide εyntheεiε reactant monomerε. Said firεt surface is then treated with appropriate reagents such as bases to hydrolyze the terminal ester group of said first alkylsilane and thuε deprotect the terminal hydroxyl group. A εolution of 3' or 5' protected oligonucleotide synthesis reactant monomers of one nucleobaεe type is then coated onto a second εurface, onto which an elaεtiomeric relief iε impreεεed to tranεfer εome of εaid reactant to the surface of high regions of said elaεtiomeric relief (with raised square regions corresponding to a subset of the raised εquare regionε of the relief uεed to form the regular array of said firεt alkylεilane) , or the entire εurface of said elastiomeric relief is directly coated with a solution of εaid reactant monomer. Said relief with reactant εituated on itε εurface iε then contacted, in an orientation and poεition which alignε the array element featureε of εaid relief with thoεe of the previouεly formed grid of the alkylεilane on εaid firεt surface, with said first surface, such that said reactant iε contacted with the expoεed hydroxylε of εaid firεt alkylεilane compound, and reactions occur linking this nucleotide monomer with the hydroxyls of said first alkylsilane compounds in the affected array elements. Such a proceεs is carried out with different relief patternε and the remaining three nucleotide monomers, such that all array elements experience the positionally controlled addition of exactly one of the four nucleotide precurεor εpecieε. Note that in thiε example, εaid firεt εurface regionε which may have optionally been treated to be poorly wetted by said reactant εolutions will serve as borders impeding the spreading of these reactants beyond targeted array elements; said well solvated linker moieties are drawn into the solutionε with which they are contacted, even where theεe are εituated on surfaces which are poorly wetted by said solutions with which they are contacted (in which case undesirable spreading of reactants to inappropriate surface regions iε prevented while εyntheεis is not frustrated) . Said first surface is then thoroughly rinsed. Capping reagents (εuch aε acylating reagentε) may optionally be applied to all of εaid first surface to prevent any further elongation of unreacted hydroxyl groups. Other standard oligonucleotide εyntheεiε εtepε, εuch aε oxidation, may then be performed as required by the particular chemistry uεed. The entire array iε then treated with reagents to deprotect the reactant monomers added during the previouε cycle (e.g. εubmerged in a εolution compriεing trichloroacetic acid in the case of trityl protecting groups, exposing a terminal hydroxyl group which is the target for further reactantε) . The εecond baεe of all array elementε iε then added, in a nucleotide addition cycle compriεing the coating of four diεtinct reliefε (for example, together εelecting all array elements but sharing none) with four distinct reactantε, and four contacting εtepε as above, followed by appropriate steps according to the chemistries used, as above. Such cycleε are repeated until the array thuε εynthesized subεtantially consists of oligonucleotides of the deεired length (i.e. n cycleε total to produce an array of n-merε) and deεired element εequence according to poεition within εaid array. Finally, εtepε deprotecting nucleobase moieties and, where applicable, deprotecting the phosphate backbone, are performed to yield the desired final molecular structure, aε in conventional oligonucleotide synthesis methods, excepting support removal stepε. Infoπnation regarding the configuration of raised array elementε, the reactant and cycle with which they are aεεociated, is preserved such that position of an array element corresponds in a known way with the sequence of reliefs and reactants used to synthesize the oligonucleotides therein, which in turn thus have the correεponding sequence composition. The denεity (number of array elementε and hence diεtinct εequenceε per unit area) of arrays thus synthesized may rival or exceed that posεible with light directed patterning method . An important advantage of this method is that conventional oligonucleotide εyntheεiε reagentε may be used.
Alternative copolymer array patterned synthesis methods: The relief forming methods deεcribed above may be used to produce a stencil capable of masking surface regions from exposure to reactants. Here, a substrate is first coated uniformly with a sacrificial layer, which is hardened or set. Then, an elaεtiomer precursor iε applied to the surface of said sacrificial layer and then molded by a negative relief. Alternatively, analogous caεting or injection molding may be utilized, or emboεεing of a polymeric film with brief poεt-etching of the embossed film may be used. Said sacrificial layer is then diεεolved to free the patterned elastiomeric stencil.
Such a stencil is used in combination with an array of microfabricated columns, said columns being of uniform height, and with a pitch smaller than the smallest stencil feature. Said stencil may either be combined with said array of microfabricated columns into one εtenciling body, or said stencil and said columns may merely be fuεed together. In either case, said stencil is juxtaposed to the substrate which is to be subjected to patterned copolymer synthesis, and said array of microfabricated columns iε preεεed onto εaid stencil, such that said columns force the εtencil εurface into good maεking contact with εaid εubεtrate. Solutionε containing reactants (e.g. deprotection reagents or monomer reactants) are then cauεed to flow through said array of microfabricated columns, such that areas not masked by said stencil are exposed to said εolutionε. Before εaid εtencil iε released from said subεtrate, thorough waεh εtepε are performed to remove reεidual reactantε or reagentε. Differently patterned εtencilε are used in predetermines sequence with different monomer typeε to produce the desired diverse copolymer array with sequence correlated to location in a predetermined manner.
In the case that said array microfabricated columns is to form one body with said stencil, these may be joined together, e.g. by coating the upper surface of said stencil with an adhesive or croεεlinking agent which will react with both the material of εaid columns and the material of said stencil. Compounds used for such joining muεt be selected so as to be inert to the reagents and solvents which will be pasεed over them. Note that said array of microfabricated columns may favorably be produced by LIGA methods or other replication methods including those of the present invention, and may favorably be composed of metals, e.g. formed by electrodeposition or electroless plating methods.
Uses of copolymer array decorated articles:
Surfaces prepared according to the above aεpect of the present invention may be used in a variety of ways including for sequencing by hybridization (where said copolymers are oligonucleotideε or oligonucleotide analogs) , sequence confirmation by hybridization, for the εcreening of small peptides and the determination of affinity interactions (aε described by Fodor et al., referenced elsewhere herein), for the detection or determination of antibodies, in clinical medical asεays, and in the screening of organic material for desired propertieε according to known methodε in combinatorial chemiεtry. Sequencing by hybridization, aε well aε other techniqueε involving binding determination, may favorably be performed by contacting εampleε with such an array, where sample moleculeε binding to said array is monitored by any of se ral known art techniques, while physical or chemical conditionε are changed and binding is recorded (e.g. by capture of signalε detected by εuch monitoring and recorded electronically) aε a function of conditionε. In a preferred inεtance of binding determination with variation of phyεical and/or chemical conditionε, the condition dependent propertieε of setε of array elements (i.e. copolymer sequences) of interest are determined by observation of condition dependent binding of known samples, and information thus obtained iε used to further refine the discrimination of correct (i.e. most highly matched) or most specific binding from incorrect or relaxed specificity binding.
For example, in this way, difficulties in εequencing by hybridization methodε ariεing from differences in the conditions for optimal binding εpecificity which correεpond to individual array elementε (oligonucleotideε), which fruεtrate attemptε to find globally optimal binding conditionε without sacrificing the specificity of many or some of the array elements in an array compriεing large numberε of diεtinct oligonucleotide elementε, may be overcome. Here, conditionε which may be varied include temperature, ionic εtrength, divalent cation concentration, preεence and/or concentration of tetramethylamonium chloride, preεence and/or concentration of denaturant compoundε εuch aε formamide or dimethylformamide, pH, and other factors known to affect the hybridization specificity and stringency of oligonucleotides or polynucleotides, may be varied individually or in combination. For example, the binding, and also diεεociation of a particular εequence with the complementary n-mer oligonucleotide will occur at a characteriεtic temperature (i.e. having a characteriεtic Tm melting temperature at which half of all homoduplex complexes disεociate) , and thiε property verifieε the correctneεε of baεe pairing.
Array Detection of Rare Molecular Species: Such arrayε may be used to detect very low quantities, approaching single molecules, of complementary molecules in samples by a few distinct methods. According to the first method, a target array iε deεigned in a hierarchial manner εuch that as monomers or εubunitε are added, they are added to subεetε of regions to which the previous monomerε or subunits were added. For example, an oligonucleotide array is produce by forming first square regions, subdivided into four square regionε each containing one of adenosine, guanosine, cytoεine or thymidine nucleotideε (e.g. through a set of four stepε, each εtep adding one nucleotide to one subdivision region) . During the succeεεive synthesiε εtepε, identical εquareε with εides one half that of those of the preceding εtep and area one quarter that of the preceding εtep are formed, again εubdivided into four square regions each containing one of the four nucleotideε, preferably in the same relative configuration as that of the preceding set of εtepε, are added, εuch that one subdiviεion εquare of the previouε step is thus subdivided into four subdivisions. Such a hierarchial subdivision procesε compriεing n repetitionε of the above sets of four steps (one for each nucleotide moiety) iε repeated to produce an array of 4n n-mers, or desired subεets thereof. For purposes of detecting a rare molecular specieε, a set of arrayε of oligonucleotideε of increaεing length, e.g. { (n-m) , (n-m+1) ... (n-l) , n}-mers in the case of single nucleotide increments, is produced as above. This set of arrayε iε designed such that each succesεive array in the series is in the mirror image of the preceding array of said series, with at least one further εyntheεiε cycle (set of synthesiε steps) further subdividing the regionε oppoεed to the smallest array elements of the preceding array. According to the expected relative abundance of the species in a εample to be detected, εaid sample is bound first to the array consisting of the smalleεt number of elements. Thus the sample is divided such that each oligonucleotide targets a region of some εubεet of moleculeε in εaid sample. Hybridization to such a lower density array by rare species will be more accurate under appropriate conditions in that fewer array elements of larger area present more molecular targetε and fewer degreeε of freedom. Thus the kinetic limitation on binding depends mainly on the diffuεional transit time from across said array. After a sample is bound to such a lower density first array, it is juxtaposed to a further subdivided mirror image second array, with only a small gap between these arrayε, which iε filled with an appropriate buffer. Conditions are changed to denature the asεociation of εample molecules with said firεt array, and then to binding conditions sufficiently stringent that εaid εample moleculeε will favorably bind to their target oligonucleotideε on said second array in preference to forming the lower energy association with their shorter targets on said first array. This proceεs resultε in εuccessive subdivision of said sample, such that each subdivision step εucceεεively narrowε the volume which muεt be traverεed by a εample molecule in order to find itε correct binding target, and further correspondingly reduces the chances that a molecule will find an incorrect target.
An analogous method within the scope of this firεt method may be applied where an array is designed, as above, such that array elements are similar (but not identical) in sequence to most of their nearest neighbors, such that spatial proximity is related to ordered sequence εimilarity. This similarity may be used to serve a εimilar "narrowing down" function accompliεhed above by juxtapoεition of hierarchially related mirror image arrayε. In the preεent caεe, conditionε affecting binding εtringency are varied εuch that after a sample is first applied, only a few contiguous base pairs are sufficient for binding. The regions bound by a sample molecule comprising a specific sequence may either be within the contiguous region compriεing many array elementε having m of the total n monomerε in common (εtarting from the firεt variable nonomer added during the firεt cycle of array syntheεis), or with m contiguous monomers offεet from the copolymer terminal from which εynthesis began, in which caεe, for each claεs of array elements with said m monomers in common, contiguouε regionε of elementε compriεing said m monomer εequence complementary to said specific sequence, where claεses are diεtinguiεhed according to the number of monomerε by which εaid m monomer εequence iε offεet from εaid firεt variable monomer, become increasingly small and increasingly diεtant from each other. Thiε reεultε in reduced dwell time in incorrect regionε (incorrect with reεpect to the exactly complementary n-mer target) but in the presentation of larger contiguous targets by regions comprising the n-mer target. In other words, said specific sequence binds to the correct region comprising the n-mer target and εimilar n-mer εequenceε according to an equilibrium expreεεion where the proportion of said specific sequences bound to correct regionε iε proportionate to the concentration of unbound εaid εpecific sequenceε timeε the relative area of said correct region (in direct analogy to the concentration of m-mers in a conventional equilibrium conεtant expression) . Conditions are then made gradually more stringent, such that more associationε between contiguouε baεe pairε are demanded for binding. Stringency (i.e. the conditionε affecting εtringency) may be oεcillated in an increaεing trend, such that sample molecules are moεt likely to home in on their final target oligonucleotideε. Those sample molecules which do not initially bind sufficiently near their targets will enjoy increased diffusional mobility as those sample molecules which have bound to said array are less likely to impede their diffusional transport through the sample εolution, e.g. after they no longer bind m-mer comprising elements further from their correct targets under more stringent conditionε.
Generalizing thiε method to any copolymer with sequence dependent binding properties, phyεical and chemical conditionε may be varied to examine the characteriεticε of, or detect the preεence of, binding activitieε corresponding to respective εetε of conditionε, or εpatial or temporal gradientε thereof.
Note that deεignationε above of terminal m-mers are chosen for clarity rather than limitation. The essence of the forgoing description concerns the hierarchially imposed proximity of array elementε with similarity which generally decreaseε with increaεing diεtance of elements in said array. While an m-mer may occur in many array elements in many diεtinct regionε of εaid array, it remainε the caεe that the m-merε in phaεe with the target n-mer sequence are grouped together in one contiguous region comprising said target n- mer, regardless of the ordering of synthesis cycles with respect to such hierarchiality.
A second method, applicable to polynucleotide εampleε, involveε the amplification of rare specieε, or εequence fragments thereof. In this case, eεtablished art methods such as the polymerase chain reaction (e.g. with PCR primers flanking the diverse target sequence) or the ligase chain reaction (e.g. for an 8-mer array, involving all 4-mer oligos under conditions εufficiently εtringent to preclude any miεmatch within said 4-mer εequenceε for binding) are used to amplify the sample target sequenceε, which may be a diverse sequence in a conserved sequence context. Thus, small quantities of molecular specieε within heterogeneouε εampleε may be amplified to a sufficient extent that detection of binding to an oligonucleotide array iε conveniently accomplished. Amplification reagentε (e.g. primerε or nucleotideε for PCR or oligonucleotides for LCR) may be labeled, for example, with one or more affinity moieties (e.g. biotin, digoxigenin, etc.,) or one or more fluorescent dye moietieε (e.g. Texaε red, rhodamine, fluoroeεcien, etc.,) or one or more other reporter groupε or portionε. For theεe amplification methodε, amplification will produce a geometrically increasing quantity of sequences matching the amplified sequences but also a related quantity (which may be biased by such known art methods as asymmetric PCR)
Spatially Controlled Macromolecular
Col localization:
Similar operations as used to synthesize copolymers on a εurface in a εpatially predetermined manner deεcribed above may similarly be used to collocalize multiple distinct macromolecule or macromolecule typeε to predetermined εurface regions. For example, such methods may be uεed to generate a very large number of poεitionally predetermined combinationε, εhapes and/or patterns of large numbers of adhesion proteins which may be used , for example, to test the effect on cell adheεion, cell motility, cell εhape or cell phyεiology in biological and clinical aεεayε, as shown by Whitesideε and co-workers for patterns consisting of a single protein specieε, as well as in other biotechnological uses, including the interfacing of cells to microelectronic deviceε and sensors. These methods may avail the protein patterning methods disclosed by Whitesideε and co- workerε47'48, or may avail other immobilization chemistries. The combination of protein patterning with combinatorial methods thus enabled permits the study of competing influenceε when different pattern, relative abundanceε, and different combinationε coexist. Note that in these cases, the binding of the macromolecules, which may or may not be macromonomers, may be according to affinity interaction or less specific weak interactions, i.e. need not be covalent binding.
The preεent aεpect of thiε invention thus depends on the uεe of a relief pattern compriεing raised surface elements to contact a solution containing one or more protein or polypeptide to portions of a firεt εurface to which εaid protein or polypeptide will adsorb, wherein contact only occurs between said raiεed surface elements of said relief pattern and said firεt surface, such that a pattern of said protein or polypeptide is depoεited in the pattern correεponding to εaid raiεed surface elements onto said first surface, wherein such patterning stepε are repeated to yield depoεitε of plural different proteins or polypeptideε at different regionε of εaid firεt εurface, εuch that different combinationε result in a spatially predetermined manner. Patterned Combinatorial Materials Deposition:
Aε reviewed above, X.-D. Xiang et al.49 have recently deεcribed a method for the production of arrayε of combinationε of material, which are then εcreened to diεcover combinations with useful properties. The patterning methods deεcribed herein may be extended to the production of such arrays but further permitting the rapid, selective deposition of materials which are not conveniently evaporated or without the use of evaporation equipment. Here, a suεpenεion of very fine particleε of a material is applied to the surface of a wettable relief, which iε preferably of elaεtiomeric compoεition. The liquid layer of said εuspension on said relief iε εufficiently thick that an adequate quantity of suεpended material per unit area iε obtained on the surface of said relief. This will depend on the viscoεity and surface energy of the liquid used. Such a εuspension coated relief iε then juxtapoεingly contacted to a porouε flat surface, which may be the surface of a filter. Depending on the composition of the material of said flat surface, capillary action may draw the particles in εaid suspenεion onto εaid flat surface, aε in εlip-casting. Material transfer of thiε kind will only occur in areas of contact with said relief, i.e. thoεe raiεed areas of said relief. Where the compoεition of the material of said flat surface does not suitably permit capillary action to be relied upon with the solution used to εuspend a material, said flat εurface muεt be that of a porous filter, so that negative pressure may be used to draw the suspenεion solution through said filter, depositing suspended material onto said filter surface. After repeated cycleε of depoεition of at least two but preferably more different suspensions of different materials, in a pattern determined by the patterned relief uεed, a patterned array of different materialε mixtureε is yielded. This may then, for example, be subjected to annealing εtepε. Thus, large numberε of different combinationε of materialε may be produced in a convenient format for teεting and characterization, including from materials which are not εuitably evaporated.
Template Patterned Synthesis of Sheet-like Complexes and Copolymers: The formation of chemical patterns on surfaces permits the use of εuch chemically patterned εurfaces as templates for the production of patterned molecular complexes and copolymers, which themselves may be used as templates for co plementarily patterned molecular complexes and copolymers. This aspect of the present invention iε analogous with other aspects of the present invention in that spatially patterned surfaceε may be replicated from an initial pattern and thereafter from each other. In the present aspect, patterned compositions of sheet-like molecules serve as molecular templateε for the definition of patterns in thus complementarily patterned εheet- like molecules in a molecular replication process analogous to the polymerase chain reaction invented by K. Mulliε for the amplification of polynucleotide moleculeε.
The foregoing pattern replication methods of the present invention and thoεe found in related art, aε well as scanning probe based patterning methods50'51'52 may be used to perform the patterned chemical derivatization of surfaces. As shown by examples from related art, such modifications may cause the preferential binding of molecular or macromolecular species to predetermined regions of said surfaceε, which are demarcated by said patterned chemical derivatization. Such surface modifications may be used, for example with the methods of G.M. Whitesides et. al. or those of W.T. Muller, P.G. Schultz, et al., to bind specific firεt affinity groupε or species to predetermined regions of said surface. Said affinity groupε or εpecieε are chosen so as to bind affinity derivatized layer forming molecules comprising a second set of affinity groups or species which are in communication with a layer forming precursor moiety (such as a polymethylene chain, or rigid rod oligomer or polymer) . The affinity moiety of εaid second εet of affinity groupε iε preferably located at one terminuε of εaid affinity derivatized layer forming moleculeε, which are preferably εubεtantially linear in εtructure. Said affinity derivatized layer forming moleculeε preferably but not neceεεarily further compriεe a moiety of third affinity group or εpecieε, which iε preferably located at the terminus opposite that at which said affinity moiety of said εecond εet of affinity groupε iε located; εaid affinity derivatized layer forming molecules may also comprise one or more (preferably two or more) distinct reactive, cross-linkable or photopolymerizable chemical functional groups, situated along said layer forming molecules such that each of said distinct reactive, croεε-linkable or photopolymerizable chemical functional groupε occurε at a particular poεition along the length of said layer forming molecules, and may, after a layered structure haε been formed by aεεociation of moieties εelected from said second set of affinity groups with said firεt affinity groupε pattered on εaid εurface, be cauεed to react to form a covalently linked network joining said layer forming molecules. Said first affinity groups and said second affinity groups are preferably chosen such that the interaction between them iε εufficiently εtrong and εpecific under a first set of conditionε but iε substantially weakened or neutralized under a second set of conditions; thus, under said first said of conditions, layer forming molecules assemble at surfaceε comprising regions of said firεt affinity group in patternε correεponding to the pattern of εaid regionε, while εaid εecond εet of conditionε serveε to εeparate layerε compriεing said affinity groupε chosen from among εaid second set of affinity groups from surfaces (including surfaces of layers) comprising said first affinity groups. Said third affinity group or species is chosen such that it does not bind to either of εaid firεt affinity groupε or εaid second set of affinity groups, but will bind to affinity groups selected from a fourth set of affinity groups, which in turn will not bind to said first affinity groups, other affinity groups selected from said fourth set of affinity groups, nor to said second affinity groupε (i.e. εaid firεt affinity groupε and affinity groups of said second εet of affinity groups bind only to each other and not to identical affinity groupε; said affinity groups choεen from εaid third εet of affinity groups and said affinity groups chosen from said fourth set of affinity groups bind only to each other and not to themεelves; e.g. sets of affinity groups may each consist of one of adenine, thymine, cytosine or guanosine nucleobases) . The composition of said surface in unpattemed (or differently derivatized regionε) iε choεen to not bind εaid affinity derivatized layer forming moleculeε. After a pattern of εaid first affinity groups is formed on εaid surface, a solution containing εaid affinity derivatized layer forming molecules is contacted with said surface under appropriate conditions and for sufficient time to permit the binding of εaid affinity derivatized layer forming molecules to all available εiteε on εaid εurface. Unbound species are washed from said surface. Thus, said layer forming molecules comprising affinity groups from said εecond set of affinity groupε are bound to εaid εurface in a pattern replicating the pattern of said firεt affinity groupε on said surface. These may then be crosε-linked via said reactive groupε, for example by the addition of suitable crosε-linking agentε, photopolymerized, or cauεed to react together by eεtabliεhing appropriate phyεical or chemical conditions such that a covalent network is formed. Note that said affinity group chosen from said third set of affinity groupε may be initially caged or protected to εuppress the formation of associationε until a deprotection step is performed. In this inεtance, affinity groupε chosen from said fourth set of affinity group may be reacted with regions of said surface not containing said firεt affinity group, in direct analogy to the complementarily patterned two component monolayers discloεed by G.M. Whiteεideε and co-workers, formed by patterning a first component monolayer and then permitting a second component compound to self-assemble into a monolayer on remaining uncoated regions. Alternatively, patternε composed of domains of multiple distinct affinity moietieε may be formed by multiple relief contacting εtepε, or by multiple spatially selective activation εtepε (exampleε include: light directed, for photodeprotectable or photoactivatable compound derivated surfaceε; εcanning probe controlled surface catalyεiε such aε the methodε of W.T. Muller, P.G. Schultz et al., or of B.J. Mclntyre, M. Salmeron and G.A. Somorjai, or other εcanning probe based patterning methods capable of directing the adsorption or reaction of molecules comprising the desired affinity moieties to the selected regions of patterned surfaces; and, the replication methods of related art or of the present invention, e.g. involving microcontact printing) . Multiple distinct molecular εpecieε comprising multiple distinct affinity moiety types or clasεeε and typeε may be patterned onto the εame εurface by repetitive cycleε of patterned activation or patterned masking followed by deposition of the respective compound (e.g. by contacting a solution of said respective compound to said patterned activated or patterned maεked εurface) εuch that deposition occurs at all available siteε. Thuε, a firεt pattern may be formed, and the regionε thuε εpecified cauεed to react with a first chemical compound by contacting εaid surface with εaid firεt chemical compound or εolutionε thereof, a εecond pattern may be formed and the regions thus specified cauεed to react with a second chemical compound by contacting said surface with said second chemical compound or solutionε thereof, and so on, until patterns of all deεired patternε of all deεired chemical compoundε (comprising the corresponding diεtinct affinity moietieε) are formed.
After the polymerization of εuch a patterned layer into a εheet- like polymer, it iε released from the initial patterned surface, e.g. by exposure to elevated temperatures. The affinity groups on each side of said sheet-like polymer may then be used, by similar steps as those used to produce said sheet-like polymer, as a template for the formation of complementarily patterned εheet-like polymerε by expoεure to appropriate affinity group terminally functionalized layer forming monomerε. Repetition of εuch layer forming εtepε thus provides geometric amplification (here for fabrication purposeε) of the patterned εheet-like polymer, in analogy to PCR, provided that the sheet-like polymer produced at each step is freed, as above, from the template surface which directed its formation.
Patternε thuε generated may be designed to facilitate orderly binding together of such layers under appropriate conditions (e.g. thermal annealing) into bi-layer or multilayer εtructureε, in analogy to the hybridization of DNA moleculeε. Such design will favorably include pattern elements that ensure proper sheet-like alignment with proper registry before bulk or random associations between surface regions of said layers are permitted to occur. For example, considering a square sheet-like εtructure, at εufficiently low concentration to preclude εignificant trimolecular or tri-complex reactions, one corner of each of two sheet-like structures to be juxtaposed may comprise thiol groups, which thus target theεe corners to each other before affinity interactions are permitted to occur. A second set of cornerε, diagonally located from εaid firεt εet, may compriεe primary hydroxylε, which are then croεεlinked by an appropriate crosslinking agent, such as a bifunctional acid anhydride compound. Thus, a firεt set of cornerε are targeted together, followed by the diagonally oppoεite εet, after which regiεtry of appropriately designed affinity patterns causes registry of the remainder of the juxtapoεed εurfaceε.
Where εetε of affinity group terminally functionalized layer forming monomerε in which different affinity groups correspond to different intermediate (intervening) structural member compoεition, and hence phyεical propertieε, layered and multilayered
Rapid Formation of Precise Thickness Multilayers: Where multilayerε compriεing many molecular layerε with monolayer precision are required, methods more rapid than εequential monolayer depoεition are deεirable. Thiε may be accompliεhed by forming a Langmuir-Blodget film on a solid surface which may later be disεolved by a εolution which will not disrupt or destabilize the overlying layer. This surface is again used to lift an interfacial layer, such that there are now two layers situated on said surface. Dissolving such a substrate permits the overlying layer to be situated at a phase boundary, and iε favorably accomplished by slowly lowering said εubεtrate into εaid εolution which diεεolveε it, such that once it is disεolved, said overlying layer is situated at εaid phaεe boundary. After the preceding εtepε, there would be two stacked monolayerε at thiε point. Conditionε (e.g. temperature, compoεition, etc.,) are adjusted such that a dissoluble solid may again be pasεed through said εolution without diεsolution, to capture said overlying layer previously situated at said phase boundary. Said surface with said overlying layers is again used repeatedly to capture layers of a precise thickness, which thicknesε is determined by the preceding liberation step. The number of capture steps and the layer thickness thus determine the precise number of monolayers added before a subεtrate iε diεεolved, liberating the resulting multilayer at said phaεe boundary. Thuε thickneεε may increaεe multiplicatively rather than in increments of a single monolayer or a single bilayer. Thus, εuch a method comprises the steps of: forming an interfacial layer, e.g. from amphiphilic molecules at an air water interface; depositing such a layer on the flat εurface, preferably of a diεεoluble solid; repeating thiε depoεition step at least one more time; liberating the multilayer thus produced from εaid εurface, preferably εuch that it iε situated at a phaεe boundary and preferably by diεεolving εaid solid in a εolution which will not disturb the structure of said multilayer; and, repeating this procesε one or more timeε with said multilayer, such that such repetition yields a multiplication in the thicknesε of the preceding multilayer. The principal limitation to thiε method iε that the εum of the area of the monolayerε of the reεultant multilayer will be conεerved with reεpect to the area of the initially formed monolayerε or bilayerε. Means other than ordinary disεolution may alεo be used to liberate multilayerε. For example, a εubεtrate used with this embodiment may be of a polymeric composition comprising photolabile linkages. Said polymeric composition comprising photolabile linkages is preferably chosen such that the photodegradation products will be soluble in the εolution uεed to form interfacial filmε or to support thus liberated films, and to not bind with or otherwise interfere with these films. Subject to these limitations, a wide range of compoεitionε are suitable for substrates. Where subεtrateε are of polymeric compoεition, theεe are moεt preferably caεt with a highly flat surface, simple examples of which include glass and freshly cleaved mica. After each addition of layerε aε deεcribed above, substrateε are floated or otherwise held at the surface of the solution to support the overlying film supported by said substrate after dissolution of said substrate, and the substrate is exposed to a sufficient intensity of light of a frequency chosen to cauεe the degradation of said photolabile linkages for a sufficient length of time to effect complete diεεolution of εaid εubεtrate, with further time being allowed aε neceεsary for degradation products to diffuse away in thiε solution from the interfacial layer.
Note that such a proceεε may alternatively avail a release layer, which may be produced aε a εelf-asεembling monolayer, for example compriεing photolabile or thermolabile linkageε situated on the surface of an insoluble εubεtrate. Here, εaid εubεtrate and releaεe layer are lowered into a solution which will εupport the overlying layer or film as a phyεical treatment diεrupting εaid releaεe layer iε effected εuch that εaid overlying layer or film iε lifted by εaid εolution from said substrate, the bonds comprised in the structure of εaid releaεe layer having been diεrupted. The composition of said releaεe layer is preferably chosen εo that the diεrupted layer subεequently produced is well wetted by the solutionε uεed, and such that the free degradation productε (thoεe not bound to the subεtrate εurface) are well solvated by the solutionε uεed.
Additional Embodiments: Molecular and Supramolecular Synthesis with Positional Control:
This embodiment describes first deviceε for the parallel poεitional εyntheεiε of molecular and εupramolecular objectε or articleε (termed workpieceε or workpiece precursors) which may also be devices, methodε for the fabrication of εaid firεt deviceε according to the general microfabrication methodε of the preεent invention, and methodε for the use of said firεt devices to effect positionally controlled molecular and supramolecular syntheεiε. Terminology uεed will include terminology from the field of art of εcanning probe microεcopy.
Surfaceε arrayε of Z-actuators, each under independent control are produced, for example, by the foregoing methodε of the present invention according to the following design. A capacitance based actuator is produced by forming electrodes on a surface which serves as a first plate of a parallel plate capacitor. Two categories of capacitive actuators are possible: unfilled (i.e. air or vacuum gap) capacitors where the coulombic force between capacitor plates ariεing from the charge separation due to applied voltage difference is opposed by forceε associated with mechanical flexure of a second capacitor plate partially or completely εuspended over said first plate εuch that a cantilever arrangement results; and filled capacitors wherein εaid firεt plate iε coated with an elaεtiomeric or other compreεsible substance, according to the molding or casting methods of the present invention, upon which a second plate of a parallel plate capacitor is formed, where compreεεion of εaid elaεtiomeric or other compressible substance opposeε the coulombic forceε resulting from charge separation acrosε the gap of εaid parallel plate capacitor. Note that for filled capacitorε, compoεition of the εubstance between capacitor plates may further be chosen according to dielectric properties in addition to mechanical propertieε. The functional characteriεticε of both categorieε of actuatorε will be highly dependent on geometrical factorε and on materialε properties, providing a large range of realizable device characteriεtics according to tradeoffs elected in design and fabrication. Each such capacitive actuator may be placed under control of a different voltage signal, under electronic control, favorably with the electronic circuitry effecting thiε control integrated into the same array device, according to conventional electronic device integration deεign practiceε and, for example, the microfabrication methodε of the preεent invention, such that each capacitive actuator may be tranεlated to a position independently of other capacitive actuators in the same array. For purposeε of the preεent deεcription, the direction normal or perpendicular to the εurface upon which said array of actuatorε iε formed shall be denoted as the Z-axiε, which is also the axis εubεtantially perpendicular to the plane of the plateε of εaid parallel plate capacitor actuatorε. An integrated array of Z-actuatorε iε thus produced on a substrate surface.
The portions of said Z-actuators furtheεt from the subatrate (i.e. the topmoεt capacitor plate) may further compriεe or be in communication with a small cantilever, the purpose of which iε to facilitate the meaεurement of forceε acting upon any tips associated with said cantilever. Said forces may conveniently be measured by reflecting a laser beam from the bottom surface (i.e. the surface nearer to said substrate, which in this instance is chosen to be transparent) and monitoring and changes in the reflected angle of said laεer beam (e.g. with a split photodiode arrangement as is commonly used as detection means in AFMε), or by other meanε which have been employed in the design of AFM cantilever deflection monitoring means (e.g. capacitive detection, tunneling detection, piezoreεiεtivity change detection, etc.,.)
In the caεe of tunneling detection, a cantilever iε coated with a conductive layer on the bottom εurface of εaid cantilever, which iε juxtapoεed with a tunneling contact. The extent (proximity) of said tunneling contact to said conductive layer on said cantilever may be self-aligned by electroplating at said tunneling contact until a desired current is observed between εaid conductive layer and εaid tunneling contact, while εaid cantilever iε at itε equilibrium (undeflected) poεition, optionally followed by a brief reverεed potential to diεεolve a very εmall portion of the depoεited metal from the εurface of said tunneling contact to establiεh a predetermined gap εize. Detection methods relying on εuch εtructureε may additionally rely on detection of variation in field emitted current between εaid conductive layer and the conductive εtructure termed a tunneling contact above in the caεe of tunneling current baεed deflection detection. The signals corresponding to the deflections thus detected may, as in conventional AFM syεtemε, be uεed to adjust the position of said Z- actuators to restore a desired set point value. As will be apparent to thoεe skilled in the arts of SPM and SPM design, other regimes of AFM and SPM will be posεible with the appropriate modificationε or extenεions of the present array format.
The externally exposed surface of the topmost plate of said parallel plate capacitor, or layers formed upon εaid externally expoεed εurface may be further patterned εuch that a confined εurface region, of predetermined εize and εurface elemental or chemical compoεition iε produced in communication with the moving portion of εaid capacitor actuator. In the εimpleεt case, a maεking pattern may be formed on the exposed surface of a gold said topmost plate εuch that only a εmall region, for example εmaller than lOOnm by lOOnm εquare of bare gold remainε exposed. This surface thuε compriεeε a localized region or pad in and X-Y array of εaid actuatorε, which may be tranεlated in the Z direction independently of other εimilar localized regions or pads of other actuator array elementε.
Patterned Molecular Tip Arrays:
In one variant of thiε embodiment, εaid square of bare gold may be uεed aε a target for the random adεorption of organothiol moleculeε or molecular complexes. Such adsorption iε preferably carried out εuch that depoεition of εaid organothiol moleculeε or molecular complexes occurs at a rate of one per pad or region, i.e. preferably exactly one per actuator, but at least on average one per actuator in the case of random deposition. In other variations, small objects may be εubstituted for said organothiol molecules or molecular complexeε, in which case the εaid externally expoεed εurface or coatingε thereupon are choεen so as to εtably bind the correεponding εaid εmall objects selected. Said small objectε may be εelected from among: macromolecules, enzymes and conjugates thereof, biological receptors, immunoglobulins or fragmentε thereof or conjugateε thereof, colloids, nanocrystalliteε, polymeric beadε, dendrimers, fullerene derivatives, nanotube derivatives, mesoscopically structured single objects or other small objects of predetermined geometries or geometrical familieε. Theεe moleculeε, molecular complexeε or other small objects serve as an extremities or projections in communication with actuator padε; theεe will be generically referred to herein aε tipε. Note that other types of tips include any of the above small objects in communication via a flexible molecular linker to a chemical functional group which binds stably to said pads, such that εaid small object iε poεitionally conεtrained according to the flexibility and tethering of εaid linker, but not precisely poεitioned. Further, another type of tip may be formed by electron beam depoεition of vaporized εubεtanceε onto εaid padε.
Note that actuator arrayε may homogeneously consiεt of a εingle pad type decorated by a εingle type of tip, multiple pad typeε (i.e. multiple pads of each type where each of said types has a different εurface chemical composition) decorated on a one-to-one composition basis by corresponding multiple small object types, or may comprise different pads decorated, according to foregoing spatially predetermined copolymer εynthesis methodε, with diεtinct oligonucleotides, polypeptides or other copolymers with diεtinct binding propertie .
Arrayε of workpiece padε are produced by forming small exposed regions of a εurface of a convenient composition on a substrate surface, such that said workpiece pads may line up with actuator pads and/or tips of the above actuator arrays. Workpiece padε may alεo comprise or overlie actuators, which may for example serve to tilt εaid workpiece padε relative to εaid tipε, or may merely compriεe pad arrayε which facilitate the colocalization of tip and workpiece, depending on the deεign of the article of fabrication and the tip or tipε uεed.
In particular, molecular tips within tip arrayε (tip flatε) εuch aε thoεe deεcribed by Drexler53 may be εelected according to the tilt of εaid workpiece pad effected by one or more actuatorε which adjuεt the angle of the plane of εaid workpiece pad relative to the normal defined by the Z-actuator of the tip pad. Note that the tip arrays described by Drexler pertain to conventional atomic force microscopes and cantilevers uεed therewith, εaid cantileverε modified to further comprise εaid tip flatε. To perform the correεponding εynthetic operationε in parallel, rather than on a εingle workpiece, the preεent invention provideε that an array of actuatorε poεitionε molecular tipε or arrayε thereof along the Z-axiε, while one or more actuators associated with each workpiece pad orients said workpiece pad to orient said workpiece pad such that one molecular tip of the opposed array is selected according to the orientation dependent proximity. Such molecular tip arrayε may, following Drexler, be εituated on a curved εurface, here each in communication with one of εaid Z- actuators, where the approach of a particular molecular tip in one of said arrays is selected according to the tilt of the workpiece relative to said one of said arrays which selects a distinct normal to said curved surface, thus selecting said molecular tip situated on said curved surface at the locus of the line normal to both said curved surface and said workpiece pad (i.e. the point of contact as said curved surface would approach said workpiece pad, asεuming convex εhape.) Thuε, proviεion of workpiece pads comprising one or more actuatorε effecting tilted orientation of said workpiece pads relative to the Z-axis translation of said Z-actuators of said tip pad arrays permits the use of arrayε of tip arrays in the parallel modification of arrays of workpieces. If such tilt iε not effected by arrayε of actuators, only one workpiece may be produced at a time benefiting from the selection of particular individual tips (one at a time) from among the multiple tips in a tip array, becauεe the appropriate workpiece εubεtrate alignment for one workpiece will preclude interaction of other workpieceε on the workpiece pad array from being juxtapoεed with the padε of εaid tip pad array. The particular advantage of being able to εelect individual tipε from an array of tipε in the modification of a workpiece accrues from the ability thereby to selectively interact εaid workpiece with a different, predetermined chemical functionality or compoεition of εaid individual tipε, while maintaining a determiniεtic relationεhip between the spatial location of said tip array with said workpiece (i.e. eliminating the need to locate a workpiece upon changeover of tipε.) An array of tilt actuatorε permitε εuch advantage to be availed in coinbination with the advantageε of paralleliεm accruing from the simultaneous positionally controlled modification of workpieces situated upon said workpiece pad array comprising said array of tilt actuatorε.
Local Vertical Positioning and Global Lateral Scanning of Probe Arrays: Where conventional scanning probe microscope designs, including microfabricated implementations thereof,54'55 comprise tips each having X, Y and Z actuators, greater ease of fabrication and greater X-Y positioning accuracy may be achieved in array format by producing an array of tips each situated on a diεtinct, independently controlled and monitored Z-actuator, where εaid array of tipε overlying εuch an array of Z-actuatorε iε translated by a single X-Y positioning means. While such an arrangement precludes independent X-Y motions of the individual tips of said array of tips, the elimination of separate X-Y actuators for each tip of εaid array of tipε permitε a higher areal denεity of tips (or tip pads in the terminology adopted above, as distinct from the molecular tips of a molecular tip array, which would be εituated on a single tip pad of said tip pad array) than would be posεible where redundancy of X-Y actuatorε occurε. Retention of independent Z-actuatorε in such a design permits maximally precise alignment of different tips with the corresponding opposed workpiece pads without variationε in interaction forceε between tipε and workpieceε which would reεult when cantilever flexion compenεates for Z positional variation of εaid workpieceε relative to εaid tipε. Where εaid X-Y positioning means is a piezoelectric tube εcanner, additional care muεt be taken in calibration and scanning voltage waveform generation to enεure that deviationε from planar orientation of the tube opening do not cause a rocking motion of the array mounted thereupon (i.e. compensation in the Z-axis at various points around the height of the tube εcanner aε a function of X and Y poεition) . Leεs conventional piezoelectric bimorph actuators or capacitor actuatorε are thuε favored in this regard, though other measures may ensure proper operation with piezoelectric tube scannerε.
Self Alignment of Molecular Tip with Molecular
Workpiece:
Where molecular tipε or other molecular positioning members are randomly situated on said tip pads of said tip pad arrays, alignment with the molecular precurεorε of workpieceε or workpieceε situated on the workpiece pads of said workpiece pad array must be accompliεhed. This is moεt favorably accompliεhed by initially poεitioning εaid molecular precursors of workpieces on εaid workpiece pad array with εaid molecular tipε or other molecular poεitioning memberε on said tip pad arrayε. Placement of εaid molecular precurεorε of workpieces or said workpieceε on εaid workpiece padε of εaid workpiece pad array by said molecular tipε or other molecular poεitioning memberε enεureε correct relative poεitioning of said workpiece precursors or workpieces relative to said molecular tips or other molecular positioning members and the reactants later carried thereupon. Thus, random variations in the precise positions of said molecular tips or other molecular positioning memberε on the padε of εaid tip pad arrayε poεe no difficulty in alignment becauεe εuch placement cauεeε correεponding variationε in the precise positions of said workpiece precursors or workpieces according to those of said molecular tips or other molecular poεitioning members, i.e. positions of molecules or complexes thereof on arrayε are replicated, and εuch replication meaεureε ensure good poεitional match between molecular tipε or molecular positioning members and workpieces.
As an example, a molecular workpiece precursor, comprising a thiol group, may be deposited onto a workpiece pad comprising an exposed gold surface by a molecular tip, such that the resulting position of asεociation of εaid thiol with said gold surface corresponds to the position on said tip pad or cantilever of said molecular tip. In a further extension of this example, said workpiece precursor may additionally comprise a cleavable linkage susceptible to a particular predetermined physical or chemical treatment, and a first reactive group, for example a hydroxyl. A εecond molecular workpiece precursor molecule, for example of identical composition, may be depoεited by the εame proceεε at a second location on said workpiece pad. Thus, two predetermined reactive groups are placed in a predetermined arrangement on εaid workpiece pad. Aε deεired, a third molecular precurεor molecule may εimilarly be positioned at a third point, in which caεe the three positioned reactive groups thuε fix a coordinate system admitting no grosε rotations (i.e. further exclude the rocking motions posεible in εtructureε anchored with only two reactive groupε fixed on a εurface.) Following the placement of εuch workpiece precurεorε, molecular componentε may be reacted with the reactive groups of said workpiece precursorε according to the methodε deεcribed below.
Note that further εelf-alignment may be achieved at an earlier εtep by replicating the flat surface of the subεtrate to be used to produce εaid workpiece pad array from that to be uεed to produced εaid tip pad array. Such a εtep will enεure that local variationε from planarity (e.g. curvature) in the surface of said tip pad array are compensated by the corresponding variationε thuε produced in the replicated workpiece pad array εubstrate surface. The necesεity of εuch εteps in practice will depend on the εize of such surfaceε and the ability to obtain or fabricate ideally flat subεtrate εurfaceε as materials for the production of tip pad arrays and workpiece pad arrays.
Constraint-Based Simplification of Positional Synthesis: A more immediately practical approach to the positional synthesiε propoεed by Drexler fabricateε structureε from structureε larger than single atoms, and avails conventional solution and solid-phase chemistry where positional control is not essential. These methods permit the synthetic fabrication of extended, complex heterogeneous structureε which would be difficult or impoεsible to produce by known art chemical methods.
Most conveniently, the size of molecular components or reactantε and the linkerε which hold theεe in communication with said molecular tips or molecular positioning means are chosen to be of a sufficiently great size that angstrom positioning accuracy is not εtrictly required to exert poεitional control over εyntheεiε. Inεtead, εaid linkers, which may for example be composed of a polymethylene (polyethylene) polymer chain or other preferably straight chain flexible polymer chain, which are attached via εome cleavable linkage to said molecular components or reactantε, conεtrainε εaid molecular componentε or reactionε to a volume defined by the linear length of εaid linkerε (and the εize of εaid molecular componentε or reactantε) with a probability diεtribution (or effective concentration aε a function of position within said volume) which may be predicted uεing Flory theory. Said volume to which εaid molecular components or reactant may be termed a conεtraint volume. Poεitional control over εynthetic reactionε involving said molecular componentε or reactantε and said workpieces or workpiece precursors inheres in the X-Y tranεlation of the substrate comprising said tip pad array and the Z-translation of εaid tip padε, εuch that the resultant three dimensional positional control tranεlates εaid conεtraint volume which containε said molecular componentε or reactantε (more εpecifically, the reactive chemical functional groupε thereupon) into proximity with the deεired reactive εiteε on εaid workpiece. In other wordε, control over translation of said constraint volumes iε uεed to εelect which reactive sites of said workpieces are reacted. Thuε, the reεolution of εuch a method is limited by the extent of said constraint volume, and workpieceε are preferably deεigned according to εaid reεolution (otherwiεe reεultant workpieces will obey the corresponding probability distribution related to the number of posεible εiteε within an approached conεtraint volume and the effective concentration diεtribution at each of said posεible sites.)
Note that such a linker may alternatively have a large central portion of their length composed of a rigid polymeric member (with terminal flexible polymeric linkages), in which case the attached molecular components or reactants are confined rather to a shell, which may be termed a constraint shell. The configuration of said constraint shell is determined by the structure of the partially rigid said linker, i.e. defining a volume beyond which the terminally situated said molecular components or reactantε may not extend and also a closest point of approach to the point on said tip pad at which said partially rigid said linker is attached beyond which said molecular components or reactants may not approach. In thiε case, the effective concentration of said molecular components or reactants within such constraint shells are correspondingly higher aε compared with the above described constraint volume corresponding to a completely flexible structure. Note, however, that greater poεitioning accuracy may be required in these inεtanceε, but εuch accuracy εhould be well within that practically achievable with current inεtrumentation. After the poεitionally conεtrained reactionε occur according to the above deεcribed εtepε, communication of said linkers and εaid workpieces, which resultε from any bondε formed by εaid reactions, may be teεted. Thiε may be accompliεhed by monitoring any forces exerted by said communication as said tip pad array iε withdrawn from εaid workpiece pad array or, alternatively, aε εaid Z-actuatorε of εaid tip padε are tranεlated away from εaid workpieceε. Said forceε may be monitored conveniently if each of εaid molecular tip (or said linker) is in communication with a cantilever, the position of which iε tracked by any known art method εuch as optical beam deflection, monitoring of a tunneling current between said cantilever (coated by a metal) and a tunneling tip, changeε in piezoreεiεtivity, changes is capacitance where an electrode situated on said cantilever forms one plate of a capacitor, etc.,.
Two poεεible cases occur. First, communication of said tip with said workpiece may be detectable aε cantilever deflection upon retraction of εaid tip from εaid workpiece, while εaid retraction doeε not sever said linkage. Alternatively, said retraction may sever said linkage. These two caseε will have different requirementε and advantageε.
In the firεt case, a linker is preferably of elastiomeric compoεition such that tension iε gradually applied to εaid workpiece aε deflection of εaid cantilever occurε during εaid withdrawal. In thiε way, gross severing of linkageε, which would be expected with applied tenεionε of less than 10 nN for single bonded linkages, are avoided. Otherwise care must be taken to ensure that applied tensions remain under a tolerable maximum to prevent such severing. In such caseε, once a reaction between said molecular component and said workpiece has occurred, the tip remainε in communication with εaid workpiece, until chemical or phyεical treatment severs the resulting linkage. Linkages (i.e. linkage composition) is chosen according to the deεired methodε of linker cleavage. For example, linkerε may compriεe bondε εuεceptible to cleavage by particular chemical reagents, may be heat labile or may be photocleavable, e.g. comprising esters to compounds such as nitroveratryloxycarbonyl compounds, such that said ester linkages are cleaved by exposure to appropriate wavelength actinic radiation.
In the second caεe, such aε the case for molecular components bound to εaid molecular tip or molecular poεitioning meanε via affinity interactionε conεiεting of weak forceε (e.g. hydrogen bondε, van der Waalε forceε, solvation energies) withdrawal will lead to severing with a predetermined applied force, which will be detected aε the correεponding cantilever deflection. In the caεe of oligonucleotide baεed affinity interactionε, the obεervationε of G.U. Lee, L.A. Chriεey and R.J. Colton56 are indicative of the kind of affinity interaction εevering forceε expected. Thiε εecond caεe, with oligonucleotide binding, iε preferable in that well characterized binding interactions may be used to effect rapid, reversible binding of molecular components to molecular tips or other molecular targets (i.e. molecular positioning moieties) comprising the complementary oligonucleotide sequence. In either case, reactant molecular components are situated on εaid molecular tipε or tip pads by contacting said tip pad array with a solution comprising the said reactant molecular components to be added to said workpieces during the subsequent reaction step and permitted to bind to the available εites on said tip pad array, i.e. to molecular tips or to the affinity groups thereupon.
Once a molecular component has reacted with the specified site on a workpiece or workpiece precursor, and has been severed from the molecular tip or molecular positioning means, if any workpieces on εaid array of workpiece pads did not evidence communication with said cantilever (i.e. negative deflection upon tip withdrawal), and yield iε to be maximized at the expense of the repetition of steps, the preceding steps from binding of molecular components to molecular tips or molecular positioning means on said tip pad array through said withdrawal step may be repeated with the same molecular component and positioning. Siteε which had previouεly been reacted will lack active reactive sites, while those which did not successfully react will experience a repeated opportunity to react with the desired species. After an arbitrary number of failures, a workpiece may be deemed to be mispositioned or otherwise refractive to correct asεembly.
Conεtraint based εimplification applieε not only to conεtraint of reactantε (i.e. molecular componentε) to tips, but also applies to the design of molecular components themselves and the design of the workpieces which they are uεed to aεεemble. A molecular component will generally be bound to a workpiece at more than one location, i.e. will generally have greater than one point of connectivity with εaid workpiece. Thiε iε moεt εimply and rapidly accomplished with only one positioning step per molecular component, with the formation of subεequent linkageε constrained by the geometries of said molecular component and the structure, at that εtep, of the workpiece. In other wordε, a positionally controlled reaction links εuch a reactant molecular component to a firεt reactive εite on a workpiece, and the εtructure of said reactant molecular component and said workpiece are such that the reactive groups on εaid reactant molecular component and εaid workpiece will react together for only one relative topological orientation of εaid reactant molecular component and εaid workpiece. For rigid εtructureε (i.e. those comprising rigid polymeric εtructureε) εimple deεign rules may yield such a result, namely, that the pairwise distances of all similar reactive groupε are different and that once a firεt bond iε formed, under poεitional constraint imposed by linkage to said tip, no incorrect second bonds to reactive groupε on εaid workpiece may form after release of said reactant molecular component from said tip. In other words, no rotation of said reactant molecular component about the first bond formed under positional conεtraint by εaid tip will yield proximity of reactive groupε situated on said reactant molecular component to correspondingly reactive groups of said workpiece except that rotation which permits the deεired reaction and hence the desired connectivity. Note that flexible components may alεo be employed according to εuch constraint-baεed deεign methodε, but here flexibility entailε that the positional constraints on reactions of these components obtaining after a first bond or linkage is formed between such flexible componentε and a workpiece will concern the maximum possible reach of other reactive groups on εaid flexible componentε. Thus, the selection of rigid or flexible components for different parts of a workpiece structure will be according to considerations concerning both the deεired structure and the preferred molecular asεemble strategy. Note, however, that with both flexible and rigid components, and also with components compriεing both flexible and rigid regionε, component εetε may be synthesized comprising different topologieε εuch as different degrees of branching, different lengthε of branch arms, different chemical functionalization of regions or location of said branch arms, etc.,.
Aside from the above deεcribed poεitionally conεtrained reactionε, workpieceε may be treated with other reactantε or chemical modifying reagentε from bulk εolution. For example, reactive groupε on molecular components may all be primary hydroxyl groupε. Once εaid molecular componentε are incorporated into workpieceε (and releaεed from said tips), and said tip array withdrawn from said workpiece pad array, said hydroxyls may be modified to be reactive to other hydroxyls, e.g. by reaction with bivalent cross-linking agents (provided in excess) comprising two acid anhydride or two acid chloride functionalities. The first such functional group of each such cross-linking agent molecule will react with each hydroxyl on said workpiece, while the second such functional groupε of each such crosε-linking agent molecule, now a reactive group on said workpiece, will only react with approaching hydroxyls of new molecular components. Of course, thiε entails that no two hydroxyls available on a workpiece may be cloεer together than the length εpanned by the reactive functionalitieε of εuch cross-linking groups. Alternatively, molecular components may carry protected functional groups, which, once εaid molecular componentε are reacted onto said workpieces and releaεed from said tips, are deprotected by contacting εaid workpiece pad array, aε in the caεe of activation with cross-linking agents, in this case with deprotection reagents. In either case, function groups which were held inert with respect to each other to prevent unwanted reactions between molecular componentε aε theεe are prepared and bound to said tipε, are activated once on εaid workpiece. In the caεe of acid chloride or acid anhydride chemiεtrieε, inert εolvent media muεt be chosen. Further, control over the stepε at which functional groupε on εaid workpieceε are activated or deprotected provides another method of control over synthesis, i.e. a method by which a subεet of functional groupε are held inert until a deεired aεεembly step, whereupon they are deprotected or activated by treatment with appropriate reagents. It will be obviouε to those εkilled in organic εyntheεiε that other reactive chemiεtrieε, as will aε multiple, concurrently uεed chemiεtrieε will be poεεible and often preferably advantageouε over the εimple example preεented here merely for purpoεeε of illustration.
Note that molecular tips and any affinity groupε thereupon may thuε be εpared from expoεure to highly reactive groupε (e.g. oligonucleotide moietieε of molecular tipε need never be expoεed to acid chloride containing compounds, which would inactivate said oligonucleotides for proper binding) .
As an example, reactive groups may be primary hydroxyls on alkyl chain linkers, rigid structural members may compriεe polyparaphenylene derivativeε57 or the phenylacetylene polymerε diεcloεed by J. Zhang, J.S. Moore et al.58, in either caεe comprising componentε with εtructures synthetically accesεible, for example, by the methods of J. Zhang, J.S. Moore et al.,.59 Affinity members for reversibly binding these components to molecular tips may comprise oligonucleotideε, oligopeptideε, peptide nucleic acid oligomerε or εmall molecule haptenε joined to εaid componentε (e.g. via methylene linkerε), according to the complementary affinity members on εaid molecular tipε. Other molecular component structures of interest in the construction of molecular devices by such asεembly methodε include rotaxaneε comprising macrocycles which themselves comprise reactive groupε, and catenaneε. Molecular componentε baεed on theεe topological compounds provide structureε uεeful aε moving partε in objectε and deviceε fabricated by theεe synthesis methodε.
Affinity groups such as oligonucleotides may be included within the structureε of εaid molecular componentε or may be poεitioned by tipε into proximity with proximity εelected reactive groupε on said workpieces which will react with linkers (e.g. at terminal hydroxyls) attached to said affinity groupε. Note that if workpieceε are to εubεequently be exposed to reactive compounds,, εaid affinity groupε may be provided with any potentially reactive functional groupε protected by protecting groupε, which may be removed in a final εtep after εuch exposure. Note that such affinity groups to be included in the final εtructure of εaid workpiece muεt thuε be protected to enεure that these are not derivatized in undesired ways by synthesis reagentε, but affinity groups used to bind reactant molecular components to said molecular tips or molecular positioning means may be sacrificed. Said affinity groups used to bind reactant molecular componentε to said molecular tips or molecular positioning means may be in communication with the remainder of the structure of the reactant molecular component which they serve to target to said molecular tipε or molecular poεitioning meanε via linkerε cleavable by predetermined phyεical and/or chemical treatmentε (e.g. hydrolyεis of an ester linkage) such that these moieties may be removed from said workpiece after they are no longer needed. Of course, such removal must be accomplished according to chemistries which do not adversely affect the remainder of the workpiece εtructure, and are thuε to be choεen according to such design considerations.
Note also that similar positional constraint methods may be used to effect positionally controlled deprotection of reactive groups, such that the poεition of a tip iε used to εeleσt which reactive group or groupε on a workpiece, out of εeveral groupε of εimilar compoεition at different locationε on εaid workpiece, will be active during a subsequent reaction step where the reactant molecular components react with εuch deprotected or activated chemical functional groupε on εaid workpieceε directly from solution. Thiε variation adhereε to the εame baεic principle of εelective poεitional modification of chemical functional groupε, here preparing their activity for subεequent εynthetic steps. For example, chemical functional groups may be primary amineε, which may be protected by esterification. Here, a molecular tip may comprise or may be linked (e.g. by known art conjugate chemistry) to a protease enzyme molecule, such as Proteinase K. Only thoεe primary amineε within the reach of such enzyme molecules tethered to a molecular tip or tip pad are suεceptible to enzymatic deprotection, and hence only the εubεet of such amines on said workpieces will be deprotected. Other catalysts such aε ribozymes, metal tip surfaceε, or metallic colloidε partially embedded in polymer matrices may similarly be used, as may tethered moleculeε which effect deprotection by non-catalytic reactionε.
Nanoscale Data Pattern Replication and Readout Methods and Means:
The large data capacitieε realizable with the variouε data storage methods comprising the use of scanning probe technology based data readout are not accompanied by a commensurate increase in the rate of data recording except where large numbers of scanned probes are used to record a data pattern onto a storage medium surface. Because one particular advantage of such storage technologies is in applicationε related to data publishing, a rapid method for the duplication of predefined bit patters readable with such readout technologies would be deεirable, aε haε been the caεe with read-only optical data εtorage technologieε.
The methodε of the preεent invention are well εuited to the fabrication of εurfaceε with inεcribed nanoεcale data patternε readable with variouε scanning probe readout meanε. Theεe include near-field photon transfer (corresponding to NFSOM), electron tunneling and electron field emisεion (correεponding to STM and field emisεion mode STM) , and surface profile detection (corresponding to AFM and variationε thereon) .
In the caεe of photon tranεfer, a transparent surface such as glasε or polycarbonate iε coated with a metal film, which iε patterned by the foregoing methodε of the preεent invention for the εpatially predetermined protection or maεking of metallic film εurfaceε from etchantε such that the desired bit pattern is formed compriεing apertures in said metallic film and intact regions in εaid metallic film which reduce or eliminate the local tranεmiεεion of light. Such a bit pattern may be read out by conventional NFSOM apparatuε or variations thereupon, or the near-field detection means described below. The patterned said metal layer may mask the local transmiεεion of photonε to a probe aperture which conductε received photonε to a detector, εaid photonε originating from a source on the oppoεite εide of the tranεparent material comprising said transparent surface, in which case said metal layer serves as a photon tranεmiεεion maεk. Alternatively, εaid pattern in said metal layer may maεk the tranεmiεsion of photons emitted due to the fluoreεcence of compoundε in said transparent material excited by photons either emanating from an NFSOM tip aperture which may be the same aperture used for the detection of fluorescentiy emitted photons or emanating from a source on the other side of εaid tranεparent εurface.
Similarly, in the case of electron tunneling transfer or electron field emisεion transfer, a metallization pattern may similarly be formed, here on an insulator, or alternatively a relief pattern may be formed in one or more metal layerε with all expoεed εurfaceε having conductive propertieε. In either caεe, a bit pattern which may be replicated by the methods of the preεent invention iε encoded in εuch a εurface relief pattern. Such a bit pattern may be read out with instruments such as STMs, microfabricated STMε or the array deviceε diεcloεed herein comprising plural Z-actuators each aεεociated with a tip, which entire array iε εcanned acroεε a juxtaposed surface by an X-Y poεitioning meanε and wherein data patternε are detected by monitoring, with electronic meanε, voltage, current or height of each tip either εingly or in the various poεεible combinationε and where εaid current may be either a tunneling current or a field emiεεion current according to particular implementation . In the caεe of AFM or other εurface height responsive data readout means, relief surfaces comprising surface patterns are replicated by casting, injection molding or embosεing methodε.
In all of these cases, an original relief comprising the desired bit patterns may be formed by scanning probe technology based patterning methods such as those of related art reviewed above.
By such methods, large numberε of replicas of an initial surface pattern encoding information may be rapidly and inexpensively produced, permitting economical data publication in high density formats readable by scanning probe based data readout technologies.
Simplified Method and Means for Data Readout bv Near- Field Optical Scanning: A CCD array, fabricated by methods of the present invention or by conventional methods may be modified to serve as a highly parallel detection means for optical data readout relying on near-field photon transmission through a small aperture. An array of waveguide elements, for example compoεed of PMMA, iε molded onto the εurface of said CCD array such that each photodiode of said CCD array will capture photons conducted by each of εaid waveguide elementε. The array comprising said waveguide elements must therefore be aligned with the photodiode array of εaid CCD. Gapε between εaid waveguideε are filled in with, for example, an opaque polymeric material, or may be electroplated provided thiε will not cauεe short circuits in said CCD, which may be designed to prevent such problems. Said waveguide element array and any material filling said gaps are formed such that a substantially flat surface resultε. A metal film, preferably of gold, of εufficient thickneεε to prevent appreciable transmission of photons iε then deposited onto εaid εurface. Said film iε then maεked εuch that only one small pinhole will be etched overlying each photodiode and associated waveguide; said pinhole will serve as an aperture limiting photon tunneling. This may be accomplished by patterning with organothiol compounds using an elastiomer, according to the method of Whiteεideε and co-workerε, followed by an acid etch step, by the related multilayer vetoing methods deεcribed herein, or by the other patterning methods disclosed herein. Thus, an array of pinholeε of predetermined size and location is fabricated with asεociated waveguideε, which conduct any photonε received at the aperture formed by εaid pinholeε to the photodiodeε of εaid CCD in a one-to-one manner.
Such an array of pinholes with associated waveguides may alεo be fabricated by LIGA methodε and variationε thereupon, and then εituated in an appropriately aligned manner onto a CCD array εurface.
Alternatively, the εame type of structure comprising an array of pinholeε with associated waveguides may be fabricated by other molding and electroforming methodε deεcribed herein and then, εimilarly, εituated in an aligned manner onto a CCD array. Such an array may be termed an aperture limited CCD array (ALCCD) .
The CCD array deviceε uεed in thiε aεpect of the present invention are preferably of high sensitivity, εuch that a reduced number of photonε must be received by each photodiode to register a signal. Such an ALCCD may be used for data readout by juxtaposing the εurface of said ALCCD comprising εaid pinhole array to a patterned metal film surface of a transparent material such glasε, such aε that described above which pattern may be replicated as described above. Said pattern metal film surface is held parallel to the εurface of εaid ALCCD compriεing εaid pinhole array, at a diεtance of leεε than one half the wavelength of the light employed and preferably at a distance of leεε than one-quarter of thiε wavelength. Smaller εeparationε are further preferred. The region of εaid patterned metal film oppoεite each pinhole on the εurface of said ALCCD is either be coated with εaid metal film or may expose the underlying said transparent material, which distinction provides for the detection of the corresponding data pattern. Thuε, the denεity achievable with thiε data readout apparatuε and method is primarily determined by the dimensionε of εaid pinhole, which limitε the photons thus received to thoεe originating from narrow expoεed regions. Said patterned metal film surface is εcanned, in a manner maintaining parallel alignment with the pinhole array surface of εaid ALCCD, εuch that an area correεponding to the εize of each CCD array element paεεeε opposite each pinhole, by repeatable positioning meanε εuch aε piezoelectric or capacitor actuatorε. In other wordε, said patterned metal film εurface iε εcanned in the X and Y while maintaining a εubεtantially conεtant Z εeparation, such that the εignal received by each photodiode of each CCD element correlateε, according to εcan timing, with a particular poεition on εaid patterned metal film surface. Scanning is conducted slowly, such that said CCD captures the bit image corresponding to a particular position of said patterned metal film surface, which is then read out from said CCD according to conventional methods associated with the use of such electronic imaging or memory devices. Thus data rates will primarily depend on the device characteristics of the CCD used. The X-Y location of said patterned metal film surface relative to said ALCCD are then translated by incremental εcanning motion of said actuator, and the bit pattern asεociated with this subεequent poεition iε then εimilarly collected.
In the εimplest instance of this method, photons originate from a source on the opposite side of said patterned metal film than said ALCCD, with transmisεion occurring through exposed regions of said transparent surface as it is masked by said patterned metal film. The number of photons received by each CCD element are limited by the relative size of said pinhole to the area of each CCD element. Thus it iε deεirable that either high intenεity εourceε are uεed to compensate for this limitation, or that incident light be focused so as to reach the pinholes εituated on the εurface of said ALCCD at a sufficient areal intenεity. This may be accomplished by the use of a microlens array, for example such as that described by H.A. Biebuyck and G.M. Whitesides,60 with appropriate focal length of the lens elements which it comprises, such that incident light is concentrated as it pasεeε through said patterned metal film surface and reaches said pinholes on said ALCCD surface. Said microlens array is thus conεtructed at a similar density aε the CCD which iε uεed, and iε aligned with the εurface of εaid ALCCD εuch that εaid pinholeε are each at the focal point of the correεponding lenε. In thiε caεe, εaid patterned metal film surface is situated between said microlenε array and εaid ALCCD, and remainε in a fixed poεition relative to εaid ALCCD aε εaid patterned metal film εurface iε εcanned between εaid microlenε array and εaid ALCCD surface.
Method and Means for the Ordered Dissection of Chromosomes, Chromatin and Polynucleotides :
A relief is formed by replication methods at the εurface of an elastiomer polymer composition, comprising a pattern of parallel lines comprising narrow raised regions generally of leεε than 200nm in width and wider εunken regions preferably of greater than 1 micron in width. The aspect ratio of said narrow raised regions may be reduced by sloping the surface of said wider sunken regions in proximity to εaid narrow raiεed regionε. Said polymer compoεition is chosen such that the reεulting molecular network compriεeε chemical functional groups which may be reacted with cross-linking reagents to attach moleculeε to the εurface of εaid relief. An appropriate croεε-linking agent compriεing a central linker of predetermined length, preferably shorter than 50nm is coupled to a nuclease enzyme (such as DNase I or a type II reεtriction endonuclease, preferably recognizing a 4 base restriction site) according to known art conjugate chemistry. Said cross-linking agent is chosen so as to link said nuclease to εaid elastiomer polymer compriεing εaid chemical functional groups. A small volume of a solution of εaid croεs-linking agent conjugated to said nuclease is spread on a surface as a thin layer in the pattern of a single stripe, preferably of lesε than a few millimeters in width. Said relief is contacted with said stripe of said conjugate on said surface, oriented with said parallel lines perpendicular to said stripe, such that nuclease molecules are linked to the εurfaces of said narrow raised regions along the region juxtaposed with εaid εtripe. The solution used to form said strip may favorably contain a dye or pigment to facilitate identification of the enzyme modified region, or other marking methods may be used to accomplish εuch identification. Thuε, a relief comprising parallel lines and trenches of predefined size and location is produced with a predefined region of raiεed featureε upon which nucleaεe molecules are εituated.
Linear or linearized DNA moleculeε or chromatin are treated εo aε to be immobilized at εpecific regionε along their length, preferably near to one distinct terminus, and optionally derivatized with beadε at the opposite terminus. For example, these may be hybridized with an oligonucleotide compriεing a firεt affinity moiety εuch aε biotin moiety and one or more pεoralen groupε (where plural groupε are preferred to favor the formation of covalent linkageε with both εtrandε of the DNA double helix) . After hybridization with εaid oligonucleotide, εaid pεoralen groupε are cauεed to react with εaid DNA to form a covalent linkageε. Streptavidin is bound to a glass surface in a narrow stripe (<1 micron in width) εhorter in length than the width of εaid εtrip of εaid conjugate above. Said DNA terminally linked to said biotin is then contacted with said narrow stripe of streptavidin and affinity binding iε permitted to occur. Then, for example, the immobilized DNA sample is optionally then briefly subjected to degradation by a 5' to 3' exonuclease, followed by treatment with a polymerase and second affinity group modified nucleotide triphosphateε to terminally decorate εaid linearized DNA moleculeε with said second affinity group. Where modification with said second affinity group is performed, said immobilized DNA sample is.then contacted with a εolution comprising microscopic or sub-micron diameter beads, which may be fluoreεcently labeled, εurface modified with receptorε (e.g. immunoglobulins, etc.) according to known art conjugate chemistry. Alignment of the immobilized DNA molecules is then performed by known art techniqueε.61 Said sample iε then εubjected to mild fluid flow in the direction perpendicular to εaid narrow stripe of εtreptavidin, aε said glasε εurface is withdrawn from the liquid (or the liquid otherwise passeε away from said DNA sample with an interface traveling in a direction perpendicular to said narrow strip of streptavidin) . The molecules of εaid DNA sample are thus aligned in the direction perpendicular to said narrow stripe of streptavidin, fully extended, and situated on said glass surface. The same method may be applied to chromatin (first order packing of DNA with histone nucleosomeε) or with condensed metaphase chromosomeε. Straightening of terminally immobilized moleculeε may alternatively be accompliεhed, where said molecules underwent the optional addition of terminal beads as above, by methods such as those of T.T. Perkins et al.62 whereby beadε εituated on DNA moleculeε are manipulated uεing an optical trap (also known as laεer tweezerε) , or, where εaid beads are of paramagnetic compoεition, by the application of an appropriately directed magnetic field of sufficient strength to straighten, but not so large as to disrupt, the sample molecular and supramolecular εtructure.
Once εample moleculeε are end-immobilized to a preciεe, predetermined region, oriented and extended by meanε εuch as those above, the relief prepared above with raiεed narrow lineε modified with nucleaεe molecules iε then contacted with εaid glaεs surface on which said sample molecules have been straightened and aligned, in an orientation such that the parallel lines of said relief are perpendicular to εaid sample molecules, where said contacting iε performed under εlight preεεure to enεure εealing of the channelε thuε temporarily formed. A gentle fluid flow is immediately established in said channelε, such that once a molecular region is freed at both ends by cleavage by said nuclease moleculeε, it is carried down said channel away from said nuclease molecules, which might otherwise further degrade it. Said flow then carrieε the cleaved molecule fragmentε to collection volumes formed in said relief structureε at the ends of εaid channels, which are arranged such that the location of the channel in which a fragment was cleaved may be determined or inferred from the location of said collection volume. Thus, the collection volumes forms a linearly ordered set, which set compriseε an ordered arrangement of fragmentε of a well defined physical length and precise ordering with respect to their original location in the in-tact sample molecules. Such a method may be repeated, as desired, with a slightly different offset of said relief with respect to said narrow stripe of streptavidin, such that different regions are occluded from the collected sample εet, to the extent that retention under εaid narrow raiεed lineε poεeε any difficultieε. Note that in all εteps, care is taken to avoid shearing sample molecules. Note further that εlight differences in the positioning of the endε of immobilized εample molecules entails that the ordering of said fragmentε in a collection volume iε better deεcribed aε by a poεitional diεtribution function with reεpect to the εequence of the original in-tact moleculeε from which εaid fragmentε derive.
Nonetheleεε, thiε εimple proceεε yieldε ordered fragment populations uεeful for eεtabliεhing phyεical mapε of genetic material and producing ordered εampleε therefrom for purpoεeε such as library cloning or subcloning or other analysiε techniques, and particularly useful in genome sequencing projects.
Two alternative genetic material diεεection methodε availing ..-lcropatterned reliefε are alεo poεεible.
In the firεt alternative, a relief compriεing parallel raiεed narrow lineε and trencheε, such as that described above, is formed. This relief is then contacted with a first surface on which are coated particles εuch aε metallic or glass colloids or hard microcrystalliteε, which are coated with chemical εpecies capable of crosεlinking εaid particleε to εaid relief. Thuε, said particles are bound to the raiεed narrow regionε of said relief. Genetic material is immobilized on a second εurface and aligned aε above, and the relief of the preεent alternative iε oriented aε above with said parallel lines perpendicular to the direction of alignment of said genetic material on said second surface and contacted with said second surface under an applied normal force. Liquid is caused to flow through the channelε formed by said trenches and said second surface as a result of the preceding contacting εtep. The relief iε then translated in the direction of said parallel lines as said normal force iε maintained and said liquid flow is maintained. Thuε, the genetic material iε abraded and therefore cut by said particles, and once cut, fragments are freed from communication with said second surface and said relief, such that said fragmentε are carried by εaid liquid flow down said channels to collection volumes. The cutting action availed in the present alternative of this embodiment is in direct analogy to the cutting of DNA molecules by an atomic force microscope tip. Said second surface may be hard, such aε glaεε, may be elaεtiomeric; εaid second surface may comprise bonded particles similar or identical to those used on said raised regions of said relief, in which case the cutting action may be compared to a εciεεoring action.
In a εecond alternative, a relief compoεed of opaque materialε, compriεing parallel lines of narrow raised regions and trencheε iε uεed, aε above, to define flow channelε when contacted under normal force with a εecond εurface, here of a εheet-like or flat material. In this alternative, said second surface iε tranεparent to radiation of the appropriate frequency range but iε coated (preferably on said second surface, or instead on the surface not contacted with said relief) with materialε which mask εaid radiation. For example, εaid flat material may be quartz coated with metallic mask materials and said radiation may be X-radiation, or said flat material may be glasε coated with any material opaque to viεible or ultraviolet light, which iε uεed for irradiation. In the caεe of X-irradiation, genetic material expoεed will be cleave directly due to double stranded breaks. In the case of ultraviolet or viεible light irradiation, εaid genetic material iε firεt bound by photochemical reagentε which will cleave DNA when activated by radiation expoεure; where εaid maεking material iε on εaid εurface proximal to εaid relief, radiation may be tranεmitted to εaid reagentε in the near field regime, permitting reεolution smaller than the wavelength used and equal to the masking material feature reεolution. The masking pattern used is deεigned such that when said relief is contacted to εaid εecond εurface, the lineε of narrow raiεed regionε are juxtapoεed directly to unmaεked regionε of said second surface. Thus, said irradiation causeε breakε of εaid genetic material at the loci of said narrow raised regionε, which are in contact with εaid second surface, εuch that the fragmentε thuε produced will be freed within the channelε formed by said second εurface and said trenches and carried away, by a fluid flow applied in said channels, to a collection volume correεponding to the reεpective channel.
Replicon Chimera Library Contiguity Analysis: Articles produced by the methods of the present invention may be used to facilitate the analysis of polynucleotide librarieε such as plasmid libraries, bacteriophage librarieε, coεmid librarieε, yeaεt autonomouε chromosome (YAC) based libraries and the like. In the field of gene discovery and genome mapping it is often convenient to work with samples, generally in DNA form, which are fragmented and incorporated into replicon moleculeε capable of being maintained, replicated, expreεsed in and purified from appropriate host organiεm . The fragmentation of genetic material which occurε in the preparation of εuch librarieε, however, introduceε difficulty into eεtablishing the order with which the fragments thus obtained occur within the original, unfragmented polynucleotide sample. Further, mapping of genetic material over millionε of baεe-pairε yieldε information uεeful in the field of geneticε independent of the uεe of polynucleotide librarieε. Therefore, a method which both provides rapid and economical genome or chromosome mapping, and is capable of determining the ordering relationεhip between the replicon chimera compriεing a library would be of great uεe. Current art methodε for accompliεhing εuch taεkε generally rely on the probing of arrays or replicas of clonal replicon-chimera isolateε, whether in the form of colony or plaque iεolateε or arrayε of εpots of genetic material from such a library, generally rely on the transcription of RNA from bacteriophage promoterε at the borderε of the replicon uεed to create the library and directed εuch that εample fragmentε are thuε tranεcribed. Generally, such transcripts are transcribed with radiolabeled ribonucleotides, and the RNA thus obtained is used to probe said arrays or replicas; those array elements or colonies or plaqueε which hybridize the radiolabeled RNA tranεcriptε thuε share common sequenceε with the fragments from which said transcripts were obtained. Thus, these methods determine which clonal isolates contain common sequenceε, and by repetition identify εerieε of clonal iεolateε which are of contiguouε origin. Because such librarieε may contain well over 104 diεtinct chimera, εuch information is rarely if ever exhaustively determined for any given library, and iε uεually only applied to small fractionε of such librarieε.
Copolymer arrayε such as those disclosed in related art or produced by the methods of the present invention may gainfully be applied to the taεk of determining the linkage relationεhip between the individual chimera of a library and alεo to chromosomal mapping thereby. In this method, each replicon must be rendered uniquely distinguishable, for example by the incorporation of a sufficiently long random nucleotide sequence (e.g. 9 variable base pairε [which need not all be contiguouε] in a replicon deεigned for uεe in preparing librarieε of which <10^ clones will be used.) Such a sequence will be referred to as a tag sequence, but it will be understood by those skilled in the relevant arts that different tagging and discrimination methods (e.g. the use of epitope library fused terminal-protein replicated viral or bacteriophage [e.g. PRD1, phi29, adenovirus], probed with peptide or antibody arrays) are poεsible, although nucleotide sequence based tagging with oligo array discrimination iε favorably convenient, and will therefore be emphaεized here. For nucleotide sequence based tagging, the tag sequence is favorably placed εuch that it is transcribed under the influence of a promoter in the replicon sequence and the appropriate purified RNA polymeraεe.
A one dimenεional array compriεing all εequenceε complementary to all poεεible tag εequenceε (or receptors capable of reversibly binding all tag moieties) iε formed by the above εpatially controlled copolymer array forming methodε. Additionally, parallel to the lineε of said one dimensional array of said tag sequenceε, a plurality of lineε of oligoε of different εequence are formed, preferably during the same patterned synthesiε steps. The sequenceε of the oligoε in each line are chosen to be useful in clasεifying the fragment incorporated in a given chimera, or the tranεcript therefrom. The hybridization of each claεεifying oligo with a εample molecule may be regarded as a positive result from a clasεification test; each fragment is teεted by the full εet of claεεifying oligoε (i.e. all lineε), εuch that for n such lines such an array will be capable of discriminating up to 2n different classification results. Clasεifying εequenceε are therefore optimally choεen to have about a 50% chance of probing any given εample fragment or tranεcript, however large numberε of claεεifying εequenceε (lineε) may obviate thiε effort. The essence of this method conεists in uniquely identifying a chimera (according to tag identity) and uniquely clasεifying one terminuε of the incorporated sample fragment, which clasεification information iε associated with the identity of the chimera to which it pertains, where the use of copolymer arrayε permitε the concurrent analyεiε of large numberε of chimeraε or repliconε. Correεpondence between tag identity and the regions of said lines of said claεsifying oligos is enforced at the appropriate step with a relief (preferably of elastiomeric composition) of parallel raised lines and trencheε, of εufficient spacing to accommodate both the tag probing oligo array elementε and the length of the polynucleotide fragmentε, juxtapoεed to the detection oligo array compriεing εaid one dimensional array compriεing all sequences complementary to all posεible tag εequenceε and εaid lineε of εaid claεsifying oligos, oriented such that said parallel raised lineε and trencheε are perpendicular to εaid lineε of said clasεifying oligoε.
A library of said chimera, in mixture form, is either transcribed or fragmented, such that for each chimera, a first fragment type comprising a tag εequence and a εufficient length of εample fragment derived εequence iε obtained. Such fragmentε are either produced in labeled form (affinity, fluoreεcent, radioactive or other label) or otherwiεe labeled before application to said array. Said lines of said clasεifying oligoε are covered to prevent contact with εaid first fragment type as a εolution containing εaid first fragment type iε contacted with a region of εaid array compriεing εaid one dimensional array compriεing all εequenceε complementary to all poεεible tag εequences, to which the moleculeε of εaid first fragment type are permitted to hybridize under sufficiently εtringent conditions to enεure high εpecificity and accuracy of tag identification. Unbound εample moleculeε are washed away. Said relief is then juxtaposed to said array in said perpendicular orientation. The hybrids formed comprising said sequences complementary to all possible tag sequences are then denatured and a flow of buffer solution is applied along the channels formed by the trenches of said relief and the surface of the substrate of said two dimensional array, and conditions permitting εtringent hybridization are then quickly re-eεtablished. Thus, the molecules of said first fragment type, which have been confined to particular channels according to the tag sequence which they comprise are swept by said flow acrosε said lines of said claεεifying oligos and which they thus have the opportunity to hybridize with if and only if they comprise sequenceε complementary to the sequenceε of any of said clasεifying oligoε. Unbound molecules are then washed away. Hybridization of sample molecules to εaid array is then detected by means which correspond to the labeling method used, and the corresponding data favorably recorded by digital computer.
These data are then used to establish the linkage relationship between chimeras according to εimilarity of classification: two chimeras possibly overlap if the clasεification of said first fragment type derived from each of them are consistent, i.e. share a εufficiently large number of claεεificationε not shared with an improbably high proportion of chimera. In other words, two sample fragments which partially overlap will be clasεified εimilarly according to the hybridization of claεεifying oligoε to the εequenceε which they εhare. Combination of data derived from enεembleε of εuch overlap information permit the reconstruction of maps of larger contiguouε regionε (εuch aε chromosomal material) from which sample fragments derive, and simultaneously yield information about which chimeras (as identified by tag sequences) are derived from particular regions of said maps.
Aε will be apparent to thoεe skilled in molecular biology and the art of recombinant DNA technology, many different tagging schemes are posεible. If care iε not taken to enεure that tag εequences differ sufficiently from sample fragment sequence, ambiguouε reεultε will be obtained, but the ambiguity of εuch results will generally be apparent, such that these results may be identified and ignored (e.g. where an improbably high number of classifying oligoε are hybridized within a given channel. ) Such ambiguities may be reduced by specifying tag and tag probing sequences by methodε εimilar to thoεe applied to the deεign of sequencing and PCR primers, i.e. methods which concern the selection of rare sequences and methods which concern the production of degenerate probes (complex mixtures of oligos conforming to some consensus sequence but each hybridizing to different targets having the same complementary consensus sequence) for rare sequences.
Molecules of said first fragment type preferably consiεt of material from one extremity of the εample fragment incorporated into the reεpective chimera. Thiε minimizeε the degree of overlap between two chimeraε classified as contiguouε and thuε minimizeε the number of chimeraε neceεsary to represent the original linear εample, reducing the work neceεεary for analyεiε. Note, however, that more redundant information may be obtained by permitting a greater degree of overlap, for example, by producing and then analyzing longer moleculeε of εaid first fragment type. Thus, two extreme caseε are poεεible: said first fragment type comprising most all of a chimera; and said firεt fragment type conεisting of a minimal length of εequence to permit useful clasεification. In practice, tradeoffε exiεt between these extremes, and intermediate cases are probably preferable according to the specific library and sample.
As will be apparent, each of εaid moleculeε of said firεt fragment type muεt compriεe εaid tag εequence and εequence from εaid original sample from which εaid library waε derived. Where εaid molecules of said firεt fragment type are limited to one terminuε of the εample fragment εequence (i.e. one εample fragment sequence region adjacent to one arm of the replicon vector used to construct the library) , the method of preparation of said first fragment type must ensure that the tag sequence associated with one terminus may be correlated with the tag εequence aεsociated with the other terminus. At leaεt two instances conforming to this requirement are posεible.
In a firεt inεtance, εaid moleculeε of εaid firεt fragment type may be prepared by reεtricting aliquotε of the library of said chimera differently. Here, a the vector from which εaid library waε prepared comprises a first unique restriction enzyme εite, which may be choεen according to infrequent occurrence in the εubject genome or original εample, on one εide of a tag εequence, and a εecond unique reεtriction enzyme site, which may be chosen according to infrequent occurrence in the subject genome or original sample, on the other side of a tag sequence. In this caεe, a first aliquot iε subjected to reεtriction digeεtion with the enzyme εpecific for εaid first unique restriction enzyme site, and a second aliquot is subjected to reεtriction digeεtion with the enzyme specific for said εecond unique enzyme εite. Additionally, fragments may be shortened by any convenient method εuch aε εhearing, random nucleaεe treatment, etc., to reεtrict the length of polynucleotide fragmentε captured by tag probe oligoε and εubsequently analyzed to convenient lengthε. Each aliquot iε analyzed εeparately according to thiε aspect of the present invention, and resultε are uεed to correlate similarly claεεed fragment εequenceε found in said first aliquot with thoεe found in εaid εecond aliquot. In a εecond inεtance, each molecule of εaid vector from which εaid library waε prepared compriεeε two different, unique tag εequenceε, each located near a terminuε of said molecule, i.e. near the locus to which a εample fragment iε ligated. Thus, each tag sequence correεpondε to a terminus of a sample fragment in the library constructed with said vector. Molecules of said first fragment type having exactly one of said two different, unique tag sequences are produced by known art methods which will be related in consideration to the design of said vector (e.g. restriction digestion, transcription from an artificial promoter, etc., according to corresponding vector featureε in relation to said two different, unique tags and the inεerted εaid sample fragment.) Said molecules of said first fragment type having exactly one of said two different, unique tag sequences are analyzed according to the above library analysiε methodε, probing in each caεe with the memberε of only one tag probe εet. Becauεe each terminus is asεociated with a different tag in each clone, the tag from one terminuε muεt be correlated with the tag of the other terminuε. Thiε εecond correlation εtep is accomplished by the εame method, binding chimera or fragmentε compriεing the vector termini with a one dimenεional tag probing oligo array complementary to the first tag εet, where lineε of different claεεifying oligoε in the array are replaced with lineε of different tag probing oligoε, correεponding to thoεe which bind tag εequenceε of the second tag set. The foregoing embodiments and exampleε have been preεented for illustration rather than limitation, with the many posεible variationε, changeε and alternative embodimentε which will be obviouε to thoεe of ordinary εkill in the relevant artε included within the εcope and εpirit of the preεent invention. Thuε, the εcope and the breadth of the preεent invention is intended to be defined by the appended claims rather than the foregoing description.
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42 Moy, V.T. et al. ; 1994. Science, 266:251. "Lee, G.U., et al. ,- 1994. Science, 266:101.
44For example, see Drexler, K.E.; 1992. Nanosystems.
45Rabani, E.M.; 1994. Unpublished resultε concerning preparation of
STM substrateε from Au filmε vacuum evaporated onto freεhly cleaved ruby mica, later annealed with acetylene flame. 46Ward, M.D.; et al.; 1994. Perεonal communication. 13 January. 47For example, εee Prime, K.L. ; Whiteεideε, G.M. ; 1991. Science,
252:1164. 48Singhvi, R. ; Whiteεides, G.M. ; Ingber, D.E.; 1994. Science,
264:696. 9Xiang, X.-D.; et al.; 1995. Science, 268:1138. 50Mϋller, W.T.; Schultz, P.G.; et al.; 1995. Ibid. 51McIntyre, B.J.; Salmeron, M. ; and G.A. Somorjai; 1994. Ibid. 5 Voelker, M.A. ; 1992. Proc. SPIE, 1762:10. 53Drexler, K.E.; 1992. Jbid. pp. 54Yao, J.J.; MacDonald, N.C.; Arney, S.C; 1992. Nanostructures and
Mesoscopic Systems. Academic Presε. pp. 25-33. 55Akamine, S.; Albrecht, T.R. ; Zdeblick, M.J.; Quate, C.F.; 1989.
IEEE Electron Device Letters, 10 (11) -.490. 56Lee, G.U.; Chriεey, L.A.; Colton, R.J.; 1994. Science, 266:111. 5 For example the soluble derivatives deεcribed by Rehahn, M. ; et al.;
1990. Makromol. Chem., 191:1991. 58Zhang, J.; Moore, J.S.; et al. ; 1992. J. Am. Chem. Soc,
114 (6):2273. Zhang, J.; Moore, J.S.; et al. ; 1992. J. Am. Chem. Soc,
114(6) -.2213. Biebuyck, H.A.; Whitesideε, G.M. ; 1994. Langmuir, 10 (8) -.2190. For exampleε, εee Perkinε, T.T.; et al.; 1995. Science, 268:83.
See alεo Bensimon, A.; et al. ; 1994. Science, 265: 2096. Perkins, T.T. ; et al. ; 1994. Science, 264:822.
whereforth, I claim:

Claims

C l aims :
1. A process for the fabrication of microscale or nanoscale deviceε compriεing the uεe of replicated patterned relief εurfaceε and a maεk forming εtep εelected from the group consisting of: multilayer vetoing of self-asεembling monolayer patternε on metallic films; polymerization of resiεt from patterned initiator compound regions; casting of polymeric resiεt patternε on εurfaces; injection molding of resist precursorε; emboεεing of a preformed resist film followed by a mild etch to eliminate reduced thicknesε regionε; curing of prepolymer εolutionε wetting underlying patternε.
2. A process according to claim 1 further comprising the incorporation of alignment patternε into εaid replicated patterned relief εurfaceε and the surfaces to which these are juxtaposed, where εaid alignment patternε are selected from the group consisting of: patterns which cause the appearance of Moire patterns when overlapped; interlocking surface relief patternε.
3. A process for the patterning of three or more distinct affinity species or reactants serving as precursors thereof on a surface comprising one or more steps of contacting said surface with one or more micropatterned relief surfaces.
4. A method according to claim 3 for the spatially controlled syntheεis of copolymer sequenceε on solid surfaces comprising the use of relief εurfaceε to effect or prevent the physical contact of reactants or reagents with regions of said solid εurfaceε.
5. A method for the formation of molecular layerε having εpatially predetermined compoεitional pattern, comprising one or more steps of asεociating terminal affinity group functionalized moleculeε with complementary affinity groupε patterned onto εurfaces or other εuch molecular layers.
6. A method for the replication of compositional patternε of molecular layerε produced according to claim 5.
7. A method for the positionally controlled synthesis of molecules or molecular complexes according to positional conεtraint deεign ruleε and compriεing the use of positioning means which directly or indirectly bind reactant moleculeε, reactant precursor molecules, catalystε, enzymeε, abzymeε, ribozymeε, deprotection reagent moleculeε, activator reagent molecules or protecting reagents, and position these relative to reactive groupε situated on a workpiece or workpiece precursor comprising one or more molecules.
8. A method according to claim 7 where said poεitioning meanε comprises a microfabricated array.
9. A method for the spatially ordered disεection of genetic material compriεing the immobilization of εaid genetic material to a predefined region of a surface.
10. A method according to claim 9 comprising the use of micropatterned surfaceε.
11. A method for the determination of the ordering of cloneε or polynucleotide mapε from a polynucleotide library compriεing the use of one or more oligonucleotide or polynucleotide arrays.
12. A method for the fabrication and replication of data patterns on surfaces for read out by data readout means comprising scanned probes comprising the use of one or more patterned relief surfaceε.
13. Method and meanε for readout of data patternε and surfaces prepared according to claim 12 comprising a charge coupled device and actuation meanε for tranεlating said εurfaceε relative to εaid charge coupled device.
14. A method according to claim 11 further compriεing the uεe of an elaεtiomeric relief compriεing raiεed parallel stripes with microscale featureε.
15. A proceεε for the patterning of two or more diεtinct materialε or precurεors thereof on a porous surface comprising one or more steps of contacting said surface with one or more micropatterned relief surfaces each of which is coated with a εuεpenεion of one of εaid diεtinct materialε.
16. A method according to claim 15 further compriεing an annealing step.
17. A method for the fabrication comprising the steps of:
(a) creating one or more elastiomeric microreliefs;
(b) coating said elastiomeric microrelief with a material from which an article is to be fabricated;
(c) juxtapoεing εaid elastiomeric microrelief of step (b) to a surface;
(d) cauεing said material from which an article is to be fabricated to polymerize or harden or set;
(e) removing said elaεtiomeric microrelief;
(f) forming or molding a εacrificial material over the surface produced in step (d) and causing said sacrificial material to polymerize or harden or set if neceεsary;
(g) repeating steps (b) through (f) until the article under fabrication is complete; and, (h) removing said sacrificial material added in any step (f).
PCT/IB1996/000912 1995-07-28 1996-07-26 Pattern formation, replication, fabrication and devices thereby WO1997006468A2 (en)

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US6558904B2 (en) 1997-02-19 2003-05-06 Clondiag Chip Technologies Gmbh Method for producing structured, self-organized molecular monolayers of individual molecular species, in particular substance libraries
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WO2000032809A2 (en) * 1998-11-27 2000-06-08 Noxxon Pharma Ag Cloning and copying on surfaces
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US6534271B2 (en) 1998-11-27 2003-03-18 Noxxon Pharma Ag Cloning and copying on surfaces
WO2000044939A1 (en) * 1999-01-29 2000-08-03 Surmodics, Inc. Replicable probe array
US6514768B1 (en) 1999-01-29 2003-02-04 Surmodics, Inc. Replicable probe array
WO2001020402A1 (en) * 1999-09-14 2001-03-22 Massachusetts Institute Of Technology Fabrication of finely featured devices by liquid embossing
US6517995B1 (en) 1999-09-14 2003-02-11 Massachusetts Institute Of Technology Fabrication of finely featured devices by liquid embossing
US7470540B2 (en) 2000-10-17 2008-12-30 Febit Ag Method and device for the integrated synthesis and analysis of analytes on a support
US6936181B2 (en) 2001-10-11 2005-08-30 Kovio, Inc. Methods for patterning using liquid embossing
US6848462B2 (en) 2001-12-06 2005-02-01 Nanostream, Inc. Adhesiveless microfluidic device fabrication
WO2003064026A1 (en) 2002-01-31 2003-08-07 Nimblegen Systems Llc Pre-patterned substrate, device and method for optical synthesis of dna probes
JP2006501807A (en) * 2002-01-31 2006-01-19 ニンブルゲン システムズ リミテッド ライアビリティ カンパニー Pre-patterned substrate, apparatus and method for optical synthesis of DNA probes
US6814859B2 (en) 2002-02-13 2004-11-09 Nanostream, Inc. Frit material and bonding method for microfluidic separation devices
US7153421B2 (en) 2002-02-13 2006-12-26 Nanostream, Inc. Frit material and bonding method for microfluidic separation devices
WO2003068401A1 (en) * 2002-02-13 2003-08-21 Nanostream, Inc. Frit material for multilayer microfluidic separation devices
US6957608B1 (en) 2002-08-02 2005-10-25 Kovio, Inc. Contact print methods
WO2005038525A1 (en) * 2003-09-26 2005-04-28 Infineon Technologies Ag Stamp device for use in soft lithography and method for producing the same
DE102004008241A1 (en) * 2004-02-19 2005-11-24 Universität Bremen Enzyme-based nanolithography
DE102004008241B4 (en) * 2004-02-19 2006-09-07 Universität Bremen Enzyme-based nanolithography
WO2006061600A1 (en) * 2004-12-07 2006-06-15 University Of Southampton Particles for use as a solid support and process for their preparation
EP1731961A1 (en) 2005-06-10 2006-12-13 Obducat AB Template replication method
US7854873B2 (en) 2005-06-10 2010-12-21 Obducat Ab Imprint stamp comprising cyclic olefin copolymer
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