CA2654470A1 - Substrates for performing analytical reactions - Google Patents
Substrates for performing analytical reactions Download PDFInfo
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- CA2654470A1 CA2654470A1 CA002654470A CA2654470A CA2654470A1 CA 2654470 A1 CA2654470 A1 CA 2654470A1 CA 002654470 A CA002654470 A CA 002654470A CA 2654470 A CA2654470 A CA 2654470A CA 2654470 A1 CA2654470 A1 CA 2654470A1
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- layer
- cladding layer
- zero mode
- core
- mode waveguide
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Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/10—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
- G01N2021/7706—Reagent provision
- G01N2021/7709—Distributed reagent, e.g. over length of guide
- G01N2021/7713—Distributed reagent, e.g. over length of guide in core
Abstract
Substrates, including zero mode waveguide substrates that have been fabri cated to provide additional f.upsilon..pi.clionai elements and/or components including increased volumes for positioning of active surfaces and or compo nents for the mitigation of negative electrochemical properties of the under lying substrates.
Description
SUBSTRATES F'GR PERI'ORMING ANALYTICAL RE-ACTIQNS
CRUSS-REFER.ENCETO RELATED APPLICATIONS
l[l(?o1l This application claims the benefit under 35 U.S.C. Y I 19(e) of l).S.S.N. 60i812,863, filed June 12, 2006, whieh is hereby expressly incorporated by referertce in its entirety.
STA"1'EMENTR.EGARDING FEDERALLY SPONSORED RESI:ARCI--I
[00021 Portions of the invention were made with ;overnment support under M~.GR[ Grant No. RoI-1-1G00371.0-01 and the government has certain rights to the invention.
BACKGRQt1ND OF THE INVENTION
[0003] The application of widcly differing scientific disciplines to biological research has yielded profound advances in the ways that biological systems are characterized and monitored, and the way in which biological disorders are treated. In particular, the combination of solid state electroiiies techiiokogies to biological research applications has provided a number oI'important advances including, e.g., niolecular array technology, i.e., DNA arrays (See, U.S. Patent No.
6,261.776), microfluidic cliip technologies (See, U.S. 13atent No. 5,976336), chemica(ly sensitive field effect transistors (Chemf`E'I's), and other valuable sensor technologies.
[00041 Zero mode tvaveguide (ZMW) technology provides yet another extension of these semiconductor fabrication technolo~;ies to different areas of research and dia~nostics. In particular, ZMW arrays have been applied in a range of biochemical analyses and particularly in the field of genetic analysis. ZMWs typically comprise a nanoscale core, well or opening disposed in an opaque cladding layer that is disposed upon a transparent substrate. Due to the narrow dimensions of the core electromagnetic radiation that is of a frequeney above a particular cut-off frequency will be prevented from propagating all the way through the core. Notwithstanding the foregoing, the radiation tvill penetrate a limited distance into the core, providing a very small illuminated volume within the core. By illuminating a very small volume, one can potentially interrogate very small quanitities of reagents, including, e.g., single molecule reactions.
100051 Bv inonitoring reactions at the ~,inole i-noIecule level, one can precisely identitv and,'or monitor agiven reaction. "t his is the b.. b,hind ti~:ularlypr mising field of's=n-:1e inolecule DNA sequencing technology that monitors the molecule by molecule synthesis of a DNA
strand in a template dependant fashion by a single polymerase enzyrne.
[00051 Despite the foregoing advances, a number of additional improvements and advances are available. The present invention provides a number of such advances and improvements.
BRIEF St;MMARY Qp "['1-iE INVENTION
(0007[ "1"he preseiii invention is generally directed to improvements in the configuration and%or manufacturing of substrates used in chemical and biochemical analyses, and particularly to substrates having optical features fabricated into such substrates, such as zero rnode ~Nraveguides (ZM W s).
[0008] In a first aspect, the invention provides a zero mode waveguide substrate that comprises a transparent substrate layer having at least a first surface, a metal cladding layer disposed upon the first surface of the transparent substrate layer and an aperture disposed through the cladding layer to the transparent substrate layer, and forming a core region surrounded by the metal cladding layer, wherein the core is dimensioned to prevent electromagnetic radiation of a frequency greater than a cut-off frequency from propagating entirely through the core. The substrate also includes a sacrificial anode disposed upon the cladding layer.
[00091 Relatedly, the invention provides methods of monitoring a reaction, comprising providing a reaction mixture within a core of a zero mode waveguide that comprises a metal cladding layer, wherein the zero mode wavcguide includes a sacrificial anode, and monitoring the reaction mixture within an illumination volume of the core.
[00101 in an additional aspect, the invention provides a zero mode WavegLtide substrate that comprises a transparent substrate layer, an opaque cladding layer, and a core, comprising an aperture disposed through the cladding layer and extending at least partially into the transparent substrate layer.
BRIEF DESCRIPTION OF THE FIGURES
[o4I i] Figure 1 provides a schematic illustration of a Zero Mode Waveguide (ZMW) disposed in a substrate.
j00121 Figure 2 provides a schematic illustration of a ZMW including a sacrificial anode layer.
[00.131 Figure 3 provides a schematic illustration of a ZMW including an increased volume e transpar~nt substrate layer.
~
100141 Figure 4 provides a schematic exemplary fabrication process for the ZMW
shown in Figure 3.
100151 Figure 5 shows an alternative exemplary fabrication process for producing the ZMW
shown in Figure 3.
100161 Figure 6 is a schematic illustratian of the comparative benefits of ZMWs including recessed surfaces.
DETAILED DESCRIPTION OF THE INVENTION
100171 The present invention is generally directed to improvements in the configuration and/or manutacturing of substrates used in chemical and biochemical a-nalyses, and particularly to substrates having optical features fabricated into such substrates, such as zero rnode waveguides (Zlvt Ws).
100181 In the context of the present invention, analytical substrates generally comprise a rigid or sen7iWrigid solid substrate, typically substantially planar in overall configuration, upon which reactions of interest may be carried out and monitored, using an associated detection system, such as a fluorescence microscope, optical imaging system, or electrical signal detection system.
Typically, such substrates may have additional functional components manufactured into the substrate or disposed upon a surface of the substrate. Such components include, e.g., molecular components, such as molecular binding moieties, e.g., antibodies, nucleic acids non-specific chemical coupling groups, binding peptides, or the like, or property altering groups sLich as hydrophobic or hydrophilic groups, to provide areas of relative hydrophobicity or hydrophilicity on the substrates. Likewise, the substrates may have structural components f'abricated onto or into the substrates, including, e.g., barriers, wells, posts, pillars, channels, troughs, electrical contacts, ÃnaslC
layers, cladding layers or the like.
100191 The present invention may^ be generally applied across a range of different substrate types, depending upon the need for the particular solution offered by the invention. For example, in a first aspect, the invention provides substrates that incliide arrays of zcro mode waveguides (ZMWs) disposed therein. ZMWs are typically characterized by a cladding cornponent that surrounds a core component, ~ here the core is dimensioned such that electromagnetic energy entering the core that is of a frequency that falls below a particular threshold or cut-off frequency, is precluded from propagating entirely through the core. Th.e result is that direction of such electrornagnetic c:neroy at the core results in a very small region of illumination within only a portion of the core. Where the core is provided as an open volume bounded by the cladding, the resu(t is an abilitv to illuminate an extremely small volume within the core.
The ability to illuminate extremely small volumes, e.g., of chemical or biochemical reactants, is of particular value in a nuinber of applications. These ZMW arrays, their fabrication and use, are described in U.S. Patent No. 6,917,726, which is incorporated herein in its entirety for all purposes.
100201 Zero mode waveguides typically comprise cross-sectional dimensions of from about nm to about 250 nm, and are preferably between about 20 and about 100 nm in cross sectional dimension. The ZMWs are typically circular in cross-section, although they may also be elongated structures, e.g., ovals, elipses, slits, grooves, or other non-circular shapes. The depth of the core of the zero rnode wavegaide, subject to the discussion herein, is typically defined, at least in part, by the thickness of the cladding layer, which will typically range from about 25 nm to about 500 ttt-n thick, and preferably, between about 50 nm and about 200 nm thick. One may select a desired depth from the full range ofapplicable depths, depending upon the desired application, f00211 The zero mode waveguides afthe invention are typically fabricated into a substrate that includes a transparent substrate layer and an opaque cladding layer deposited upon the transparent stibstrate. The core portion of the waveguides conlprises an aperture disposed through the clad.ding fayer to the transparent layer to define a well having an open upper end and a lower end that is capped by the transparent substrate. A schematic illustration of such waveguides is shown in Figure 1. As shown, the overall substrate 100 includes a waveguide core 102 disposed within opaque cladding layer 104 and continuing through to the transparent substrate layer 106.
t00221 ZMW fabrication can be carried out by a number of methods. However in preferred aspects, a layered fabrication strategy is used that provides a thin metal film over a transparent planar substrate, e.g., glass, fused silica, quartz, alumina, transparent polymers, or the like.
Defining the cores is usually carried out by providing an appropriate resist layer over the transparent substrate and developing the resist layer to yield pillars or posts as a negative image of the desired Lvaveguide core.s. An opaque film. such as a thin metal film or semiconductor film layer, is then deposited over the substrate to provide the cladding layer. and the resist pillars are removed to yield apertures or cores, in their place, surrounded by the cladding layer. Typical metal claciding layers include any of a number of metal films that may be processed using conventional metal deposition techniclues, such as sputtering and/or evaporation. Such metals include aluminum, gold, platinum, chrQrniun7, and the like as well as combinations of these, e.g., as multilayer depositions.
Semiconductor cladding layers are also useful in a variety of applications and include, e.g., any of a variety of Ãiroup I ll-V, lV, andror II-Vl serrriconductors that may be deposited upon the substrate usirig, e.g., vapor deposition.
100231 Defining the resist pillars r-nay be carried out by a variety of methods known in the art of serniconductor processing, including, e.g., photolithographic methods, c-beam lithography techniques, nano-imprint lithography, or the like.
[0024] In at least a first aspect, the overall structure of the zero mode waveguide may incorporate additional functionality that, in preferred cases., is applied as additional lavers to the overall substrate.
[0025] As noted above, preferred zero mode waveguides comprise metal cladding layers disposed over transparent substrate materials. In the application of such metallized substrates to cher-nical andlor biochemical operations, they wi11 inevitably be exposed to environments that can be somewhat damaging to the metal, including, e.g., relatively high salt concentrations and/or non-neutral pi-I environments, e.g., acidic or basic conditions. All of these factors add to the tendency for such materials to be susceptible to corrosion. ln particular, exposure of these metallic components to high salt environirrents can create an opportunitv for galvanic corrosion to occur.
Accordingly, in at least one aspect, the present invention provides a zero mode waveguide substrate that includes a sacrificial or galvanic anode as a component of the substrate.
Such galvanic anodes may be integral to the substrate, e.g., deposited as integral to or as a discrete layer upon a substrate, or they may be added as a separate component to the reaction rnixture, e.g., as particles, strips or wires. Any of a variety of different metals can be employed as the galvanic anode, including, e.g., zinc, magnesium, aluminum, iron, nickel or any other metal that functions as a sacrificial anode for the underlying cladding layer metal. It will be apprcciated that selection of an optimized metal anode layers rnay depend to some extent upon the make-up of the underlying cladding layer.
Additionally, because the environment to which a device is exposed will vary according to the needs of an application, the reduction potentials of metals will vary from their standard values. As such, numerous materials can be used as a sacrificial anode. For a cladding material with a half-cell potential X (under the conditions used), and an anode materials with a half-cell potential Y. a metal can be used as a sacrificial anode if Y is less than X. In some cases, either the c,ladd'sng material or the anode material or both tvill spontaneouslv- undergo a change to another surface state with new half cell potentials X' and Y , and if'Y' is less than X' then these materials are usable as sacrificial anodes. Preferred materials in the capacity of sacrificial anode r-naterials irrclude: Magnesium, Zinc, Beryllium, Cadmium, Uranium, Aluminum, Indium, ".I-in, Lead, Iron, Nickel, Copper, Chromium, i tantalum, an:d tungsten. In parlicu[ar[y preferred aspects, a zinc laver is deposited along with or on top of the metal cladding layer. A variety of metal film deposition techniques may be employecl to provide the anode layer over the cladding la}-er, including sputtering, evaporation, pÃasina vapor deposition, and the (ike.
100261 In a related enibodii-nent, the additional (ayer may be electrically insulated from the cladding layer via a nonconductive [ayer disposed between the anode layer and the cladding layer.
In this embodiment, an electrical potential can be applied directly to the anode layer, thus removing the restriction that the anode material have a particular reduction potential.
In this embodiment, the invention provides an electrical contact to the cladding layer, and a separate electrical contact to the anode [ayer. The nonconductive layer can be applied in a separate step, or it can be a native oxide of one of the other materials to be applied. For example, the native oxide ofalumin.um is non-conductive, and methods are known in the art for forming an insulating layer of aluminum oxide on a conductive aluminuin layer.
100271 In the context of the manufacturing processes set forth above, the zinc [aver may generally be co-deposited with the metal cladding layer or deposited as a subsequent layer on top of the metal claddiiig layer. Once the resist pillars are removed. the waveguide core would be disposed through both the galvanic anode and t-netal cladding layers, "l'ypically the anode layer will be from about 0.1 nm to about 100 nm thick, preferably from about I to about 100 nm thick, fron7 a.bou! I to about 50 nm thick and in some cases from about 10 to about 50 nm thick. An example of a waveguide substrate in accordance with this aspect ofthe invention is shown in Figure 2. As shown, and with reference to Figure 1, the overall ZMW device 200 again includes a core 102 disposed in cladding layer 104 that is, in turn, disposed on the surface of transparent substrate 106.
In addition to the foregoing, however, a sacrificial anode layer 208 is disposed upon the upper surface of the cladding layer 104, and serves to prevent excessive galvanic corrosion of the metal cladding laver during application.
100281 In alternative aspects, a discrete anode component may be added to the waveguide arrav in other than a standard deposition process. In particular, an appropriate metal particle, strip or the like rnay, be deposited upon the waveguide array. provided it is of sufficient size, e.g., suflecieritlv large or s.ufticic:ntlv srnall. that it does not interfere with the waveguide functions, e.p., biocking the openings of excessive numbers of the cores.
10029) In another alternate structural configuration. the zero mode waveguides are provided with a space or volume at the end of the core that e-xtends beyond the cladding layer into the transparent stibstrate. In particular, by providing an additional extension of the volume created by the core, one can obtain a number of advantaaes over a zero mode waveguide where the volume and core are co-extensive, e.g., as in F-i -7ure 1. An example of a waveguide having this alternative structure is illustrated in Figure 3. As shown, a zero mode waveguide sLibstrate 300 includes a core 302 disposed through a claddin4.; laver 304, that is deposited upon a transparent substrate 306. The wavegLlide structure also inclLides a recess 308, well or extension of the core volume into the transparent substrate 306 to increase the volume of the overall waveguide structure. In particular, in the case of ZMWs that include modified surfaces, e.g., having engineered surfaces that include reactive molecules and/or linker molecules and/or protective surface treatments, i.e., to prevent nonspecific surface associations, the resulting active surface may be moved further away from th.e underlying substrate surface. As a result, the reactions of interest, e.g., being carried out at the reactive surface, may fall at the edges or outside of the optimal observation volume of a ZMW. As such, by recessing the underlying substrate surface, one can better position a reactive surface within the observation volume. This benefit is schematically illustrated in Figure 6.
[0030] In a non-recessed ZMW, as illustrated in Panel I of Figure 6. As shown, a ZMW
substrate 600 includes a core 602) disposed within a cladding layer 604 through to the transparent substrate layer 606. A reactive surface as indicated by Xs 608, e.g., for performing a desired reaction of interest such as a nucleic acid polymerization reaction, is provided within a ZNMW core 602, and is coupled to the underlying substrate 606 sLirface via linker molecules 610. E3eca.use of the size of the litikers 610, and potentially other surface modifications, the reactive surface 608 may be disposed at the edge or outside of the observation volume (shown bounded by dashed line 612).
By recessing the lower surface of the underlying substrate, however, one can alleviate this potential issue. In particular, as shown in Figure 6, panel II, the ZMW 620 includes a recessed lower surface 624 at the base of the core 622 that results in reactive surface 608 falling well within the observation volume. This provides a zero mode waveguide having, inter alia, a more readily tunable observation volume, e.g., adjustable beyond the thickness of the cladding layer alone, higher signal level for optics disposed below the waveguides, resulting in improved signal to noise ratios.
100311 In addition to the aforementioned advantages, the increase in the volume of the detection region provides an advantage in the statistics of occupancy of molecules of interest in a detection volu.me. In some instances it is desirable for an assay to have one and only one molecule under observatic3n in each confinement. In the case where the bottom of the claddiraa and the floor ~
of the device are in the same place, the confinement has a profile that i.s exponentially decreasing with a short attenuation length. The result is that it is rare that a molecule will be positioned in the detection zone and that a second molecule will not be present higher up in the detection zone such that it creates background noise to the first molecule. By lowering the floor, the profile changes to einulate a step function, ~vhere there is a portion of the device that is "in"
the detection zone with relativeEq unifor-n observation efficiency, and another part of the device that is "out" of the detection volume kk, ith the very fast exponential decay separating the two regions. In this latter situation the probability ofobtairting by random chance the arrangement that there is one rnolecLile in the detectioii zone and no inolecules in the exponential decay region is greatly increased. An additional advantage of the relative uniformity provided by the deep well is that the level of fluorescence output of a single molecule is made Iess dependent on position, and therefore inare uniform in time when there is some freedom for fluctuation due to a flexible linker tethering the fluorophore to the device.
100321 The recess or well 308 at the transparent substrate 306 is typically ofcornparable cross sectional dimensions as the core, e.g., diarneter, but may vary in its depth depending upon the desired application and fianctionality.
100331 Fabrication of the modified zero mode waveguides according to this aspect of the invention may be carried out by a nuinber of methods. For example, in a first preferred aspect, the well is fabricated as an additional deposition step in the process of ZMW
fabrication, that takes place prior to deposition of the metal cladding layer. In particular, usin ;
the negative iniage of the resist pillars, a layer of transparent material, e.g., of'the same or similar composition to the underlying traiisparent substrate, is deposited over the transparent substrate. flThe metal cladding layer, and optionally, any additional layers, such as a galvanic anode laver, is then deposited over it.
Once the pillars are removed frorn the structure, a waveguide core is left in the cladding layer with an additional volume that extends into the transparent substrate layer. This process is schematically illustrated in Figure 4.
]',00341 As shown in Figure 4, a transparent substrate 400 is provided having an Lipper surface 402. A resist layer 404 is then deposited over the surface 402. The resist is exposed and developed to yield a negative image of the desired. waveguide cares, shown as pillars 406. A]ayer of additional transparent material 408 is then deposited upon the exposed surface 4022 of the substrate to build up the transparent substrate layer around the pillar 406.
The transparent inaterial is preferably the same or similar in composition to the underlying transparent substrate 400. For ~
example, in the case of glass substrates, an SiO2 layer may be deposited over the substrate surface using any of a variety oftechnic{ues known in the field ofsemiconductor manufacturing, including, e.g., use of spin on glass, plasma enhanced chemical vapor deposition, vapor deposition of silicon followed by oxidation to SiO-2 (e.g., thermal oxidation). A metal cladding layer 410 is then deposited over the transparent layer 408. The resist pillars are then removed from the overall substrate using, e.g., a lift-off or "knock-off" process, yielding the open core 412 of the vvaveguide structures disposed in the cladding layer. Additionally, the core volume extends into the additional transparent layer 408, to provide added volume to the core.
100351 Cn an alternative process, the waveguide core rnay be used as a masking layer for an additional etching step. In particular, a ZMW is fabricated using the processes set forth above. "1"he metal claddint) layer then functions as a masking layer for a subsequent etching step. I.n this context, the etchant used and the amount of eteh time are selected so as to yield a desired geometry of the etched space. For example, where a substrate is etched using anisotropic reactive ion etching (R.IE), the resulting feature will have vertical side walls and the same aerial view as the opening in the cladding. In this instance, inethods are known in the art for etching the substrate Inaterial with a tolerable degree of selectivity to the cladding material. In the event that etching occurs isotropically (either using wet chelnical etching or R.IE), the time may be controlled to avoid yielding an etched volume that is substantially larger than the waveguide dimensions.
Alternatively or additionally, one may use more crystalline substrates, e.g., quartz, or the like, where etching is more anisotropic despite the etching conditions, yielding a more contined etched space.
Alternatively, dry etching techniques may be used. A schematic illustration of this process is shown in Figure 5.
100361 As shown in Figure 5, a ZMW substrate 500 includes a ZMW core 502 disposed through a metal cladding layer 504 disposed upon the transparent substrate 506, whicli in this aspect ot'the invention comprises an inorganic material, e.g., glass, quartz or fusedsilic-a. In accordance with t)iis aspect of the invention, the cladding Eayer doubles as a masking layer for a subsequent etching step. In particular, the su.bstrate is exposed to an appropriate etchant (as indicated by arroNvs 508) which etches additional depth into the core volume, e.g., as shown by shaded region 508 and depression 5 10. In some cases, it may be desirable to utilize metals in the cladding Iayer that are more routinely employed in lithographi:c techniques, such as chromium., gold, or the like, as they may have greater tolerance of the harsher etching environments than other metals or may function better as masking materials, providing higher resolution features.
CRUSS-REFER.ENCETO RELATED APPLICATIONS
l[l(?o1l This application claims the benefit under 35 U.S.C. Y I 19(e) of l).S.S.N. 60i812,863, filed June 12, 2006, whieh is hereby expressly incorporated by referertce in its entirety.
STA"1'EMENTR.EGARDING FEDERALLY SPONSORED RESI:ARCI--I
[00021 Portions of the invention were made with ;overnment support under M~.GR[ Grant No. RoI-1-1G00371.0-01 and the government has certain rights to the invention.
BACKGRQt1ND OF THE INVENTION
[0003] The application of widcly differing scientific disciplines to biological research has yielded profound advances in the ways that biological systems are characterized and monitored, and the way in which biological disorders are treated. In particular, the combination of solid state electroiiies techiiokogies to biological research applications has provided a number oI'important advances including, e.g., niolecular array technology, i.e., DNA arrays (See, U.S. Patent No.
6,261.776), microfluidic cliip technologies (See, U.S. 13atent No. 5,976336), chemica(ly sensitive field effect transistors (Chemf`E'I's), and other valuable sensor technologies.
[00041 Zero mode tvaveguide (ZMW) technology provides yet another extension of these semiconductor fabrication technolo~;ies to different areas of research and dia~nostics. In particular, ZMW arrays have been applied in a range of biochemical analyses and particularly in the field of genetic analysis. ZMWs typically comprise a nanoscale core, well or opening disposed in an opaque cladding layer that is disposed upon a transparent substrate. Due to the narrow dimensions of the core electromagnetic radiation that is of a frequeney above a particular cut-off frequency will be prevented from propagating all the way through the core. Notwithstanding the foregoing, the radiation tvill penetrate a limited distance into the core, providing a very small illuminated volume within the core. By illuminating a very small volume, one can potentially interrogate very small quanitities of reagents, including, e.g., single molecule reactions.
100051 Bv inonitoring reactions at the ~,inole i-noIecule level, one can precisely identitv and,'or monitor agiven reaction. "t his is the b.. b,hind ti~:ularlypr mising field of's=n-:1e inolecule DNA sequencing technology that monitors the molecule by molecule synthesis of a DNA
strand in a template dependant fashion by a single polymerase enzyrne.
[00051 Despite the foregoing advances, a number of additional improvements and advances are available. The present invention provides a number of such advances and improvements.
BRIEF St;MMARY Qp "['1-iE INVENTION
(0007[ "1"he preseiii invention is generally directed to improvements in the configuration and%or manufacturing of substrates used in chemical and biochemical analyses, and particularly to substrates having optical features fabricated into such substrates, such as zero rnode ~Nraveguides (ZM W s).
[0008] In a first aspect, the invention provides a zero mode waveguide substrate that comprises a transparent substrate layer having at least a first surface, a metal cladding layer disposed upon the first surface of the transparent substrate layer and an aperture disposed through the cladding layer to the transparent substrate layer, and forming a core region surrounded by the metal cladding layer, wherein the core is dimensioned to prevent electromagnetic radiation of a frequency greater than a cut-off frequency from propagating entirely through the core. The substrate also includes a sacrificial anode disposed upon the cladding layer.
[00091 Relatedly, the invention provides methods of monitoring a reaction, comprising providing a reaction mixture within a core of a zero mode waveguide that comprises a metal cladding layer, wherein the zero mode wavcguide includes a sacrificial anode, and monitoring the reaction mixture within an illumination volume of the core.
[00101 in an additional aspect, the invention provides a zero mode WavegLtide substrate that comprises a transparent substrate layer, an opaque cladding layer, and a core, comprising an aperture disposed through the cladding layer and extending at least partially into the transparent substrate layer.
BRIEF DESCRIPTION OF THE FIGURES
[o4I i] Figure 1 provides a schematic illustration of a Zero Mode Waveguide (ZMW) disposed in a substrate.
j00121 Figure 2 provides a schematic illustration of a ZMW including a sacrificial anode layer.
[00.131 Figure 3 provides a schematic illustration of a ZMW including an increased volume e transpar~nt substrate layer.
~
100141 Figure 4 provides a schematic exemplary fabrication process for the ZMW
shown in Figure 3.
100151 Figure 5 shows an alternative exemplary fabrication process for producing the ZMW
shown in Figure 3.
100161 Figure 6 is a schematic illustratian of the comparative benefits of ZMWs including recessed surfaces.
DETAILED DESCRIPTION OF THE INVENTION
100171 The present invention is generally directed to improvements in the configuration and/or manutacturing of substrates used in chemical and biochemical a-nalyses, and particularly to substrates having optical features fabricated into such substrates, such as zero rnode waveguides (Zlvt Ws).
100181 In the context of the present invention, analytical substrates generally comprise a rigid or sen7iWrigid solid substrate, typically substantially planar in overall configuration, upon which reactions of interest may be carried out and monitored, using an associated detection system, such as a fluorescence microscope, optical imaging system, or electrical signal detection system.
Typically, such substrates may have additional functional components manufactured into the substrate or disposed upon a surface of the substrate. Such components include, e.g., molecular components, such as molecular binding moieties, e.g., antibodies, nucleic acids non-specific chemical coupling groups, binding peptides, or the like, or property altering groups sLich as hydrophobic or hydrophilic groups, to provide areas of relative hydrophobicity or hydrophilicity on the substrates. Likewise, the substrates may have structural components f'abricated onto or into the substrates, including, e.g., barriers, wells, posts, pillars, channels, troughs, electrical contacts, ÃnaslC
layers, cladding layers or the like.
100191 The present invention may^ be generally applied across a range of different substrate types, depending upon the need for the particular solution offered by the invention. For example, in a first aspect, the invention provides substrates that incliide arrays of zcro mode waveguides (ZMWs) disposed therein. ZMWs are typically characterized by a cladding cornponent that surrounds a core component, ~ here the core is dimensioned such that electromagnetic energy entering the core that is of a frequency that falls below a particular threshold or cut-off frequency, is precluded from propagating entirely through the core. Th.e result is that direction of such electrornagnetic c:neroy at the core results in a very small region of illumination within only a portion of the core. Where the core is provided as an open volume bounded by the cladding, the resu(t is an abilitv to illuminate an extremely small volume within the core.
The ability to illuminate extremely small volumes, e.g., of chemical or biochemical reactants, is of particular value in a nuinber of applications. These ZMW arrays, their fabrication and use, are described in U.S. Patent No. 6,917,726, which is incorporated herein in its entirety for all purposes.
100201 Zero mode waveguides typically comprise cross-sectional dimensions of from about nm to about 250 nm, and are preferably between about 20 and about 100 nm in cross sectional dimension. The ZMWs are typically circular in cross-section, although they may also be elongated structures, e.g., ovals, elipses, slits, grooves, or other non-circular shapes. The depth of the core of the zero rnode wavegaide, subject to the discussion herein, is typically defined, at least in part, by the thickness of the cladding layer, which will typically range from about 25 nm to about 500 ttt-n thick, and preferably, between about 50 nm and about 200 nm thick. One may select a desired depth from the full range ofapplicable depths, depending upon the desired application, f00211 The zero mode waveguides afthe invention are typically fabricated into a substrate that includes a transparent substrate layer and an opaque cladding layer deposited upon the transparent stibstrate. The core portion of the waveguides conlprises an aperture disposed through the clad.ding fayer to the transparent layer to define a well having an open upper end and a lower end that is capped by the transparent substrate. A schematic illustration of such waveguides is shown in Figure 1. As shown, the overall substrate 100 includes a waveguide core 102 disposed within opaque cladding layer 104 and continuing through to the transparent substrate layer 106.
t00221 ZMW fabrication can be carried out by a number of methods. However in preferred aspects, a layered fabrication strategy is used that provides a thin metal film over a transparent planar substrate, e.g., glass, fused silica, quartz, alumina, transparent polymers, or the like.
Defining the cores is usually carried out by providing an appropriate resist layer over the transparent substrate and developing the resist layer to yield pillars or posts as a negative image of the desired Lvaveguide core.s. An opaque film. such as a thin metal film or semiconductor film layer, is then deposited over the substrate to provide the cladding layer. and the resist pillars are removed to yield apertures or cores, in their place, surrounded by the cladding layer. Typical metal claciding layers include any of a number of metal films that may be processed using conventional metal deposition techniclues, such as sputtering and/or evaporation. Such metals include aluminum, gold, platinum, chrQrniun7, and the like as well as combinations of these, e.g., as multilayer depositions.
Semiconductor cladding layers are also useful in a variety of applications and include, e.g., any of a variety of Ãiroup I ll-V, lV, andror II-Vl serrriconductors that may be deposited upon the substrate usirig, e.g., vapor deposition.
100231 Defining the resist pillars r-nay be carried out by a variety of methods known in the art of serniconductor processing, including, e.g., photolithographic methods, c-beam lithography techniques, nano-imprint lithography, or the like.
[0024] In at least a first aspect, the overall structure of the zero mode waveguide may incorporate additional functionality that, in preferred cases., is applied as additional lavers to the overall substrate.
[0025] As noted above, preferred zero mode waveguides comprise metal cladding layers disposed over transparent substrate materials. In the application of such metallized substrates to cher-nical andlor biochemical operations, they wi11 inevitably be exposed to environments that can be somewhat damaging to the metal, including, e.g., relatively high salt concentrations and/or non-neutral pi-I environments, e.g., acidic or basic conditions. All of these factors add to the tendency for such materials to be susceptible to corrosion. ln particular, exposure of these metallic components to high salt environirrents can create an opportunitv for galvanic corrosion to occur.
Accordingly, in at least one aspect, the present invention provides a zero mode waveguide substrate that includes a sacrificial or galvanic anode as a component of the substrate.
Such galvanic anodes may be integral to the substrate, e.g., deposited as integral to or as a discrete layer upon a substrate, or they may be added as a separate component to the reaction rnixture, e.g., as particles, strips or wires. Any of a variety of different metals can be employed as the galvanic anode, including, e.g., zinc, magnesium, aluminum, iron, nickel or any other metal that functions as a sacrificial anode for the underlying cladding layer metal. It will be apprcciated that selection of an optimized metal anode layers rnay depend to some extent upon the make-up of the underlying cladding layer.
Additionally, because the environment to which a device is exposed will vary according to the needs of an application, the reduction potentials of metals will vary from their standard values. As such, numerous materials can be used as a sacrificial anode. For a cladding material with a half-cell potential X (under the conditions used), and an anode materials with a half-cell potential Y. a metal can be used as a sacrificial anode if Y is less than X. In some cases, either the c,ladd'sng material or the anode material or both tvill spontaneouslv- undergo a change to another surface state with new half cell potentials X' and Y , and if'Y' is less than X' then these materials are usable as sacrificial anodes. Preferred materials in the capacity of sacrificial anode r-naterials irrclude: Magnesium, Zinc, Beryllium, Cadmium, Uranium, Aluminum, Indium, ".I-in, Lead, Iron, Nickel, Copper, Chromium, i tantalum, an:d tungsten. In parlicu[ar[y preferred aspects, a zinc laver is deposited along with or on top of the metal cladding layer. A variety of metal film deposition techniques may be employecl to provide the anode layer over the cladding la}-er, including sputtering, evaporation, pÃasina vapor deposition, and the (ike.
100261 In a related enibodii-nent, the additional (ayer may be electrically insulated from the cladding layer via a nonconductive [ayer disposed between the anode layer and the cladding layer.
In this embodiment, an electrical potential can be applied directly to the anode layer, thus removing the restriction that the anode material have a particular reduction potential.
In this embodiment, the invention provides an electrical contact to the cladding layer, and a separate electrical contact to the anode [ayer. The nonconductive layer can be applied in a separate step, or it can be a native oxide of one of the other materials to be applied. For example, the native oxide ofalumin.um is non-conductive, and methods are known in the art for forming an insulating layer of aluminum oxide on a conductive aluminuin layer.
100271 In the context of the manufacturing processes set forth above, the zinc [aver may generally be co-deposited with the metal cladding layer or deposited as a subsequent layer on top of the metal claddiiig layer. Once the resist pillars are removed. the waveguide core would be disposed through both the galvanic anode and t-netal cladding layers, "l'ypically the anode layer will be from about 0.1 nm to about 100 nm thick, preferably from about I to about 100 nm thick, fron7 a.bou! I to about 50 nm thick and in some cases from about 10 to about 50 nm thick. An example of a waveguide substrate in accordance with this aspect ofthe invention is shown in Figure 2. As shown, and with reference to Figure 1, the overall ZMW device 200 again includes a core 102 disposed in cladding layer 104 that is, in turn, disposed on the surface of transparent substrate 106.
In addition to the foregoing, however, a sacrificial anode layer 208 is disposed upon the upper surface of the cladding layer 104, and serves to prevent excessive galvanic corrosion of the metal cladding laver during application.
100281 In alternative aspects, a discrete anode component may be added to the waveguide arrav in other than a standard deposition process. In particular, an appropriate metal particle, strip or the like rnay, be deposited upon the waveguide array. provided it is of sufficient size, e.g., suflecieritlv large or s.ufticic:ntlv srnall. that it does not interfere with the waveguide functions, e.p., biocking the openings of excessive numbers of the cores.
10029) In another alternate structural configuration. the zero mode waveguides are provided with a space or volume at the end of the core that e-xtends beyond the cladding layer into the transparent stibstrate. In particular, by providing an additional extension of the volume created by the core, one can obtain a number of advantaaes over a zero mode waveguide where the volume and core are co-extensive, e.g., as in F-i -7ure 1. An example of a waveguide having this alternative structure is illustrated in Figure 3. As shown, a zero mode waveguide sLibstrate 300 includes a core 302 disposed through a claddin4.; laver 304, that is deposited upon a transparent substrate 306. The wavegLlide structure also inclLides a recess 308, well or extension of the core volume into the transparent substrate 306 to increase the volume of the overall waveguide structure. In particular, in the case of ZMWs that include modified surfaces, e.g., having engineered surfaces that include reactive molecules and/or linker molecules and/or protective surface treatments, i.e., to prevent nonspecific surface associations, the resulting active surface may be moved further away from th.e underlying substrate surface. As a result, the reactions of interest, e.g., being carried out at the reactive surface, may fall at the edges or outside of the optimal observation volume of a ZMW. As such, by recessing the underlying substrate surface, one can better position a reactive surface within the observation volume. This benefit is schematically illustrated in Figure 6.
[0030] In a non-recessed ZMW, as illustrated in Panel I of Figure 6. As shown, a ZMW
substrate 600 includes a core 602) disposed within a cladding layer 604 through to the transparent substrate layer 606. A reactive surface as indicated by Xs 608, e.g., for performing a desired reaction of interest such as a nucleic acid polymerization reaction, is provided within a ZNMW core 602, and is coupled to the underlying substrate 606 sLirface via linker molecules 610. E3eca.use of the size of the litikers 610, and potentially other surface modifications, the reactive surface 608 may be disposed at the edge or outside of the observation volume (shown bounded by dashed line 612).
By recessing the lower surface of the underlying substrate, however, one can alleviate this potential issue. In particular, as shown in Figure 6, panel II, the ZMW 620 includes a recessed lower surface 624 at the base of the core 622 that results in reactive surface 608 falling well within the observation volume. This provides a zero mode waveguide having, inter alia, a more readily tunable observation volume, e.g., adjustable beyond the thickness of the cladding layer alone, higher signal level for optics disposed below the waveguides, resulting in improved signal to noise ratios.
100311 In addition to the aforementioned advantages, the increase in the volume of the detection region provides an advantage in the statistics of occupancy of molecules of interest in a detection volu.me. In some instances it is desirable for an assay to have one and only one molecule under observatic3n in each confinement. In the case where the bottom of the claddiraa and the floor ~
of the device are in the same place, the confinement has a profile that i.s exponentially decreasing with a short attenuation length. The result is that it is rare that a molecule will be positioned in the detection zone and that a second molecule will not be present higher up in the detection zone such that it creates background noise to the first molecule. By lowering the floor, the profile changes to einulate a step function, ~vhere there is a portion of the device that is "in"
the detection zone with relativeEq unifor-n observation efficiency, and another part of the device that is "out" of the detection volume kk, ith the very fast exponential decay separating the two regions. In this latter situation the probability ofobtairting by random chance the arrangement that there is one rnolecLile in the detectioii zone and no inolecules in the exponential decay region is greatly increased. An additional advantage of the relative uniformity provided by the deep well is that the level of fluorescence output of a single molecule is made Iess dependent on position, and therefore inare uniform in time when there is some freedom for fluctuation due to a flexible linker tethering the fluorophore to the device.
100321 The recess or well 308 at the transparent substrate 306 is typically ofcornparable cross sectional dimensions as the core, e.g., diarneter, but may vary in its depth depending upon the desired application and fianctionality.
100331 Fabrication of the modified zero mode waveguides according to this aspect of the invention may be carried out by a nuinber of methods. For example, in a first preferred aspect, the well is fabricated as an additional deposition step in the process of ZMW
fabrication, that takes place prior to deposition of the metal cladding layer. In particular, usin ;
the negative iniage of the resist pillars, a layer of transparent material, e.g., of'the same or similar composition to the underlying traiisparent substrate, is deposited over the transparent substrate. flThe metal cladding layer, and optionally, any additional layers, such as a galvanic anode laver, is then deposited over it.
Once the pillars are removed frorn the structure, a waveguide core is left in the cladding layer with an additional volume that extends into the transparent substrate layer. This process is schematically illustrated in Figure 4.
]',00341 As shown in Figure 4, a transparent substrate 400 is provided having an Lipper surface 402. A resist layer 404 is then deposited over the surface 402. The resist is exposed and developed to yield a negative image of the desired. waveguide cares, shown as pillars 406. A]ayer of additional transparent material 408 is then deposited upon the exposed surface 4022 of the substrate to build up the transparent substrate layer around the pillar 406.
The transparent inaterial is preferably the same or similar in composition to the underlying transparent substrate 400. For ~
example, in the case of glass substrates, an SiO2 layer may be deposited over the substrate surface using any of a variety oftechnic{ues known in the field ofsemiconductor manufacturing, including, e.g., use of spin on glass, plasma enhanced chemical vapor deposition, vapor deposition of silicon followed by oxidation to SiO-2 (e.g., thermal oxidation). A metal cladding layer 410 is then deposited over the transparent layer 408. The resist pillars are then removed from the overall substrate using, e.g., a lift-off or "knock-off" process, yielding the open core 412 of the vvaveguide structures disposed in the cladding layer. Additionally, the core volume extends into the additional transparent layer 408, to provide added volume to the core.
100351 Cn an alternative process, the waveguide core rnay be used as a masking layer for an additional etching step. In particular, a ZMW is fabricated using the processes set forth above. "1"he metal claddint) layer then functions as a masking layer for a subsequent etching step. I.n this context, the etchant used and the amount of eteh time are selected so as to yield a desired geometry of the etched space. For example, where a substrate is etched using anisotropic reactive ion etching (R.IE), the resulting feature will have vertical side walls and the same aerial view as the opening in the cladding. In this instance, inethods are known in the art for etching the substrate Inaterial with a tolerable degree of selectivity to the cladding material. In the event that etching occurs isotropically (either using wet chelnical etching or R.IE), the time may be controlled to avoid yielding an etched volume that is substantially larger than the waveguide dimensions.
Alternatively or additionally, one may use more crystalline substrates, e.g., quartz, or the like, where etching is more anisotropic despite the etching conditions, yielding a more contined etched space.
Alternatively, dry etching techniques may be used. A schematic illustration of this process is shown in Figure 5.
100361 As shown in Figure 5, a ZMW substrate 500 includes a ZMW core 502 disposed through a metal cladding layer 504 disposed upon the transparent substrate 506, whicli in this aspect ot'the invention comprises an inorganic material, e.g., glass, quartz or fusedsilic-a. In accordance with t)iis aspect of the invention, the cladding Eayer doubles as a masking layer for a subsequent etching step. In particular, the su.bstrate is exposed to an appropriate etchant (as indicated by arroNvs 508) which etches additional depth into the core volume, e.g., as shown by shaded region 508 and depression 5 10. In some cases, it may be desirable to utilize metals in the cladding Iayer that are more routinely employed in lithographi:c techniques, such as chromium., gold, or the like, as they may have greater tolerance of the harsher etching environments than other metals or may function better as masking materials, providing higher resolution features.
Claims (13)
1. A zero mode waveguide substrate, comprising:
a transparent substrate layer having at least a first surface;
a metal cladding layer disposed upon the first surface of the transparent substrate layer;
an aperture disposed through the cladding layer to the transparent substrate layer, and forming a core region surrounded by the metal cladding layer, wherein the core is dimensioned to prevent electromagnetic radiation of a frequency greater than a cut-off frequency from propagating entirely through the core; and a sacrificial anode disposed upon the cladding layer.
a transparent substrate layer having at least a first surface;
a metal cladding layer disposed upon the first surface of the transparent substrate layer;
an aperture disposed through the cladding layer to the transparent substrate layer, and forming a core region surrounded by the metal cladding layer, wherein the core is dimensioned to prevent electromagnetic radiation of a frequency greater than a cut-off frequency from propagating entirely through the core; and a sacrificial anode disposed upon the cladding layer.
2. The zero mode waveguide substrate of claim 1, wherein the sacrificial anode is attached to the metal cladding layer.
3. The zero mode waveguide substrate of claim 1, wherein the sacrificial anode comprises a metal layer disposed on an upper surface of the cladding layer.
4. The zero mode waveguide of claim 1, wherein the sacrificial anode comprises a metal selected from Zn, Mg, Al and Fe.
5. The zero mode waveguide substrate of claim 1, wherein the sacrificial anode comprises a zinc layer disposed on an upper surface of the cladding layer.
6. The zero mode waveguide of claim 1, wherein the sacrificial anode comprises a metal particle, wire or strip.
7. The zero mode waveguide of claim 6, wherein the sacrificial anode is deposited upon but not attached to the cladding layer.
8. A zero mode waveguide substrate, comprising:
a transparent substrate layer an opaque cladding layer; and a core, comprising an aperture disposed through the cladding layer and extending at least partially into the transparent substrate laver.
a transparent substrate layer an opaque cladding layer; and a core, comprising an aperture disposed through the cladding layer and extending at least partially into the transparent substrate laver.
9. The zero mode waveguide of claim 8, wherein the transparent substrate layer comprises at least first and second transparent layers, the second transparent layer being disposed upon the first transparent layer, and the cladding layer being disposed upon the second transparent laver, wherein the aperture extends through the cladding layer and the second transparent layer.
10. The zero mode waveguide substrate of claim 9, wherein the cladding layer comprises a metal.
11. The zero mode waveguide of claim 8, further comprising a reactive surface disposed within the core and at least partially within a portion of the core extending into the transparent substrate layer.
12. The zero mode waveguide of claim 11, wherein the reactive surface comprises a polymerase enzyme.
13. A method of monitoring a reaction, comprising:
providing a reaction mixture within a core of a zero mode waveguide that comprises a metal cladding layer, wherein the zero mode waveguide includes a sacrificial anode;
and monitoring the reaction mixture within an illumination volume of the core.
providing a reaction mixture within a core of a zero mode waveguide that comprises a metal cladding layer, wherein the zero mode waveguide includes a sacrificial anode;
and monitoring the reaction mixture within an illumination volume of the core.
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Also Published As
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|---|---|
| CN101915957B (en) | 2012-12-12 |
| WO2007149724A2 (en) | 2007-12-27 |
| US7907800B2 (en) | 2011-03-15 |
| AU2007261114B2 (en) | 2012-07-12 |
| AU2007261114A1 (en) | 2007-12-27 |
| US20080095488A1 (en) | 2008-04-24 |
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| CN101915957A (en) | 2010-12-15 |
| EP2027253A2 (en) | 2009-02-25 |
| US7486865B2 (en) | 2009-02-03 |
| WO2007149724A3 (en) | 2008-07-31 |
| CN101467082B (en) | 2011-12-14 |
| EP2857829B1 (en) | 2017-08-09 |
| US20090325166A1 (en) | 2009-12-31 |
| CN101467082A (en) | 2009-06-24 |
| EP2027253A4 (en) | 2014-04-30 |
| CA2654470C (en) | 2015-02-03 |
| JP2009539411A (en) | 2009-11-19 |
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