CA2360000C - Target molecule attachment to surfaces - Google Patents

Target molecule attachment to surfaces Download PDF

Info

Publication number
CA2360000C
CA2360000C CA2360000A CA2360000A CA2360000C CA 2360000 C CA2360000 C CA 2360000C CA 2360000 A CA2360000 A CA 2360000A CA 2360000 A CA2360000 A CA 2360000A CA 2360000 C CA2360000 C CA 2360000C
Authority
CA
Canada
Prior art keywords
groups
target molecule
compound
substrate
photoreactive
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CA2360000A
Other languages
French (fr)
Other versions
CA2360000A1 (en
Inventor
Ralph A. Chappa
Sheau-Ping Hu
Dale G. Swan
Melvin J. Swanson
Patrick E. Guire
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Surmodics Inc
Original Assignee
Surmodics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Surmodics Inc filed Critical Surmodics Inc
Publication of CA2360000A1 publication Critical patent/CA2360000A1/en
Application granted granted Critical
Publication of CA2360000C publication Critical patent/CA2360000C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • 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
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/84Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40Y being a hydrogen or a carbon atom
    • C07C323/42Y being a carbon atom of a six-membered aromatic ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/61Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/63Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by introduction of halogen; by substitution of halogen atoms by other halogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • B01J2219/00279Features relating to reactor vessels
    • B01J2219/00306Reactor vessels in a multiple arrangement
    • B01J2219/00313Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315Microtiter plates
    • B01J2219/00317Microwell devices, i.e. having large numbers of wells
    • 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
    • B01J2219/00531Sheets essentially square
    • 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
    • B01J2219/00608DNA chips
    • 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
    • B01J2219/0061The surface being organic
    • 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
    • B01J2219/00621Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • 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
    • B01J2219/00626Covalent
    • 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
    • B01J2219/00635Introduction of reactive groups to the surface by reactive plasma treatment
    • 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
    • B01J2219/00659Two-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
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00677Ex-situ synthesis followed by deposition on the substrate
    • 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
    • B01J2219/00709Type of synthesis
    • B01J2219/00711Light-directed synthesis
    • 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
    • B01J2219/00709Type of synthesis
    • B01J2219/00716Heat activated synthesis
    • 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
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11Compounds covalently bound to a solid support
    • 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
    • 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

Abstract

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

Description

TARGET MOLECULE ATTACHMENT TO SURFACES
TECHNICAL FIELD
The present invention relates to methods for attaching target molecules such as oligonucleotides (oligos) to a surface, and to compositions for use in such methods.
In another aspect, the invention relates to the resultant coated surfaces themselves. In yet another aspect, the invention relates to the use of photochemical and thermochemical means to attach molecules to a surface.

BACKGROUND OF THE INVENTION
The immobilization of deoxyribonucleic acid (DNA) onto support surfaces has =
become an important aspect in the development of DNA-based assay systems as well as for other purposes, including the development of microfabricated arrays for DNA
analysis. See, for instance, "Microchip Arrays Put DNA on the Spot", R.
Service, Science 282(5388):396-399, 16 October 1998; and "Fomenting a Revolution, in Miniature", I. Amato, Science 282(5388): 402-405, 16 October 1998.
See also, "The Development of Microfabricated Arrays of DNA Sequencing and Analysis", O'Donnell-Maloney et al., TIBTECH 14:401-407 (1996). Generally, such procedures are carried out on the surface of microwell plates, tubes, beads, microscope slides, silicon wafers or membranes. Certain approaches, in particular, have been developed to enable or improve the likelihood of end-point attachment of a synthetic oligonucleotide to a surface. End-point attachment (i.e., with the nucleic acid sequence attached through one or the other terminal nucleotide) is desirable because the entire length of the sequence will be available for hybridization to another nucleic acid sequence. This is particularly advantageous for the detection of single base pair changes under stringent hybridization conditions.
Hybridization is the method used most routinely to measure nucleic acids by base pairing to probes immobilized on a solid support. When combined with amplification techniques such as the polymerase chain reaction (PCR) or ligase chain reaction (LCR), hybridization assays are a powerful tool for diagnosis and research.
Microwell plates, in particular, are convenient and useful for assaying relatively large numbers of samples. Several methods have been used for immobilization of nucleic acid probes onto microwell plates. Some of these involve adsorption of unmodified or modified oligonucleotides onto polystyrene plates. Others involve covalent immobilization. Various methods have also been used to increase the sensitivity of hybridization assays. Polymeric capture probes (also known as target molecules) and detection probes have been synthesized and used to obtain sensitivities down to 10' DNA molecules/ml. Another method used branched oligonucleotides to increase the sensitivity of hybridization assays. Yet another method used a multi-step antibody-enhanced method. Other types of nucleic acid probes such as ribonucleic acid (RNA), complementary DNA (cDNA) and peptide nucleic acids (PNA's) have also been immobilized onto microwell plates for hybridization of PCR products in diagnostic applications. Furthermore, PCR primers have been immobilized onto microwell plates for solid phase PCR.
Only a relative few approaches to immobilizing DNA, to date, have found their way into commercial products. One such product is known as "NucleoLink'", and is available from Nalge Nunc International (see, e.g., Nunc Tech Note Vol.
3, No.
17). In this product, the DNA is reacted with a carbodiimide to activate 5'-phosphate groups which then react with functional groups on the surface. Disadvantages of this approach are that it requires the extra step of adding the carbodiimide reagent as well as a five hour reaction time for immobilization of DNA, and it is limited to a single type of substrate material.
As another example, Pierce has recently introduced a proprietary DNA
immobilization product known as ReactiBindTM DNA Coating Solutions" (see "Instructions - Reacti-BindTM DNA Coating Solution" 1/1997). This product is a solution that is mixed with DNA and applied to surfaces such as polystyrene or polypropylene. After overnight incubation, the solution is removed, the surface washed with buffer and dried, after which it is ready for hybridization.
Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it does have some limitations. For example, Applicants were not able to demonstrate useful immobilization of DNA onto polypropylene using the manufacturer's instructions. Furthermore, this product requires large amounts of DNA. The instructions indicate that the DNA should be used at a concentration between 0.5 and 5 g/ml.
Similarly, Costar sells a product called "DNABIN1DTM" for use in attaching DNA to the surface of a well in a microwell plate (see, e.g., the DNABINIDTM
"Application Guide"). The surface of the DNABINDTM plate is coated with an uncharged, nonpolymeric heterobifunctional reagent containing an N-oxysuccinimide (NOS) reactive group. This group reacts with nucleophiles such as primary amines.
The heterobifunctional coating reagent also contains a photochemical group and spacer arm which covalently links the reactive group to the surface of the polystyrene plate. Thereafter, amine-modified DNA can be covalently coupled to the NOS
surface. The DNA is modified by adding a primary amine either during the synthesis process to the nascent oligomer or enzymatically to the preformed sequence.
Since the DNABINDTM product is polystyrene based, it is of limited use for those applications that require elevated temperatures such as thermal cycling.
These various products may be useful for some purposes, or under certain circumstances, but all tend to suffer from one or more drawbacks and constraints. In particular, they either tend to require large amounts of oligonucleotide, render background noise levels that are unsuitably high and/or lack versatility.
International Patent Application No. PCT/US98/20140, assigned to the assignee of the present application, describes and claims, inter alia, a reagent composition for attaching a target molecule to the surface of a substrate, the reagent composition comprising one or more groups for attracting the target molecule to the reagent, and one or more thermochemically reactive groups for forming covalent bonds with corresponding functional groups on the attracted target molecule.
Optionally, the composition further provides photogroups for use in attaching the composition to a surface. In one embodiment, for instance, a plurality of photogroups and a plurality of cationic groups (in the form of quaternary ammonium groups) are attached to a hydrophilic polymer backbone. This polymer can then be coimmobilized with a second polymer backbone that provides the above-described thermochemically reactive groups (e.g., N-oxysuccinimide ("NOS") groups) for immobilization of target molecules.
While reagent compositions having both attracting groups and thermochemically reactive groups, as described in the above-captioned PCT
application, remain useful and preferred for many applications, Applicants also find that the attracting groups may not be required under all circumstances. For instance, one suitable process for preparing activated slides for microarrays includes the steps of coating the slides with a reagent composition of a type described in the PCT
application (and particularly, one having both attracting groups as well as photoreactive and thermochemically reactive groups). The polymers are attached to the slide by activation of the photoreactive groups, following by the application of small volumes (e.g., several nanoliters or less) of target molecules (e.g., oligonucleotides) using precision printing techniques.
Once applied, the solvent used to deliver the oligonucleotide is dried (as the oligonucleotides are attracted to the bound polymer), and the slide incubated under conditions suitable to permit the thermochemical coupling of the oligonucleotide to the bound polymer. Thereafter, however, any unbound oligonucleotide is typically washed off of the slide. Applicants have found, however, that there occasionally remains a detectable trail of unbound oligonucleotide, referred to as a "comet effect", leading away from the spot. This trail is presumably due to the attractive forces within the bound polymer present on the slide surface that surrounds the spot, serving to tie up the generally negatively charged oligonucleotide as it is washed from the spot. This trail, in turn, can provide undesirable and unduly high levels of background noise.
Applicants have found that under such circumstances (e.g., the application of small volumes directly to a generally flat surface) polymeric reagents are preferably provided without the presence of such attracting groups (though with the thermochemically reactive groups and optional photogroups). Suitable reagents of this type are disclosed in the above-captioned co-pending PCT application.
Such reagents, in turn, can be used to coat oligonucleotides in a manner that provides an improved combination of such properties as reduced background, small spot size (e.g., increased contact angle), as compared to polymeric reagents having charged attracting groups.
SUMMARY OF THE INVENTION
The present invention provides a method and reagent composition for covalent attachment of target molecules onto the surface of a substrate, such as microwell plates, tubes, beads, microscope slides, silicon wafers or membranes. In one embodiment, the method and composition are used to immobilize nucleic acid probes onto plastic materials such as microwell plates, e.g., for use in hybridization assays.
In a preferred embodiment the method and composition are adapted for use with substantially flat surfaces, such as those provided by microscope slides and other plastic, silicon hydride, or organosilane-pretreated glass or silicone slide support surfaces. The reagent composition can then be used to covalently attach a target molecule such as a biomolecule (e.g., a nucleic acid) which in turn can be used for specific binding reactions (e.g., to hybridize a nucleic acid to its complementary strand).
Support surfaces can be prepared from a variety of materials, including but not limited to plastic materials selected from the group consisting of crystalline thermoplastics (e.g., high and low density polyethylenes, polypropylenes, acetal resins, nylons and thermoplastic polyesters) and amorphous thermoplastics (e.g., polycarbonates and poly(methyl methacrylates). Suitable plastic or glass materials provide a desired combination of such properties as rigidity, toughness, resistance to long term deformation, recovery from deformation on release of stress, and resistance to thermal degradation.
A reagent composition of the invention contains one or more thermochemically reactive groups (i.e., groups having a reaction rate dependent on temperature). Suitable groups are selected from the group consisting of activated esters (e.g., NOS), epoxide, azlactone, activated hydroxyl and maleimide groups.
Optionally, and preferably, the composition can also contain one or more photoreactive groups. Additionally, the reagent may comprise one or more hydrophilic polymers, to which the thermochemically reactive and/or photoreactive groups can be pendent. The photoreactive groups (alternatively referred to herein as "photogroups") can be used, for instance, to attach reagent molecules to the surface of the support upon the application of a suitable energy source such as light.
The thermochemically reactive groups, in turn, can be used to form covalent bonds with appropriate and complementary functional groups on the target molecule.
Generally, the reagent molecules will first be attached to the surface by activation of the photogroups, thereafter the target molecule, (e.g., an oligonucleotide) is contacted with the bound reagent under conditions suitable to permit it to come into binding proximity with the bound polymer. The target molecule is thermochemically coupled to the bound reagent by reaction between the reactive groups of the bound reagent and appropriate functional groups on the target molecule. The thermochemically reactive groups and the ionic groups can either be on the same polymer or, for instance, on different polymers that are coimmobilized onto the surface. Optionally, and preferably, the target molecule can be prepared or provided with functional groups tailored to groups of the reagent molecule. During their synthesis, for instance, the oligonucleotides can be prepared with functional groups such as amines or sulfhydryl groups.
The invention further provides a method of attaching a target molecule, such as an oligo, to a surface, by employing a reagent as described herein. In turn, the invention provides a surface having nucleic acids attached thereto by means of such a reagent, as well as a material (e.g., microwell plate) that provides such a surface. In yet another aspect, the invention provides a composition comprising a reagent(s) of this invention in combination with a target molecule that contains one or more functional groups reactive with the thermochemically reactive group(s) of the reagent.
Using such reagents, applicants have found that capture probes can be covalently immobilized to a variety of surfaces, including surfaces that would not otherwise adsorb the probes (such as polypropylene and polyvinylchloride). The resulting surfaces provide signals comparable to or better than those obtained with modified oligonucleotides adsorbed onto polystyrene or polycarbonate.
The present immobilization reagent and method can be used in amplification methods in a manner that is simpler than those previously reported, and can also provide improved surfaces for the covalent immobilization of nucleophile-derivatized nucleic acids. In addition to immobilized probes for amplification methods and hybridization assays, the reagents of this invention may provide improved nucleic acid immobilization for solid phase sequencing and for immobilizing primers for PCR and other amplification techniques.
In accordance with an aspect of the present invention, there is provided a method of attaching a target molecule to a substrate, the method comprising:
a) providing a reagent composition comprising a polymeric backbone, and more than two thermochemically reactive groups attached to the polymeric backbone, wherein the thermochemically reactive groups are configured and arranged to form covalent bonds with functional groups on the target molecule; b) coating and immobilizing the reagent composition on the substrate to form a bound composition; c) providing a solution comprising target molecule having one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition; d) applying one or more discrete small sample volume spots of the solution to the substrate;
and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without use of attracting groups to - 7a -attract the target molecule to the bound reagent.
In accordance with another aspect of the present invention, there is provided an activated slide for binding target molecule in a sample, the slide comprising:
a) a substrate; and b) a bound composition attached to the substrate, wherein the bound composition comprises a polymeric backbone having more than one thermochemically reactive groups attached thereto, and wherein the bound composition is configured and arranged to form covalent bonds with functional groups on the target molecule without use of attracting groups to attract the target molecule to the bound composition.
In accordance with another aspect of the present invention, there is provided a microarray comprising a substrate, a bound composition disposed on the substrate, and target molecule covalently coupled with the bound composition, wherein the microarray is prepared by a method comprising: a) providing a reagent composition comprising a polymeric backbone, and more than one thermochemically reactive groups attached to the polymeric backbone; b) coating and immobilizing the reagent composition on the substrate to form a bound composition; c) providing a solution comprising target molecule, wherein the target molecule comprises one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition;
d) applying one or more discrete spots of up to about 1 nanoliter of the solution to the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without the use of attracting groups.
In accordance with an aspect of the present invention, there is provided a method of attaching a target molecule to a substrate, the method comprising: a) providing a reagent composition comprising a polymeric backbone, and more than two thermochemically reactive groups attached to the polymeric backbone, wherein the thermochemically reactive groups are configured and arranged to form covalent bonds with functional groups on the target molecule; b) coating and immobilizing the reagent composition on the substrate to form a bound composition; c) providing a solution comprising target molecule having one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition; d) applying one or more discrete spots of up to about 1 nanoliter of the solution to the substrate; and e) - 7b -allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without use of attracting groups to attract the target molecule to the bound reagent.
In accordance with another aspect of the present invention, there is provided an activated slide for binding target molecule in a sample, the slide comprising:
a) a substrate; and b) a bound composition attached to the substrate, wherein the bound composition comprises a polymeric backbone having more than two thermochemically reactive groups attached thereto, and wherein the bound composition is configured and arranged to form covalent bonds with functional groups on the target molecule without use of attracting groups to attract the target molecule to the bound composition.
In accordance with another aspect of the present invention, there is provided a microarray comprising a substrate, a bound composition disposed on the substrate, and target molecule covalently coupled with the bound composition, wherein the microarray is prepared by a method comprising: a) providing a reagent composition comprising a polymeric backbone, and more than two thermochemically reactive groups attached to the polymeric backbone; b) coating and immobilizing the reagent composition on the substrate to form a bound composition; c) providing a solution comprising target molecule, wherein the target molecule comprises one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition;
d) applying one or more discrete spots of up to about 1 nanoliter of the solution on the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without the use of attracting groups.
DETAILED DESCRIPTION
A preferred reagent molecule of the present invention comprises a hydrophilic backbone bearing one or more thermochemically reactive groups useful for forming a covalent bond with the corresponding functional group of the target molecule, together with one or more photoreactive groups useful for attaching the reagent to a surface.
In another embodiment of the invention, it is possible to immobilize nucleic acid sequences without the use of the photoreactive group. For instance, the surface of , - 7c -the material to be coated can be provided with thermochemically reactive groups, which can be used to immobilize hydrophilic polymers having thermochemically reactive groups as described above. For example, a surface may be treated with an ammonia plasma to introduce a limited number of reactive amines on the surface of the material. If this surface is then treated with a hydrophilic polymer having thermochemically reactive groups (e.g., NOS groups), then the polymer can be immobilized through reaction of the NOS groups with corresponding amine groups on the surface. Preferably, the reactive groups on the polymer are in excess relative to the corresponding reactive groups on the surface to insure that a sufficient number of these thermochemically reactive groups remain following the immobilization to allow coupling with the nucleic acid sequence.
While not intending to be bound by theory, it appears that by virtue of the small spot size, as well as the kinetics and fluid dynamics encountered in the use of reduced spot sizes, the oligonucleotide is able to come into binding proximity with the bound reagent without the need for the attracting groups described above. When used for preparing microarrays, e.g., to attach capture molecules (e.g., oligonucleotides or cDNA) to the microarray surface, such capture molecules are generally delivered to the surface in a volume of less than about 1 nanoliter per spot, using printing pins adapted to form the spots into arrays having center to center spacing of about 200 p.m to about 500 pm.
Given their small volumes, the printed target arrays tend to dry quickly, thus further affecting the coupling kinetics and efficiency. Unlike the coupling of DNA
from solution and onto the surface of coated microplate wells, oligonucleotides printed in arrays of extremely small spot sizes tend to dry quickly, thereby altering the parameters affecting the manner in which the oligonucleotides contact and couple with the support. In addition to the design and handling of the printing pins, other factors can also affect the spot size, and in turn, the ultimate hybridization signals, including: salt concentrations, type of salts and wetting agents in the printing buffer;
hydrophobic/hydrophilic properties of the surfaces; the size and/or concentration of the oligonucleotide; and the drying environments.
As described herein (e.g., in Examples 25, 26 and 28), coatings of reagents having both photogroups and thermochemically reactive groups ("Photo-PA-I
PolyNOS"), as well as reagents having those groups together with attracting groups (a mixture of "Photo-PA-PolyNOS/Photo-PA-PolyQuat") both provided useful and specific immobilization of amine-modified DNA, with the choice between the two approaches being largely dependent on the choice of substrate (e.g., flat slide as opposed to microwell).
In a preferred embodiment, the reagent composition can be used to prepare activated slides having the reagent composition photochemically immobilized thereon. The slides can be stably stored and used at a later date to prepare microarrays by immobilizing amine-modified DNA. The coupling of the capture DNA to the surface takes place at pH 8-9 in a humid environment following printing the DNA
solution in the form of small spots.
Activated slides of the present invention are particularly well suited to replace conventional (e.g., silylated) glass slides in the preparation of microarrays using manufacturing and processing protocols, reagents and equipment such as micro-spotting robots (e.g., as available from Cartesian), and a chipmaker micro-spotting device (e.g., as available from TeleChem International). Suitable spotting equipment and protocols are commercially available, such as the "Arrayli" TM ChipMaker 3 spotting device. This product is said to represent an advanced version of earlier micro-spotting technology, employing 48 printing pins to deliver as many as 62,000 samples per solid substrate.
The use of such an instrument, in combination with conventional (e.g., poly-1-lysine coated) slides, is well known in the art. See, for instance, US Patent No.
5,087522 (Brown et al.) "Methods for Fabricating Microarrays of Biological Samples", and the references cited therein.
For instance, the method and system of the present invention can be used to provide a substrate, such as a glass slide, with a surface having one or more microarrays. Each microarray preferably provides at least about 100/cm2 (and preferably at least about 1000/cm2) distinct target molecules (e.g., polynucleotide or polypeptide biopolymers) in a surface area of less than about 1 cm'. Each distinct target molecule 1) is disposed at a separate, defined position in the array, 2) has a length of at least 10 subunits, 3) is present in a defined amount between about 0.1 femtomoles and about 10 nanomoles, and 4) is deposited in selected volume in the volume range of about 0.01 nanoliters to about 100 nanoliters. These regions (e.g., discrete spots) within the array can be generally circular in shape, with a typical diameter of between about 10 microns and about 500 microns (and preferably between about 20 and about 200 microns). The regions are also preferably separated from other regions in the array by about the same distance (e.g., center to center spacing of about 20 microns to about 1000 microns) . A plurality of analyte-specific regions can be provided, such that each region includes a single, and preferably different, analyte specific reagent ("target molecule").
Those skilled in the art, given the present description, will be able to identify and select suitable reagents depending on the type of target molecule of interest.
Target molecules include, but are not limited to, plasmid DNA, cosmid DNA, bacteriophage DNA, genomic DNA (includes, but not limited to yeast, viral, bacterial, mammalian, insect), RNA, cDNA, PNA, and oligonucleotides.
A polymeric backbone can be either synthetic or naturally occurring, and is preferably a synthetic polymer selected from the group consisting of oligomers, homopolymers, and copolymers resulting from addition or condensation polymerization. Naturally occurring polymers, such as polysaccharides, polypeptides can be used as well. Preferred backbones are biologically inert, in that they do not provide a biological function that is inconsistent with, or detrimental to, their use in the manner described.
Such polymer backbones can include acrylics such as those polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate, acrylamide and methacrylamide, vinyls such as polyvinyl pyrrolidone and polyvinyl alcohol, nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide and polyhexamethylene dodecanediamide, polyurethanes and polyethers (e.g., polyethylene oxides).
The polymeric backbones of the invention are chosen to provide hydrophilic backbones capable of bearing the desired number and type of thermochemically reactive groups, and optionally photogroups, the combination dependent upon the reagent selected. The polymeric backbone is also selected to provide a spacer between the surface and the thennochemically reactive groups. In this manner, the reagent can be bonded to a surface or to an adjacent reagent molecule, to provide the other groups with sufficient freedom of movement to demonstrate optimal activity. The polymer backbones are preferably hydrophilic (e.g., water soluble), with polyacrylarnide and polyvinylpyrrolidone being particularly preferred polymers.
Reagents of the invention carry one or more pendent latent reactive (preferably photoreactive) groups covalently bound (directly or indirectly) to the polymer backbone.
Photoreactive groups are defined herein, and preferred groups are sufficiently stable to be stored under conditions in which they retain such properties. See, e.g., U.S. Patent No. 5,002,582, Latent Photoreactive groups respond to specific applied external stimuli to undergo active specie generation with resultant covalent bonding to an adjacent chemical The photoreactive groups generate active species such as free radicals and 25 particularly nitrenes, carbenes, and excited states of ketones upon absorption of electromagnetic energy. Photoreactive groups may be chosen to be responsive to various portions of the electromagnetic spectrum, and photoreactive groups that are responsive to e.g., ultraviolet and visible portions of the spectrum are preferred and may be referred to herein occasionally as "photochemical group" or "photogroup".
Photoreactive aryl ketones are preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles (i.e., heterocyclic analogs of anthrone such as those having N, 0, or S in the 10- position), or their substituted (e.g., ring substituted) derivatives. The functional groups of such ketones are preferred since they are readily capable of undergoing the activation/inactivation/reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive moiety, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes intersystem crossing to the triplet state. The excited triplet state can insert into carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support surface, for example), thus creating a radical pair. Subsequent collapse of the radical pair leads to formation of a new carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is not available for bonding, the ultraviolet light-induced excitation of the benzophenone group is reversible and the molecule returns to ground state energy level upon removal of the energy source. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency.
Hence, photoreactive aryl ketones are particularly preferred.
The azides constitute a preferred class of photoreactive groups and include arylazides (C6R5N3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides (-CO-N3) such as benzoyl azide and p-methylbenzoyl azide, azido formates (-0-00-1\13) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (-S02-N3) such as benzenesulfonyl azide, and phosphoryl azides (R0)2P0N3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. Diazo compounds constitute another class of photoreactive groups and include diazoalkanes (-CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (-00-CHN2) such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (-0-CO-CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetates (-CO-0-) such as t-butyl alpha diazoacetoacetate. Other photoreactive groups include the diazirines (-CHN2) such as 3-trifluoromethy1-3-phenyldiazirine, and ketenes (-CH=C=0) such as ketene and diphenylketene. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency.
Upon activation of the photoreactive groups, the reagent molecules are covalently bound to each other and/or to the material surface by covalent bonds through residues of the photoreactive groups. Exemplary photoreactive groups, and their residues upon activation, are shown as follows.
Photoreactive Group Residue Functionality aryl azides amine R-NH-R' acyl azides amide R-CO-NH-R' azidoformates carbamate R-O-CO-NH-R' sulfonyl azides sulfonamide R-S02-NH-R' phosphoryl azides phosphoramide (R0)2P0-NH-R' diazoalkanes new C-C bond diazoketones new C-C bond and ketone diazoacetates new C-C bond and ester beta-keto-alpha-diazoacetates new C-C bond and beta-ketoester aliphatic azo new C-C bond diazirines new C-C bond ketenes new C-C bond photoactivated ketones new C-C bond and alcohol Those skilled in the art, given the present description, will be able to identify and select suitable thermochemically reactive groups to provide for covalent immobilization of appropriately derivatized nucleic acid sequences. For example, an amino derivatized nucleic acid sequence will undergo a covalent coupling reaction with an activated ester such as a NOS ester to provide an amide linking group.

Similar activated esters such p-nitrophenyl and pentafluorophenyl esters would also provide amide links when reacted with amine groups. Those skilled in the art would also recognize numerous other amine-reactive functional groups such as isocyanates, thioisocyanates, carboxylic acid chlorides, epoxides, aldehydes, alkyl halides and sulfonate esters, such as mesylate, tosylate and tresylate, each of which could serve as the thermochemically reactive group.
In another example, the nucleic acid sequence can be derivatized with a sulfhydryl group using techniques well known in the art. The corresponding thermochemically reactive group would be, for example, a maleimide ring structure or an a-iodoacetamide. Either of these structures would react readily to provide a covalent linkage with the sulfhydryl derivatized nucleic acid sequence.
The functionalized polymers of this invention can be prepared by appropriate derivatization of a preformed polymer or, more preferably, by polymerization of a set of comonomers to give the desired substitution pattern. The latter approach is preferred because of the ease of changing the ratio of the various comonomers and by the ability to control the level of incorporation into the polymer. A
combination of these two approaches can also be used to provide optimal structures.
In a preferred embodiment, for instance, monomers are prepared having a polymerizable group at one end of the molecule, separated by a spacer group from a photoreactive or thermochemically reactive group at the other end. For example, polymerizable vinyl groups such as acrylamides, acrylates, or maleimides can be coupled through a short hydrocarbon spacer to an activated ester such as a NOS
ester or to a photoreactive group such as a substituted benzophenone. These compounds can be prepared and purified using organic synthesis techniques well known to those skilled in the art. Some of desired monomers are commercially available, such as MAPTAC, N{3-(dimethylamino)propyl]methacrylamide (DMAPMA), and N-(3-aminopropyl)methacrylamide hydrochloride (APMA), these compounds providing quaternary ammonium salts, tertiary amines, and primary amines respectively along the backbone of the polymer.
Polymers and copolymers can be prepared from the above monomers as well, using techniques known to those skilled in the art. Preferably, these monomers and copolymers undergo free radical polymerization of vinyl groups using azo initiators such as 2,2'-azobisisobutyronitrile (AIBN) or peroxides such as benzoyl peroxide.
The monomers selected for the polymerization are chosen based on the nature of the final polymer product. For example, a photoreactive polymer containing a NOS
group is prepared from a monomer containing the photoreactive group and a second monomer containing the activated NOS ester.
The composition of the final polymer can be controlled by mole ratio of the monomers charged to the polymerization reaction. Typically these functionalized monomers are used at relatively low mole percentages of the total monomer content of the polymerization reaction with the remainder of the composition consisting of a monomer which is neither photoreactive nor thermochemically reactive toward the nucleic acid sequence. Examples of such monomers include, but are not limited to, acrylamide and N-vinylpyrrolidone. Based on the relative reactivities of the monomers used, the distribution of the monomers along the backbone is largely random.
In some cases, the thermochemically reactive group on the backbone of the polymer can itself act as a polymerizable monomer, if present during polymerization, thus requiring the introduction of that group in a second step following the initial formation of the polymer. For example, the preparation of a photoreactive polymer having maleimide along the backbone can be accomplished by an initial preparation of a polymer containing both photoreactive groups and amine groups using the techniques described above, followed by reaction of the amine groups with a heterobifunctional molecule containing a maleimide group and an isocyanate connected by a short hydrocarbon spacer. A wide variety of such polymer modification techniques are available using typical organic reactions known to those skilled in the art.
The invention will be further described with reference to the following non-limiting Examples. It will be apparent to those skilled in the art that many changes can be made in the embodiments described without departing from the scope of the present invention. Thus the scope of the present invention should not be limited to the embodiments described in this application, but only by embodiments described by the language of the claims and the equivalents of those embodiments. Unless otherwise indicated, all percentages are by weight. Structures of the various "Compounds"
identified throughout these Examples can be found in Table 13 included below.
NMR
analyses were run on a 80 Mhz spectrometer unless otherwise stated.
EXAMPLES
Example 1 Preparation of 4-Benzoylbenzoyl Chloride (BBA-C1) (Compound I) 4-Benzoylbenzoic acid (BBA), 1.0 kg (4.42 moles), was added to a dry 5 liter Morton flask equipped with reflux condenser and overhead stirrer, followed by the addition of 645 ml (8.84 moles) of thionyl chloride and 725 ml of toluene.
Dimethylformamide, 3.5 ml, was then added and the mixture was heated at reflux for 4 hours. After cooling, the solvents were removed under reduced pressure and the residual thionyl chloride was removed by three evaporations using 3 x 500 ml of toluene. The product was recrystallized from 1:4 toluene : hexane to give 988 g (91 % yield) after drying in a vacuum oven. Product melting point was 92-94 C.
Nuclear magnetic resonance (NMR) analysis at 80 MHz ('H NMR (CDC13)) was consistent with the desired product: aromatic protons 7.20-8.25 (m, 9H). All chemical shift values are in ppm downfield from a tetramethylsilane internal standard. The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for instance, in Example 3.
Example 2 Preparation of N-(3-Aminopropyl)methacrylamide Hydrochloride (APMA) (Compound II) A solution of 1,3-diaminopropane, 1910 g (25.77 moles), in 1000 ml of CH2C12 was added to a 12 liter Morton flask and cooled on an ice bath. A
solution of t-butyl phenyl carbonate, 1000 g (5.15 moles), in 250 ml of CH2C12 was then added dropwise at a rate which kept the reaction temperature below 15 C. Following the addition, the mixture was warmed to room temperature and stirred 2 hours. The reaction mixture was diluted with 900 ml of CH2C12 and 500 g of ice, followed by the slow addition of 2500 ml of 2.2 N NaOH. After testing to insure the solution was basic, the product was transferred to a separatory funnel and the organic layer was removed and set aside as extract #1. The aqueous was then extracted with 3 X
ml of CH2C12, keeping each extraction as a separate fraction. The four organic extracts were then washed successively with a single 1250 ml portion of 0.6 N
NaOH
beginning with fraction #1 and proceeding through fraction #4. This wash procedure was repeated a second time with a fresh 1250 ml portion of 0.6 N NaOH. The organic extracts were then combined and dried over Na2SO4. Filtration and evaporation of solvent to a constant weight gave 825 g of N-mono-t-B0C-1,3-diaminopropane which was used without further purification.
A solution of methacrylic anhydride, 806 g (5.23 moles), in 1020 ml of CHC13 was placed in a 12 liter Morton flask equipped with overhead stirrer and cooled on an ice bath. Phenothiazine, 60 mg, was added as an inhibitor, followed by the dropwise addition of N-mono-t-B0C-1,3-diaminopropane, 825 g (4.73 moles), in 825 ml of CHC13. The rate of addition was controlled to keep the reaction temperature below 10 C at all times. After the addition was complete, the ice bath was removed and the mixture was left to stir overnight. The product was diluted with 2400 ml of water and transferred to a separatory funnel. After thorough mixing, the aqueous layer was removed and the organic layer was washed with 2400 ml of 2 N NaOH, insuring that the aqueous layer was basic. The organic layer was then dried over Na2SO4 and filtered to remove drying agent. A portion of the CHC13 solvent was removed under reduced pressure until the combined weight of the product and solvent was approximately 3000 g. The desired product was then precipitated by slow addition of 11.0 liters of hexane to the stirred CHC13 solution, followed by overnight storage at 4 C. The product was isolated by filtration and the solid was rinsed twice with a solvent combination of 900 ml of hexane and 150 ml of CHC13. Thorough drying of the solid gave 900 g of N-[1\l'-(t-butyloxycarbony1)-3-aminopropyl]-methacrylamide, m.p. 85.8 C by DSC. Analysis on an NMR spectrometer was consistent with the desired product: 1H NMR (CDC13) amide NH's 6.30-6.80, 4.55-5.10 (m, 2H), vinyl protons 5.65, 5.20 (m, 2H), methylenes adjacent to N 2.90-3.45 (m, 4H), methyl 1.95 (m, 3H), remaining methylene 1.50-1.90 (m, 2H), and t-butyl 1.40 (s, 9H).
A 3-neck, 2 liter round bottom flask was equipped with an overhead stirrer and gas sparge tube. Methanol, 700 ml, was added to the flask and cooled on an ice bath.
While stirring, HC1 gas was bubbled into the solvent at a rate of approximately 5 liters/minute for a total of 40 minutes. The molarity of the final HC1/Me0H
solution was determined to be 8.5 M by titration with 1 N NaOH using phenolphthalein as an indicator. The N-[N'-(t-butyloxycarbony1)-3-aminopropyl]methacrylamide, 900 g (3.71 moles), was added to a 5 liter Morton flask equipped with an overhead stirrer and gas outlet adapter, followed by the addition of 1150 ml of methanol solvent.
Some solids remained in the flask with this solvent volume. Phenothiazine, 30 mg, was added as an inhibitor, followed by the addition of 655 ml (5.57 moles) of the 8.5 M HC1IMe0H solution. The solids slowly dissolved with the evolution of gas but the reaction was not exothermic. The mixture was stirred overnight at room temperature to insure complete reaction. Any solids were then removed by filtration and an additional 30 mg of phenothiazine were added. The solvent was then stripped under reduced pressure and the resulting solid residue was azeotroped with 3 X 1000 ml of isopropanol with evaporation under reduced pressure. Finally, the product was dissolved in 2000 ml of refluxing isopropanol and 4000 ml of ethyl acetate were added slowly with stirring. The mixture was allowed to cool slowly and was stored at 4 C overnight. Compound II was isolated by filtration and was dried to constant weight, giving a yield of 630 g with a melting point of 124.7 C by DSC.
Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (D20) vinyl protons 5.60, 5.30 (m, 2H), methylene adjacent to amide N 3.30 (t, 2H), methylene adjacent to amine N 2.95 (t, 2H), methyl 1.90 (m, 3H), and remaining methylene 1.65-2.10 (m, 2H). The final compound was stored for use in the preparation of a monomer used in the synthesis of photoactivatable polymers as described, for instance, in Example 3.
Example 3 Preparation of N-P-(4-Benzoylbenzamido)propyllmethacrylamide (BBA-APMA) (Compound III) Compound 11 120 g (0.672 moles), prepared according to the general method described in Example 2, was added to a dry 2 liter, three-neck round bottom flask equipped with an overhead stirrer. Phenothiazine, 23-25 mg, was added as an inhibitor, followed by 800 ml of chloroform. The suspension was cooled below on an ice bath and 172.5 g (0.705 moles) of Compound I, prepared according to the general method described in Example 1, were added as a solid. Triethylamine, 207 ml (1.485 moles), in 50 ml of chloroform was then added dropwise over a 1-1.5 hour time period. The ice bath was removed and stirring at ambient temperature was continued for 2.5 hours. The product was then washed with 600 ml of 0.3 N HC1 and 2 x 300 ml of 0.07 N HC1. After drying over sodium sulfate, the chloroform was removed under reduced pressure and the product was recrystallized twice from 4:1 toluene : chloroform using 23-25 mg of phenothiazine in each recrystallization to prevent polymerization. Typical yields of Compound III were 90% with a melting point of 147-151 C. Analysis on an NMR spectrometer was consistent with the desired product: '1-1 NMR (CDC13) aromatic protons 7.20-7.95 (m, 9H), amide NH

6.55 (broad t, 1H), vinyl protons 5.65, 5.25 (m, 2H), methylene adjacent to amide N's 3.20-3.60 (m, 4H), methyl 1.95 (s, 3H), and remaining methylene 1.50-2.00 (m, 2H).
The final compound was stored for use in the synthesis of photoactivatable polymers as described, for instance, in Examples 9-11.
Example 4 Preparation of N-Succinimidyl 6-Maleimidohexanoate (MAL-EAC-NOS) (Compound IV) A functionalized monomer was prepared in the following manner, and was used as described in Examples 9 and 12 to introduce activated ester groups on the backbone of a polymer. 6-Aminohexanoic acid, 100 g (0.762 moles), was dissolved in 300 ml of acetic acid in a three-neck, 3 liter flask equipped with an overhead stirrer and drying tube. Maleic anhydride, 78.5 g (0.801 moles), was dissolved in 200 ml of acetic acid and added to the 6-aminohexanoic acid solution. The mixture was stirred one hour while heating on a boiling water bath, resulting in the formation of a white solid. After cooling overnight at room temperature, the solid was collected by filtration and rinsed with 2 x 50 ml of hexane. After drying, the typical yield of the (Z)-4-oxo-5-aza-2-undecendioic acid was 158-165 g (90-95%) with a melting point of 160-165 C. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (DMSO-d,) amide proton 8.65-9.05 (m, 1H), vinyl protons 6.10, 6.30 (d, 2H), methylene adjacent to nitrogen 2.85-3.25 (m, 2H), methylene adjacent to carbonyl 2.15 (t, 2H), and remaining methylenes 1.00-1.75 (m, 6H).
(Z)-4-0xo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), acetic anhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, were added to a 2 liter three-neck round bottom flask equipped with an overhead stirrer.
Triethylamine, 91 ml (0.653 moles), and 600 ml of THF were added and the mixture was heated to reflux while stirring. After a total of 4 hours of reflux, the dark mixture was cooled to <60 C and poured into a solution of 250 ml of 12 N HC1 in 3 liters of water.
The mixture was stirred 3 hours at room temperature and then was filtered through a filtration pad (Celite 545, J.T. Baker, Jackson, TN) to remove solids. The filtrate was extracted with 4 x 500 ml of chloroform and the combined extracts were dried over sodium sulfate. After adding 15 mg of phenothiazine to prevent polymerization, the solvent was removed under reduced pressure. The 6-maleimidohexanoic acid was recrystallized from 2:1 hexane : chloroform to give typical yields of 76-83 g (55-60%) with a melting point of 81-85 C. Analysis on a NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) maleimide protons 6.55 (s, 2H), methylene adjacent to nitrogen 3.40 (t, 2H), methylene adjacent to carbonyl 2.30 (t, 2H), and remaining methylenes 1.05-1.85 (m, 6H).
The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100 ml of chloroform under an argon atmosphere, followed by the addition of 41 ml (0.47 mol) of oxalyl chloride. After stirring for 2 hours at room temperature, the solvent was removed under reduced pressure with 4 x 25 ml of additional chloroform used to remove the last of the excess oxalyl chloride. The acid chloride was dissolved in 100 ml of chloroform, followed by the addition of 12 g (0.104 mol) of N-hydroxysuccinimide and 16 ml (0.114 mol) of triethylamine. After stirring overnight at room temperature, the product was washed with 4 x 100 ml of water and dried over sodium sulfate. Removal of solvent gave 24 g of product (82%) which was used without further purification. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) maleimide protons 6.60 (s, 2H), methylene adjacent to nitrogen 3.45 (t, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), and remaining methylenes 1.15-2.00 (m, 6H).
The final compound was stored for use in the synthesis of photoactivatable polymers as described, for instance, in Examples 9 and 12.
Example 5 Preparation of N-Succinimidyl 6-Methacrylamidohexanoate (MA-EAC-NOS) (Compound V) A functionalized monomer was prepared in the following manner, and was used as described in Example 11 to introduce activated ester groups on the backbone of a polymer. 6-Aminocaproic acid, 4.00 g (30.5 mmol), was placed in a dry round bottom flask equipped with a drying tube. Methacrylic anhydride, 5.16 g ( 33.5 mmol), was then added and the mixture was stirred at room temperature for four hours. The resulting thick oil was triturated three times with hexane and the remaining oil was dissolved in chloroform, followed by drying over sodium sulfate.
After filtration and evaporation, a portion of the product was purified by silica gel flash chromatography using a 10% methanol in chloroform solvent system. The appropriate fractions were combined, 1 mg of phenothiazine was added, and the solvent was removed under reduced pressure. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) carboxylic acid proton 7.80-8.20 (b, 1H), amide proton 5.80-6.25 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.00-3.45 (m, 2H), methylene adjacent to carbonyl 2.30 (t, 2H), methyl group 1.95 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H).
6-Methacrylamidohexanoic acid, 3.03 g (15.2 mmol), was dissolved in 30 ml of dry chloroform, followed by the addition of 1.92 g (16.7 mmol) of N-hydroxysuccinimide and 6.26 g (30.4 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction was stirred under a dry atmosphere overnight at room temperature. The solid was then removed by filtration and a portion was purified by silica gel flash chromatography. Non-polar impurities were removed using a chloroform solvent, followed by elution of the desired product using a 10% tetrahydrofuran in chloroform solvent. The appropriate fractions were pooled, 0.2 mg of phenothiazine were added, and the solvent was evaporated under reduced pressure. This product, containing small amounts of 1,3-dicyclohexylurea as an impurity, was used without further purification. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) amide proton 5.60-6.10 (b, 1H), vinyl protons 5.20 and 5.50 (m, 2H), methylene adjacent to nitrogen 3.05-3.40 (m, 2H), succinimidyl protons 2.80 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), methyl 1.90 (m, 3H), and remaining methylenes 1.10-1.90 (m, 6H). The final compound was stored for use in the synthesis of photoactivatable polymers as described, for instance, in Example 11.
Example 6 Preparation of 4-Bromomethylbenzophenone (BMBP)(Compound VI) 4-Methylbenzophenone, 750 g (3.82 moles), was added to a 5 liter Morton flask equipped with an overhead stirrer and dissolved in 2850 ml of benzene.
The solution was then heated to reflux, followed by the dropwise addition of 610 g (3.82 moles) of bromine in 330 ml of benzene. The addition rate was approximately 1.5 ml/min and the flask was illuminated with a 90 watt (90 joule/sec) halogen spotlight to initiate the reaction. A timer was used with the lamp to provide a 10% duty cycle (on 5 seconds, off 40 seconds), followed in one hour by a 20% duty cycle (on seconds, off 40 seconds). At the end of the addition, the product was analyzed by gas chromatography and was found to contain 71% of the desired Compound VI, 8% of the dibromo product, and 20% unreacted 4-methylbenzophenone. After cooling, the reaction mixture was washed with 10 g of sodium bisulfite in 100 ml of water, followed by washing with 3 x 200 ml of water. The product was dried over sodium sulfate and recrystallized twice from 1:3 toluene : hexane. After drying under vacuum, 635 g of Compound VI were isolated, providing a yield of 60% and having a melting point of 112-114 C. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H) and benzylic protons 4.48 (s, 2H). The final compound was stored for use in the preparation of a photoactivatable chain transfer agent as described in Example 7.
Example 7 Preparation of N-(2-Mercaptoethyl)-3.5-bis(4-benzoylbenzyloxy)benzamide (Compound VII) 3,5-Dihydroxybenzoic acid, 46.2 g (0.30 mol), was weighed into a 250 ml flask equipped with a Soxhlet extractor and condenser. Methanol, 48.6 ml, and concentrated sulfuric acid, 0.8 ml, were added to the flask and 48 g of 3A
molecular sieves were placed in the Soxhlet extractor. The extractor was filled with methanol and the mixture was heated at reflux overnight. Gas chromatographic analysis of the resulting product showed a 98% conversion to the desired methyl ester. The solvent was removed under reduced pressure to give approximately 59 g of crude product.
The product was used in the following step without further purification. A
small sample was previously purified for NMR analysis, resulting in a spectrum consistent with the desired product: 'El NMR (DMSO-d6) aromatic protons 6.75 (d, 2H) and 6.38 (t, 1H), and methyl ester 3.75 (s, 3H).
The entire methyl ester product from above was placed in a 2 liter flask with an overhead stirrer and condenser, followed by the addition of 173.25 g (0.63 mol) of Compound VI, prepared according to the general method described in Example 6, g (1.50 mol) of potassium carbonate, and 1200 ml of acetone. The resulting mixture was then refluxed overnight to give complete reaction as indicated by thin layer chromatography (TLC). The solids were removed by filtration and the acetone was evaporated under reduced pressure to give 49 g of crude product. The solids were diluted with 1 liter of water and extracted with 3 x 1 liter of chloroform.
The extracts were combined with the acetone soluble fraction and dried over sodium sulfate, yielding 177 g of crude product. The product was recrystallized from acetonitrile to give 150.2 g of a white solid, a 90% yield for the first two steps. Melting point of the product was 131.5 C (DSC) and analysis on an NMR spectrometer was consistent with the desired product: '1-1 NMR (CDC13) aromatic protons 7.25-7.80 (m, 18H), 7.15 (d, 2H), and 6.70 (t, 1H), benzylic protons 5.05 (s, 4H), and methyl ester 3.85 (s, 3H).
The methyl 3,5-bis(4-benzoylbenzyloxy)benzoate, 60.05 g (0.108 mol), was placed in a 2 liter flask, followed by the addition of 120 ml of water, 480 ml of methanol, and 6.48 g (0.162 mol) of sodium hydroxide. The mixture was heated at reflux for three hours to complete hydrolysis of the ester. After cooling, the methanol was removed under reduced pressure and the sodium salt of the acid was dissolved in 2400 ml of warm water. The acid was precipitated using concentrated hydrochloric acid, filtered, washed with water, and dried in a vacuum oven to give 58.2 g of a white solid (99% yield). Melting point on the product was 188.3 C (DSC) and analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (DMSO-d6) aromatic protons 7.30-7.80 (m, 18H), 7.15 (d, 2H), and 6.90 (t, 1H), and benzylic protons 5.22 (s, 4H).
The 3,5-bis(4-benzoylbenzyloxy)benzoic acid, 20.0 g (36.86 mmol), was added to a 250 ml flask, followed by 36 ml of toluene, 5.4 ml (74.0 mmol) of thionyl chloride, and 281.11 of N,N-dimethylformamide. The mixture was refluxed for four hours to form the acid chloride. After cooling, the solvent and excess thionyl chloride were removed under reduced pressure. Residual thionyl chloride was removed by four additional evaporations using 20 ml of chloroform each. The crude material was recrystallized from toluene to give 18.45 g of product, an 89% yield. Melting point on the product was 126.9 C (DSC) and analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) aromatic protons 7.30-7.80 (m, 18H), 7.25 (d, 2H), and 6.85 (t, 1H), and benzylic protons 5.10 (s, 4H).
The 2-aminoethanethiol hydrochloride, 4.19 g (36.7 mmol), was added to a 250 ml flask equipped with an overhead stirrer, followed by 15 ml of chloroform and 10.64 ml (76.5 mmol) of triethylamine. After cooling the amine solution on an ice bath, a solution of 3,5-bis(4-benzoylbenzyloxy)benzoyl chloride, 18.4 g (32.8 mmol), in 50 ml of chloroform was added dropwise over a 50 minute period. Cooling on ice was continued 30 minutes, followed by warming to room temperature for two hours.
The product was diluted with 150 ml of chloroform and washed with 5 x 250 ml of 0.1 N hydrochloric acid. The product was dried over sodium sulfate and recrystallized twice from 15:1 toluene : hexane to give 13.3 g of product, a 67% yield.
Melting point on the product was 115.9 C (DSC) and analysis on an NMR spectrometer was consistent with the desired product.: 'H NMR (DMSO-d6) aromatic protons 7.20-7.80 (m, 18H), 6.98 (d, 2H), and 6.65 (t, 1H), amide NET 6.55 (broad t, 1H), benzylic protons 5.10 (s, 4H), methylene adjacent to amide N 3.52 (q, 2H), methylene adjacent to SH 2.10 (q, 2H), and SH 1.38 (t, 1H). The final compound was stored for use as a chain transfer agent in the synthesis of photoactivatable polymers as described, for instance, in Example 12.
Example 8 Preparation of N-Succinimidyl 11-(4-Benzoylbenzamido)undecanoate (BBA-AUD-NOS) (Compound VIII) Compound 1(50 g, 0.204 mol), prepared according to the general method described in Example 1, was dissolved in 2500 ml of chloroform, followed by the addition of a solution of 43.1 g (0.214 mol) of 11-aminoundecanoic acid and 60.0 g (1.5 mol) of sodium hydroxide in 1500 ml of water. The mixture was stirred vigorously for one hour in a 5 liter Morton flask to insure thorough mixing of the two layers. The mixture was acidified with 250 ml of concentrated hydrochloric acid and stirred an additional 30 minutes. The organic layer was separated and the aqueous was extracted with 3 x 500 ml of chloroform. The combined organic extracts were dried over sodium sulfate, filtered, and evaporated to give a solid. The product was recrystallized from toluene to give 68.37 g (82 %) of 11-(4-benzoylbenzamido)undecanoic acid with a melting point of 107-109 C. Analysis on an NMR spectrometer was consistent with the desired product: 'H NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H), amide NH 6.30 (broad t, 1H), methylene adjacent to amide N 3.35 (m, 2H), methylene adjacent to carbonyl 2.25 (t, 2H), and remaining methylenes 1.00-1.80 (m, 16H).
The 11-(4-benzoylbenzamido)undecanoic acid, 60.0 g (0.146 mol), was dissolved with warming in 1200 ml of anhydrous 1,4-dioxane in an oven-dried ml flask. After cooling to room temperature, 17.7 g ( 0.154 mol) of N-hydroxysuccinimide and 33.2 g (0.161 mol) of 1,3-dicyclohexylcarbodiimide were added to the solution and the mixture was stirred overnight under a dry atmosphere.
The solids were then removed by filtration, rinsing the filter cake with 1,4-dioxane.
The solvent was then removed under vacuum and the product was recrystallized twice from ethanol. After thorough drying in a vacuum oven, 53.89 g (73 % yield) of a white solid were obtained with a melting point of 97-99 C. Analysis on an NMR
spectrometer was consistent with the desired product: 'H NMR (CDC13) aromatic protons 7.20-7.80 (m, 9H), amide NH 6.25 (broad t, 1H), methylene adjacent to amide N 3.35 (m, 2H), methylenes on succinimidyl ring 2.75 (s, 4H), methylene adjacent to carbonyl 2.55 (t, 2H), and remaining methylenes 1.00-1.90 (m, 16H).
Example 9 Preparation of Copolymer of Acrylamide. BBA-APMA. and MAL-EAC-NOS
(Random Photo PA-PolyNOS) (Compounds IX. A-D) A photoactivatable copolymer of the present invention was prepared in the following manner. Acrylamide, 4.298 g (60.5 mmol), was dissolved in 57.8 ml of tetrahydrofuran (THF), followed by 0.219 g ( 0.63 mmol) of Compound III, prepared according to the general method described in Example 3, 0.483 g (1.57 mmol) of Compound IV, prepared according to the general method described in Example 4, 0.058 ml (0.39 mmol) of N,N,N',N'-tetramethylethylenediamine (TEMED), and 0.154 g (0.94 mmol) of 2,2'-azobisisobutyronitrile (AIBN). The solution was deoxygenated with a helium sparge for 3 minutes, followed by an argon sparge for an additional 3 minutes. The sealed vessel was then heated overnight at 60 C to complete the polymerization. The solid product was isolated by filtration and the filter cake was rinsed thoroughly with THF and CHC13. The product was dried in a vacuum oven at 30 C to give 5.34 g of a white solid. NMR analysis (DMSO-d6) confirmed the presence of the NOS group at 2.75 ppm and the photogroup load was determined to be 0.118 mmol BBA/g of polymer. The MAL-EAC-NOS composed 2.5 mole % of the polymerizable monomers in this reaction to give Compound IX-A.
The above procedure was used to prepare a polymer having 5 mole %
Compound IV. Acrylamide, 3.849 g (54.1 mmol), was dissolved in 52.9 ml of THF, followed by 0.213 g ( 0.61 mmol) of Compound VI, prepared according to the general method described in Example 3, 0.938 g (3.04 mmol) of Compound IV, prepared according to the general method described in Example 4, 0.053 ml (0.35 mmol) of TEMED and 0.142 g (0.86 mmol) of AIBN. The resulting solid, Compound IX-B, when isolated as described above, gave 4.935 g of product with a photogroup load of 0.101 mmol BBA/g of polymer.
The above procedure was used to prepare a polymer having 10 mole %
Compound IV. Acrylamide, 3.241 g (45.6 mmol), was dissolved in 46.4 ml of THF, followed by 0.179 g ( 0.51 mmol) of Compound III, prepared according to the general method described in Example 3, 1.579 g (5.12 mmol) of Compound IV, prepared according to the general method described in Example 4, 0.047 ml (0.31 mmol) of TEMED and 0.126 g (0.77 mmol) of AIBN. The resulting solid, Compound IX-C, when isolated as described above, gave 4.758 g of product with a photogroup load of 0.098 mmol BBA/g of polymer.
A procedure similar to the above procedure was used to prepare a polymer having 2.5 mole % Compound IV and 2 mole % Compound III. Acrylamide, 16.43 g (231.5 mmol); Compound III, prepared according to the general method described in Example 3, 1.70 g (4.85 mmol); Compound IV, prepared according to the general method described in Example 4, 1.87 g (6.06 mmol); and THF (222 ml) were stirred in a round bottom flask with an argon sparge at room temperature for 15 minutes.
TEMED, 0.24 ml (2.14 mmol), and AIBN, 0.58 g (3.51 mmol), were added to the reaction. The reaction was then refluxed for 4 hours under an atmosphere of argon.
The resulting solid, Compound IX-D, when isolated as described above, gave 19.4 g of product with a photogroup load of 0.23 mmol BBAJg of polymer.
Example 10 Preparation of Copolymer of Acrylamide. BBA-APMA, and [3-(Methacryloylamino)propyl]trimethylammonium Chloride (Random Photo PA-PolyQuat) (Compounds X. A-B) A photoactivatable copolymer of the present invention was prepared in the following manner. Acrylamide, 10.681 g (0.150 mol), was dissolved in 150 ml of dimethylsulfoxide (DMSO), followed by 0.592 g (1.69 mmol) of Compound III, prepared according to the general method described in Example 3, 3.727 g (16.90 mmol) of [3-(methacryloylamino)propyl]trimethylammonium chloride (MAPTAC), delivered as 7.08 ml of a 50% aqueous solution, 0.169 ml (1.12 mmol) of TEMED
and 0.333 g (2.03 mmol) of AIBN. The solution was deoxygenated with a helium sparge for 4 minutes, followed by an argon sparge for an additional 4 minutes.
The sealed vessel was then heated overnight at 55 C to complete the polymerization. The DMSO solution was diluted with water and dialyzed against deionized water using 12,000-14,000 molecular weight cutoff tubing. Lyophilization of the resulting solution gave 14.21 g of a white solid. NMR analysis (D,O) confirmed the presence of the methyl groups on the quaternary ammonium groups at 3.10 ppm and the photogroup load was determined to be 0.101 mmol BBA/g of polymer. The Compound III constituted 1 mole % of the polymerizable monomer in this reaction to give Compound X-A.
The above procedure was used to prepare a polymer having 2 mole % of Compound III. Acrylamide, 10.237 g (0.144 mol), was dissolved in 145 ml of DMSO, followed by 1.148 g (3.277 mmol) of Compound III, prepared according to the general method described in Example 3, 3.807 g (17.24 mmol) of MAPTAC, delivered as 7.23 ml of a 50% aqueous solution, 0.164 ml (1.09 mmol) of TEMED
and 0.322 g (1.96 mmol) of AIBN. Workup as described above gave 12.54 g of product (Compound X-B) with a photogroup load of 0.176 mmol BBA/g of polymer.
Example 11 Preparation of Copolymer of Acrylamide, BBA-APMA. MA-EAC-NOS. and [3-(Methacryloylamino)propyl]trimethylammonium Chloride (Random Photo PA-PolyNOS-Poly Quat) (Compound XI) A photoactivatable copolymer of the present invention was prepared in the following manner. The water in the commercially available 50% aqueous MAPTAC
was removed by azeotropic distillation with chloroform. The aqueous MAPTAC
solution, 20 ml containing 10.88 g of MAPTAC, was diluted with 20 ml of DMSO
and 100 ml of chloroform. This mixture was refluxed into a heavier-than-water liquid-liquid extractor containing anhydrous sodium sulfate for a total of 80 minutes.
A slow flow of air was maintained during the reflux to inhibit polymerization of the monomer. At the end of the reflux, the excess chloroform was removed under reduced pressure to leave a DMSO solution of MAPTAC at an approximate concentration of 352 mg/ml.
Acrylamide, 1.7 g (23.90 mmol), was dissolved in 57.7 ml of dimethylsulfoxide (DMSO), followed by 0.215 g (0.614 mmol) of Compound III, prepared according to the general method described in Example 3, 1.93 ml (0.677 g, 3.067 mmol) of the above MAPTAC/DMSO solution, 0.91 g (3.068 mmol) of Compound V, prepared according to the general method described in Example 5, and 0.060 g (0.365 mmol) of AIBN. The solution was deoxygenated with a helium sparge for 4 minutes, followed by an argon sparge for an additional 4 minutes. The sealed vessel was then heated overnight at 55 C to complete the polymerization. The polymer was isolated by pouring the reaction mixture into 600 ml of diethyl ether.
The solids were separated by centrifuging and the product was washed with 200 ml of diethyl ether and 200 ml of chloroform. Evaporation of solvent under vacuum gave 3.278 g of product with a photoload of 0.185 mmol BBA/g of polymer.
Example 12 Copolymer of Acrylamide and MAL-EAC-NOS using N-(2-Mercaptoethyl)-3,5-bis(4-benzoylbenzyloxy)benzamide (End-point Diphoto PA-PolyNOS) (Compound XII) A photoactivatable copolymer of the present invention was prepared in the following manner. Acrylamide, 3.16 g (44.5 mmol), was dissolved in 45.0 ml of tetrahydrofuran, followed by 0.164 g (1 mmol) of AIBN, 0.045 ml (0.30 mmol) of TEMED, 0.301 g (0.5 mmol) of Compound VII, prepared according to the general method in Example 7, and 1.539 g (5 mmol) of Compound IV, prepared according to the general method described in Example 4. The solution was deoxygenated with a helium sparge for 4 minutes, followed by an argon sparge for an additional 4 minutes.
The sealed vessel was then heated overnight at 55 C to complete the polymerization.
The precipitated polymer was isolated by filtration and was washed with chloroform.
The final product was dried in a vacuum oven to provide 4.727 g of polymer having a photogroup load of 0.011 mmol BBA/g of polymer.
Example 13 Copolymer of N-[3-(Dimethylamino)propyl]methacrylamide and BBA-APMA
(Random Photo Poly Tertiary Amine) (Compound XIII) A photoactivatable copolymer of the present invention was prepared in the following manner. N[3-(Dimethylamino)propyl]methacrylamide, 33.93 g (0.2 mol), was dissolved in 273 ml of DMSO, followed by 16.6 ml of concentrated HC1 and 6.071 g (17.3 mmol) of Compound III, prepared according to the general method described in Example 3. Finally, 0.29 ml (1.93 mmol) of TEMED, 0.426 g (2.6 mmol) of AIBN, and 100 ml of water were added to the reaction mixture. The solution was deoxygenated with a helium sparge for 10 minutes and the head space was then filled with argon. The sealed vessel was heated overnight at 55 C to complete the polymerization. The product was then dialyzed against deionized water for several days using 12,000-14,000 MWCO tubing. The product was filtered following dialysis to remove any solids and was lyophilized to give 47.27 g of a solid product.
The polymer was determined to have a photoload of 0.33 mmol BBA/g of polymer.
Example 14 Preparation of N-succinimidyl 5-oxo-6-aza-8-nonenoate (Allyl-GLU-NOS) (Compound XIV) A functional monomer was prepared in the following manner, and was used in Example 15 to introduce activated ester groups on the backbone of the polymer.
Glutaric anhydride, 20 g (0.175 mole),was dissolved in 100 ml chloroform. The glutaric anhydride solution was cooled to < 10 C using an ice bath. Allyl amine, 10 g (0.177 mole), was dissolved in 50 ml chloroform and added to the cooled solution of glutaric anhydride with stirring. The addition rate of ally! amine was adjusted to keep the reaction temperature < 10 C. After the allyl amine addition was completed, the reaction solution was allowed to come to room temperature while stirring overnight.
After removing the solvent, the 5-oxo-6-aza-8-nonenoic acid isolated amounted to 31.4g (105% crude) with a dual DSC melting point of 35.1 C and 44.9 C. NMR
analysis at 300 MHz was consistent with the desired product: 1H NMR (CDC13) amide proton 6.19 (b, 1H), vinyl protons 5.13, 5.81 (m, 3H), methylene adjacent to amide N
3.85 (m, 2H), methylenes adjacent to carbonyls 2.29, 2.39 (t, 4H), and central methylene 1.9. (m, 2H).
The 5-oxo-6-aza-8-nonenoic acid, 20.54g (0.12 mole), N-hydroxysuccinimide (NHS), 15.19 g (0.13 mole), and 204 ml dioxane were placed in a 1 L 3-necked round bottom flask equipped with an overhead stirrer and an addition funnel.
Dicyclohexylcarbodiimide ("DCC"), 29.7 g (0.144 mole), was dissolved in 50 ml dioxane and placed in the addition funnel. The DCC solution was added with stirring to the acid/NHS solution over 20 minutes, and the resulting mixture was allowed to stir at room temperature overnight. The reaction mixture was filtered on a Biichner funnel to remove dicyclohexylurea (DCU). The solid was washed with 2 x 100 ml dioxane. The solvent was evaporated to give 41.37 g residue, which was washed with 4 x 75 ml hexane. After the solvents were removed, the yield of crude NOS
ester was 41.19 g. One recrystallization of the crude NOS product from toluene gave a 60 %
yield with a DSC melting point of 90.1 C. NMR analysis at 300 MHz was consistent with the desired product: 11-I NMR (CDC13) amide proton 6.02 (b, 1H), vinyl protons 5.13, 5.80 (m, 3H), methylene adjacent to amide N 3.88 (m, 2H), succinimidyl protons 2.83 (s, 4H), methylenes adjacent to carbonyls 2.31, 2.68 (t, 4H), and central methylene 2.08 (m, 2H). The final compound was stored for use in the synthesis of photo activatable polymers as described in Example 15.
Example 15 Preparation of Copolymer of Vinylpyrrolidinone. BBA-APMA, and Allyl-GLU-NOS
(Random Photo PVP-PolyNOS)(Compound XV) A photoactivatable copolymer of the present invention was prepared in the following manner. Vinylpyrrolidinone, 4.30 g (38.73 mmol), was dissolved in 5.2 ml of DMSO along with 0.14 g (0.41 mmol) of Compound III, prepared according to the general method described in Example 3, 0.55 g (2.06 mmol) Compound XIV, prepared according to the general method described in Example14, by combining .08 g (0.49 mmol) of AIBN and 0.005 ml (0.033 mmol) of TEMED. The solution was deoxygenated with a helium sparge for 3 minutes. The head space was replaced with argon, and the vessel was sealed for an overnight heating at 55 C. The viscous solution was diluted with 15 ml chloroform, and then precipitated by pouring into 200 ml diethyl ether. The precipitate was dissolved in 15 ml chloroform, and precipitated a second time in 200 ml ether. The product was dried in a vacuum oven at 30 C
to give 4.79 g of a white solid. NMR analysis (CDCL3) confirmed the presence of the NOS group at 2.81 ppm and the photogroup load was determined to be 1.1 mmol BBA/g of polymer. The Allyl-GLU-NOS composed 5.0 mole % of the polymerizable monomers in this reaction to give Compound XV.
Example 16 Comparison of Random Photo PA-PolyNOS (Compound IX-C) with Random Photo PA-PolyNOS-PolyQuat (Compound XI) on Polystyrene (PS) Microwell Plates Compound IX-C and Compound XI were separately dissolved in deionized water at 5 mg/ml. The PS plates (PS, Medium Bind, Corning Costar, Cambridge, MA) containing 100 IA of Compound IX-C and Compound XI in separate wells were illuminated with a Dymax lamp (model no. PC-2, Dymax Corporation, Torrington, CT) which contained a Heraeus bulb (W.C. Heraeus GmbH, Hanau, Federal Republic of Germany). The illumination duration was for 1.5 minutes at a intensity of 1-2mW/cm2 in the wavelength range of 330-340 nm. The coating solution was then discarded and the wells were air dried for two hours. The plates were then illuminated for an additional one minute. The coated plates were used immediately to immobilize oligonucleotides stored in a sealed pouch for up to 2 months.
The 50 base oligomer (-mer) capture probe 5'-N11,-GTCTGAGTCGGAGCCAGGGCGGCCGCCAACAGCAGGAGCAGCGTGCACGG-3' (ID 1) (synthesized with a 5'-amino-modifier containing a C-12 spacer) at 10 pmoles/well was incubated in PS wells in 50 mM phosphate buffer, pH 8.5, 1 mM EDTA at 37 C
for one hour. The hybridization was performed as follows using the complementary 5'-Biotin-CCGTGCACGCTGCTCCTGCTG11TGGCGGCCGCCCTGGCTCCGACTC AGAC -3' (ID 3) detection probe or non-complementary 5'-Biotin-CGGTGGATGGAGCAGGAGGGGCCC
GAGTATTGGGAGCGGGAGACA CAGAA -3' (ID 4) oligo, both of which were synthesized with a 5'-biotin modification.
The plates with immobilized capture probe were washed with phosphate buffered saline (PBS, 10 mM Na2PO4, 150 mM NaC1, pH 7.2) containing 0.05% Tween 20 using a Microplate Auto Washer (model EL 403H, Bio-Tek Instruments, Winooski, VT). The plates were then blocked at 55 C for 30 minutes with hybridization buffer, which consisted of 5X
SCC (0.75 M NaC1, 0.075 M citrate, pH 7.0), 0.1% lauroylsarcosine, 1% casein, and 0.02%
sodium dodecyl sulfate. When the detection probe was hybridized to the capture probe, 50 frnole of detection probe in 100 1 were added per well and incubated for one hour at 55 C.
The plates were then washed with 2X SSC containing 0.1% sodium dodecyl sulfate for 5 minutes at 55 C. The bound detection probe was assayed by adding 100111 of a conjugate of streptavidin and horseradish peroxidase (SA-HRP, Pierce, Rockford, IL) at 0.5 g/ml and incubating for 30 minutes at 37 C. The plates were then washed with PBS/Tween, followed by the addition of peroxidase substrate (H202 and tetramethylbenzidine, Kirkegard and Perry Laboratories, Gaithersburg, MD) and measurement at 655 nm on a microwell plate reader (model 3550, Bio-Rad Labs, Cambridge, MA). The plates were read at 10 minutes.
The results listed in Table 1 indicate that microwell plates coated with Compound IX-C did not effectively immobilize amine-derivatized capture probes. However, by comparison Compound XI, as a coating, provided significant binding and good hybridization signals.
Compound IX-C reagent most likely passivated the surfaces and prevented the association of capture oligos. In contrast when Compound XI was used, the oligonucleotide was attracted to the surface by ionic interactions where it could then be covalently bonded with the NOS
groups.
Table 1: Hybridization Signals (A655) from PS Microwell Plates Coated with Compound IX-C and Compound XI.
Compound IX-C Compound XI
Complementary 0.187 0.031 1.666 0.064 Detection Probe Non-complementary 0.127 0.016 0.174 0.005 Detection Probe Example 17 Coating of Various Microwell Plates with a Mixture of Random Photo PA-PolyNOS

(Compound IX-B) and Random Photo PA-PolyQuat (Compound X-B) A coating solution containing a mixture of 5 mg/ml of Compound IX-B and 0.5 mg/ml of Compound X-B was prepared in deionized water. This mixture was used to treat polypropylene (PP, Corning Costar, Cambridge, MA), PS, polycarbonate (PC, Corning Costar, Cambridge, MA) and polyvinyl chloride (PVC, Dynatech, Chantilly, VA) multiwells as described in Example 16. A 30-mer capture oligonucleotide5'- NH2-GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC -3' (ID2), (synthesized with a 5'-amino-modifier containing a C-12 spacer) at 0.03, 0.1, 0.3, 1, 3, or 10 pmole/well was incubated at 4 C overnight. The hybridization was performed as previously described in Example 16 using complementary ID 3 detection oligonucleotide or non-complementary ID 4 oligo. Since PP plates are not optically transparent, the contents of each well were transferred to PS wells after a 20 minute incubation with the chromogenic substrate. The hybridization signals were measured in the PS plates. The other plates were read without transferring at 10 minutes.
Signal levels are only comparable within the same substrate group due to the different geometries of microwell plates made from different materials. Table 2 lists the hybridization signals and shows the relationship between the intensity of the hybridization signals and the amount of capture probe applied to various microwell plates coated with a mixture of Compound IX-B and Compound X-B. On PP and PVC plates, adsorption of probes was very low and the coatings with the polymeric reagents improved the signals dramatically. The signal increased with increasing capture probe added to the coated wells, but leveled off at approximately 3 pmole/well capture. The plateau in the amount of signal generated was not due to a saturating level of hybridization, but rather to the limits of the color change reaction in the colorimetric assay.
Oligonucleotide derivatives adsorb efficiently onto uncoated PS and PC
microwell plates and result in specific hybridization signals. Cros et al.
(U.S. Patent No. 5,510,084) also reported that amine-functionalized oligonucleotides adsorbed satisfactorily onto polystyrene microwell plates by unknown mechanisms.
However, there is marked variability in the amount of adsorption on uncoated PS plates among different lots (Chevier et al. FEMS 10:245, 1995).
Table 2: Hybridization Signals (A655) From Various Microwell Plate Materials Coated With a Mixture of Compound IX-B and Compound X-B.
Capture OligonucleotideAdded (pmole/well) 0.03 0.1 0.3 1 3 10 Comp NC Comp NC Comp NC Comp NC Comp NC Comp NC
PP
Uncoated0.083 0.082 0.076 0.072 0.076 0.074 0.088 0.074 0.070 0.067 0.078 0.073 Coated 0.541 0.099 1.070 0.099 1.769 0.091 2.283 0.094 2.582 0.141 2.490 0.320 PVC
Uncoated 0.074 0.079 0.081 0.075 0.097 0.078 0.137 0.076 0.215 0.081 0.337 0.092 Coated 0.423 0.116 0.875 0.110 1.326 0.112 1.583 0.142 1.628 0.186 1.604 0.332 PS
Uncoated0.235 0.099 0.435 0.091 0.827 0.090 1.205 0.093 1.380 0.093 1.404 0.136 Coated 0.435 0.121 0.801 0.105 1.177 0.116 1.401 0.132 1.470 0.132 1.487 0.302 PC
Uncoated0.676 0.248 1.364 0.244 2.103 0.256 2.701 0.266 2.745 0.295 2.930 0.388 Coated 1.034 0.327 1.602 0.306 2.136 0.295 2.218 0.287 2.380 0.342 2.500 0.572 Comp.: Complementary detection probe was added for hybridization.
NC: Non-complementary detection probe was added for hybridization.
Example 18 Evaluation of End-point Diphoto PA-polyNOS (Compound XII) and Random Photo PA-PolyQuat (Compound X-B) on PP and PVC Microwell Plates A coating solution containing a mixture of 5 mg/ml of Compound XII and 0.5 mg/ml of Compound X-B was prepared with deionized water. This mixture of the two reagents was used to coat PP and PVC microwell plates under conditions comparable to those described in Example 16. The 30-mer ID 2 capture oligonucleotide at 0.03, 0.1, 0.3, 1, 3, or 10 pmole/well in 0.1 ml was incubated at 40 C overnight. The hybridization was performed as described in Example 16 using complementary ID 3 detection oligonucleotide or non-complementary ID 4 oligo.
The hybridization signals listed in Table 3 demonstrate the relationship between the intensity of the hybridization signals and the amount of capture probe applied to PP
and PVC microwell plates coated with a mixture of Compound XII and Compound X-B. The signal increased with increasing capture oligonucleotides added to the coated wells, but leveled off at approximately 1 pmole/well. The signal-to-noise ratio (from complementary vs. non-complementary detection probes) was as high as 26 and 11 for coated PP and PVC surfaces, respectively.
Table 3: Hybridization Signals (A655) From PP and PVC Plates Coated With Mixture of Compound XII and Compound X-B.
pmole/well PP Microwell plates PVC Microwell plates Capture Added Comp. Non-comp. Comp. Non-comp.
Detection Detection 0.03 0.153 0.008 0.070 0.007 0.289 0.029 0.094 0.020 0.1 0.537 0.042 0.075 0.009 0.759 0.054 0.104 0.014 0.3 1.206 0.106 0.080 0.003 1.262 0.023 0.117 0.011 1 2.157 0.142 0.081 0.003 1.520 0.044 0.189 0.064 3 2.624 0.162 0.108 0.012 1.571 0.031 0.179 0.016 10 2.921 0.026 0.200 0.018 1.625 0.040 0.286 0.021 Example 19 Sequential Coating with Random Photo PA-PolyQuat (Compound X-B) and BBA-AUD-NO$
(Compound VIII) Compound X-B at 0.1 mg/ml in deionized water was incubated in PP and PVC
wells for 20 minutes. The plates were illuminated as previously described in Example 16 with the solution in the wells for 1.5 minutes. The solution was discarded and the wells were dried. Compound VIII at 0.5 mg/ml in isopropyl alcohol (IPA) was incubated in the Compound X-B coated wells for 5 minutes. The solution was then removed, the plate dried and illuminated as described in Example 16 for one minute after the wells were dried. The 30-mer ID 2 capture oligonucleotide at 0.03, 0.1, 0.3, 1, 3, or 10 pmole/well in 0.1 ml was incubated at 4 C overnight. The hybridization was performed as described in Example 16 using complementary ID 3 detection oligonucleotide or non-complementary ID 4 oligo. Table 4 contains the hybridization signals and shows the relationship between the intensity of the hybridization signals and the amount of capture probe applied to PP and PVC microwell plates coated with Compound X-B followed by Compound VIII coating. The signal increased with increasing capture probe added to the coated wells, but leveled off at approximately 1 pmole/well capture oligo. The signals were up to 29- and 11- fold higher for coated PP and PVC surfaces, respectively, as compared to the uncoated controls.
Table 4: Hybridization Signals (A655) From PP and PVC Microwell Plates Coated With Compound X-B Followed by Compound VIII Coating.
pmole/well PP Microwell plates PVC Microwell plates Capture Added Uncoated Coated Uncoated Coated 0.03 0.083 0.003 0.157 0.004 0.074 0.004 0.244 0.014 0.1 0.076 0.003 0.544 0.006 0.081 0.005 0.694 0.065 0.3 0.076 0.006 1.095 0.015 0.097 0.010 1.113+0.033 1 0.088 0.006 1.676 0.030 0.137 0.016 1.304 0.027 3 0.070 0.010 1.865 0.057 0.215 0.023 1.237+0.013 10 0.078 0.009 2.274 0.005 0.337 0.024 1.182 0.041 Example 20 Comparision of Random Photo PA-PolyQuat (Compound X-A) with a Mixture of Random Photo PA-PolyNOS (Compound IX-A) and Random Photo PA-PolyQuat (Compound X-A) Compound X-A at 0.5 or 0.1 mg/ml was incubated in PP microwell plates for minutes. The plates were then illuminated as described in Example 16. A
coating solution containing a mixture of Compound IX-A and Compound X-A was prepared at two ratios, 5/0.5 mg/ml and 0.5/0.1 mg/ml of Compound IX-A/Compound X-A in 10 deionized water to coat PP microwell plates. The solution was incubated in the wells for 10 minutes and the wells were illuminated as described in Example 16. The mer ID 2 capture oligonucleotide at 1 pmole/well was incubated in each well at for one hour. The hybridization was done as described in Example 16 using complementary ID 3 detection oligonucleotide or non-complementary ID 4 oligo.
The results listed in Table 5 indicate that the coating containing the combination of Compound IX-A and Compound X-A gave higher signals as compared to those from Compound X-A coating alone.
Table 5: Hybridization Signals (A655) From Compound X-A Coated PP Microwell Plates.
Ratio of Compound IX- Comp. Detection Non-comp. Detection A/Compound X-A
(mg/ml) 5/0.5 1.436 0.056 0.077 0.001 0/0.5 0.454 0.149 0.052 0.006 0.5/0.1 1.346 0.044 0.062 0.003 0/0.1 0.192 0.082 0.055 0.002 Example 21 Comparision of Non-modified Oligonucleotide vs. Amine-Modified Oligonucleotide on Random Photo PA-PolyNOS (Compound IX-B) and Random Photo PA-PolyQuat (Compound X-B) on Coated Microwell Plates A coating solution containing a mixture of Compound IX-B (5 mg/ml) and Compound X-B (0.5 mg/ml) was prepared in deionized water to coat PP, PS and PVC
microwell plates. The solution was incubated for approximately 10 minutes and illuminated as described in Example 16. The 30-mer capture 5'-NI-12-TTCTGTGTCTCC CGCTCCCAATACTCGGGC-3' (ID 5) oligonucleotide at 1 pmole/well was coupled to the wells in 50 mM phosphate buffer, pH 8.5, 1 mM
EDTA at 4 C overnight. The hybridization was performed as described in Example 16 using complementary detection oligonucleotide ID 4 or non-complementary oligonucleotide ID 3. To determine the effect of the amine-functionality of the capture oligo, a non-modified 30-mer capture probe 5'-ITCTGTGTCTCC
CGCTCCCAATACTCGGGC-3' (ID 6) (with no amine) was also added to the coated surfaces and tested. The results shown in Table 6 indicate that when an oligonucleotide without the 5'-amine modification was used as the capture probe on Compound IX-B/Compound X-B coated surfaces, the hybridization signal was less than 30% of that with amine modification.
Table 6: Signals (A655) Generated From Hybridization Reactions With Either ID
5 or ID 6 Oligonucleotides on Compound IX-B/ Compound X-B Coated Microwell Plates.
No Capture Added Non-modified Capture Amine-modified Capture Comp. Non-comp. Comp. Non-comp. Comp. Non-comp.
Detection Detection Detection Detection Detection Detection PP
Uncoated 0.032 0.001 0.036 0.004 0.033 0.001 0.036 0.001 0.037 0.005 0.033 0.001 Coated 0.038 0.002 0.040 0.001 0.555 0.041 0.044 0.001 1.915 0.029 0.066 0.003 PVC
Uncoated 0.248 0.049 0.176 0.008 0.259 0.049 0.128 0.013 0.404 0.100 0.118+0.025 Coated 0.115 0.027 0.090 0.014 0.379 0.028 0.091 0.014 1.319 0.027 0.101 0.017 PS
Uncoated 0.084 0.013 0.089 0.014 0.668 0.047 0.085 0.023 1.269 0.034 0.106+0.024 Coated 0.080 0.006 0.081 0.023 0.364 0.010 0.089 0.015 1.437 0.012 0.098+0.005 Example 22 Oligonucleotide Loading Densities on Microwell Plates Coated with Random Photo PA-PolyNOS (Compound IX-A) and Random Photo PA-PolyQuat (Compound X-A) Radiolabeled assays were performed to determine oligonucleotide loading densities and to verify results from the colorimetric assay system. In this Example, combination coatings of Compound IX-A and Compound X-A were performed on PVC wells as described in Example 16. The ID 2 and ID 5 30-mer capture oligonucleotides were immobilized on coated wells. A radiolabeled ID 2 probe was used to determine the loading density of immobilized capture oligonucleotides on the well surface. A radiolabeled ID 3 detection probe, which was complementary to ID 2, but not to ID 5, was used to measure hybridization reactions of the immobilized capture probes. Oligonucleotides ID 2 and ID 3 were radiolabeled at the 3'-end using terminal transferase (Boehringer Mannheim, Indianapolis, IN) and a-32P-ddATP
(3000 Ci/mmole, Amersham, Arlington Heights, IL) according to the manufacturer's specifications. 'Fs-labeled ID 2 and unlabeled ID 2 and ID 5 capture probes were incubated in coated wells at 50 pmole/well for 2.25 hours at room temperature.
The plates were washed and blocked as described in Example 16.
The wells with the unlabeled capture probes were hybridized with the 3213-labeled ID 3 detection probe in hybridization buffer for 1 hour at 55 C. Wells containing the 3213-labeled capture probe were incubated in hybridization buffer without the ID 3 probe. After washing three times with 2X SSC containing 0.1%
SDS
for 5 minutes at 55 C and three times with PBS/0.05% Tween, the plates were cut into individual wells and dissolved in tetrahydrofuran. The amount of radioactivity in each well was measured by scintillation counting in Aquasol-2 Fluor (DuPont NEN, Boston, MA). The results in Table 7 show that both Compound IX-A and Compound X-A were required to give good immobilization of capture probe. Also, increasing the concentrations of Compound IX-A and Compound X-A increased the amount of the capture oligonucleotide immobilized. At the highest concentrations tested, the signal to noise ratio was greater than 3000 to 1.
Table 7: Densities of Immobilized Capture Oligonucleotide and Hybridized 32P-Detection Oligo.
Mixture of Coating Reagents Immobilized Hybridized Hybridized Compound Compound capture comp.detection non-comp.detection IX-A (mg/mi) X-A (mg/ml) fmole/well fmole/well fmole/well 0 0 41.3 2.3 0.6 0 0.05 - 37.5 10.9 0.7 0.55 0 32.6 5.4 0.6 1 0.1 344.1 308.8 26.4 0.1 0.1 285.7 222.2 55.7 1 0.001 52.8 26.2 0.6 0.1 0.001 73.5 20.8 13.1 1.19 0.05 280.4 256.9 1.1 0.55 0.12 401.9 379.1 0.7 0.55 0.05 338.0 315.1 1.6 2 0.5 1633.4 1108.4 0.3 Example 23 Comparison between Random Photo-Polytertiary Amine (Compound XIII), Random Photo-PA-PolyNOS (Compound IX-A) and a Mixture of Random Photo PA-PolyNOS (Compound IX-A) and Random Photo-Polytertiary Amine (Compound XIII) Compound XIII at 0.02 mg/ml in deionized water was incubated in PP
microwell plates for 10 minutes. The wells were illuminated as described in Example 16. Compound IX-A was coated on PP wells at 2 mg/ml in deionized water as described for Compound XIII. A coating solution containing a mixture of 2 mg/ml Compound IX-A and 0.02 mg/ml Compound XIII in deionized water was prepared and coated as described for Compound XIII. The 30-mer ID 2 capture oligonucleotide at 5 pmole/well was incubated in each well at 37 C for one hour.
The hybridization was done as described in Example 16 using complementary ID 3 detection oligonucleotide and non-complementary ID 4 oligonucleotide. The contents of each well were transferred to PS wells after a 10 minute incubation with the peroxidase substrate. The results listed in Table 8 indicate that the combination of Compound IX-A and Compound XIII gave higher signals compared to those from Compound IX-A or Compound XIII coating alone.
Table 8: Hybridization Signals (A655) From Coated PP Microwell Plates.
Coating Comp. Detection Non-comp. Detection Compound IX-A 0.057 0.001 0.052 0.006 Compound XIII 0.746 0.042 0.081 0.009 Compound IX-A/Compound XIII 1.195 0.046 0.078 0.014 Mixture Example 24 Nucleic Acid Sequence Immobilization on an Amine Derivatized Surface A copolymer of the present invention is prepared in the following manner.
Acrylamide, 5.686 g (80.0 mmol), is dissolved in 100 ml of DMSO, followed by the addition of 3.083 g (10.0 mmol) of Compound IV, prepared according to the general method described in Example 4, and 2.207 g (10.0 mmol) of MAPTAC, delivered as a dry DMSO solution prepared according to the general method described in Example 11. TEMED, 0.134 ml (0.89 mmol), and AIBN, 0.197 g (1.20 mmol), are added to the mixture and the system is deoxygenated with a helium sparge for 5 minutes, followed by an argon sparge for an additional 5 minutes. The sealed vessel is heated at 55 C to complete the polymerization. The polymer is isolated by pouring the reaction mixture into 800 ml of diethyl ether and centrifuging to separate the solids.
The product is washed with 200 ml of diethyl ether, followed by 200 ml of chloroform. The polymer is dried under vacuum to remove remaining solvent.
A polymer surface is derivatized by plasma treatment using a 3:1 mixture of methane and ammonia gases. (See, e.g., the general method described in U.S.
Patent 5,643,580). A mixture of methane (490 SCCM) and ammonia (161 SCCM) are introduced into the plasma chamber along with the polymer part to be coated.
The gases are maintained at a pressure of 0.2-0.3 ton and a 300-500 watt glow discharge is established within the chamber. The sample is treated for a total of 3-5 minutes under these conditions. Formation of an amine derivatized surface is verified by a reduction in the water contact angle compared to the uncoated surface.
The amine derivatized surface is incubated for 10 minutes at room temperature with a 10 mg/ml solution of the above polymer in a 50 mM phosphate buffer, pH
8.5.
Following this reaction time, the coating solution is removed and the surface is washed thoroughly with deionized water and dried thoroughly. Immobilization of oligomer capture probe and hybridization is performed as described in Example 16.
Example 25 Immobilization and Hybridization of Oligonucleotides on Photo-Polymeric NOS
Coated Glass Slides - Comparison of coatings with and with out Photo PA
PolyQuat (Compound X-A) Soda lime glass microscope slides (Erie Scientific, Portsmouth, New Hampshire) were silane treated by dipping in a mixture of p-tolyldimethylchlorosilane (T-Silane) and N-decyldimethylchlorosilane (D-Silane, United Chemical Technologies, Bristol, Pennsylvania), 1% each in acetone, for 1 minute. After air drying, the slides were cured in an oven at 120 C for one hour. The slides were then washed with acetone followed by DI water dipping. The slides were further dried in oven for 5-10 minutes.
Compound IX-A, IX-D, and XV at various concentrations and with or without Compound X-A, were sprayed onto the silane treated slide, which were then illuminated using a Dymax lamp (25 mjoule/cm2 as measured at 335 nm with a 10 nm band pass filter on an International Light radiometer) while wet, washed with water, and dried. Oligonucleotides were printed on the slides using an X, Y, Z motion controller to position a 0.006" id blunt end needle filled with oligonucleotide solution.
Two oligonucleotides were immobilized to the prepared slides. One containing an amine on the 3' end and Cy3 fluorescent tag (Amersham, Arlington Heights, Illinois) on the 5' end, 5' Cy3-GTCTGAGTCGGAGCCAGGGCGGCCGCCAAC-NH2-3' (ID
7) (amino modifier has a C12 spacer) and the other containing an amine on the 5' end, -44 -5'-NH2-TTCTGTGTCTCCCGCTCCCAATACTCGGGC-3' (ID 5) ) (amino modifier has a C12 spacer). They were printed at a concentration of 8 pmole/ill in 50 mM
sodium phosphate pH 8.5 containing 10% sodium sulfate and 1 mM EDTA. Slides were placed overnight on a rack in a sealed container with saturated sodium chloride to maintain a relative humidity of 75%. Slides printed with (ID 7) were then washed for 5 minutes in PBS/0.05% Tween-20, for 90 minutes in blocking buffer (0.2 M
Tris with 10 mM ethanolamine) at 50 C, and for 2 hours in wash buffer (5X SSC, 0.1%
N-lauryl sarcosine, and 0.1% sodium dodecyl sulfate). Slides were washed twice with water and spun in a centrifuge to dry. They were than scanned using a General Scanning Scan-Array 3000 fluorescence scanner (Watertown, Massachusetts) and the average intensities of the resulting spots were measured. Slides printed with (ID 5) were washed for 5 minutes in PBS/0.05% Tween-20 and for 30 minutes in blocking buffer (0.2 M Tris with 10 mM ethanolamine) at 50 C. The slides were finally washed with water and dried in a centrifuge.
Fluorescently labeled complementary oligonucleotide, 5'-Cy3-CGGTGGATGGAGCAGGAGGGGCCCGAGTATTGGGAGCGGGAGACACAGA
A-3' (ID 8), was hybridized to the slides by placing 1011.1 of hybridization solution (4X SSC, 0.1% N-laurylsarcosine, 2 mg/ml tRNA) on the slide and placing a cover slip on top. The slides were then kept at 50 C high humidity (75%) to prevent drying out of the hybridization solution. Slides were then rinsed with 4X SSC, 2X SSC
preheated to 50 C for 2 minutes, 2X SSC for 2 minutes, and then twice into 0.1X
SSC for 2 minutes each. Slides were spun dry in a centrifuge. They were then scanned using a General Scanning fluorescence scanner. Average intensities of the resulting spots and background levels were measured. The results listed in Table 9 show that the coatings without compound X-A immobilize slightly less oligonucleotide but hybridization of a fluorescent oligonucleotide results in slightly higher signal. The resulting background is less on coatings which do not contain compound X-A. It also shows that polymers containing PVP backbone compound (i.e. Compound XV) are effective at immobilizing DNA and give good hybridization results.
Table 9: Immobilization and Hybridization of Oligonucleotides to Glass Microscope Slides.
Compound Poly-NOS Cmpd X-A
immobilized hybridization % BBA % NOS conc g/1 conc g/1 ID 7 signal' ID 8 signal' bkg S/N
Compound IX-A 1.25 0 39151 38512 45 856 Compound IX-A 1 0.25 42598 35674 88 405 Compound IX-A 2.5 0 35153 31061 34 914 Compound IX-A 2 0.5 44233 24735 75 332 Compound IX-D 1.25 0 30655 41669 45 926 Compound IX-D 1 0.25 38594 34300 99 346 Compound IX-D 2.5 0 41226 48976 67 736 Compound IX-D 2 0.5 46444 22743 123 185 Compound XV 1.25 0 28228 50248 34 1478 Compound XV 1 0.25 31544 47321 97 488 1 Laser power set at 60% and photomultiplier tube set at 60%
2 Laser power set at 80% and photomultiplier tube set at 80%
Example 26 Hybridization of Immobilized PCR products on Coated Glass Slides with OligonucleotideDetection Probe. Comparison between Random Photo-PA-PolyNOS
(Compound IX-A) and a Mixture of Random Photo-PA-PolyNOS (Compound IX-A) and Random Photo-PA-PolyQuat (Compound X-A).
Glass slides were coated with organosilane as described in Example 25.
Compound IX-A at 1.25 mg/ml in water or a mixture of 1 mg/ml Compound IX-A
and 0.25 mg/ml Compound X-A in water was coated onto silane treated glass slides as described in Example 25.
PCR products from P-galactosidase gene were custom prepared by ATG
Laboratories, Inc. (Eden Prairie). Primer with 5'-amine modification on the sense strand and unmodified primer on the anti-sense strand were used to prepare double-stranded- PCR products at 0.5 and 1 kilobase (kb) pair length. The control DNAs without amine were also made. The DNAs at concentration 0.2 g/ 1 in 80 mM
sodium phosphate buffer, pH 8.5, and 8% sodium sulfate were printed on the activated slides using microarraying spotting pins from TeleChem International (San Jose, CA).
The coupling was allow to proceed in a sealed container with 75% humidity overnight at room temperature.
To evaluate the signals from immobilized PCR products on microarrays, the slides were placed in boiling water for 2 minutes to denature double-stranded DNA
and to remove the non-attached strand. The slides were then incubated with 50 mM
ethanolamine in 0.1 M Tris buffer, pH 9 at 50 C for 15 minutes to block residual reactive groups on the surfaces. The slides were then incubated with pre-hybridization solution under glass cover slips at 50 C for 15 minutes to decrease the non-specific backgrounds. The pre-hybridization solution contained 5X SSC, 5X
Denhardt's solution (0.1 mg/ml each of bovine serum albumin, Ficoll and PVP), 0.1 mg/ml salmon sperm DNA and 0.1% SDS. The hybridization was then performed with 20 fmole/[11 of a fluorescent complementary detection oligo, 5'-Cy3-ACGCCGA
GTTAACGCCATCA (ID 9), in the pre-hybridization solution overnight at 45 C.
Slides were then washed and the hybridization signals scanned as described in Example 25.
The results listed in Table 10 indicate that the glass slides coated with Compound IX-A and mixture of Compound IX-A/X-A had comparable signals.
Amine-containing PCR product had at least 30-fold higher hybridization signals than non-modified DNA. The low level of signals with un-modified DNA was probably due to side reactions between amines on the heterocyclic bases to the activated surfaces.
Table 10: Hybridization Signals With Immobilized 0.5 Kb DNA And a Complementary Detection Oligonucleotide ID 9 on Compound IX-A/Compound X-A
Coated Glass Slides.
Coating Amine-primer Non-modified primer PCR product PCR product Compound IX-A 10,385 2,379 341 61 Compound IX-A and 16,858 4,008 341 79 Compound X-A Mixture Example 27 Hybridization of Immobilized PCR products on Coated Glass Slides with Oligonucleotide Detection Probe - Comparison between SurModics and other Commercial Slides.
PCR products from cDNA clones can be attached to the positively charged glass surfaces, such as polylysine; DeRisi, et. al., (Science, 278, 680-686, 1997), and a covalent approach having aldehyde groups has been reported by Schena (Schena et.al., Proc. Natl. Acad. Sci. USA, 93, 10614-10619). In this example PCR
products were attached to those surfaces and the hybridization signals were compared with the coatings from this invention. SurModics glass slides were coated with mixture of Compound IX-A and Compound X-A as described in Example 25. Silylated glass slides that have reactive aldehyde groups for immobilizing amine-functionalized DNA
was manufactured by GEL Associates, Inc. (Houston, TX). Polylysine glass slides were purchased from Sigma.
PCR products at 1 kb length from P-galactosidase at 1.5 pmole/1.11 in 50 mM
sodium phosphate buffer, pH 8.5, 1 mM EDTA and 3% sodium sulfate were printed onto silylated slides, polylysine slides and SurModics coated slides using 0.006" id needle as described in Example 25. The SurModics slides were then incubated in 75% relative humidity chamber for 2 days, denatured by submerging in boiling water bath for 2 minutes, and blocked with 10 mM ethanolamine, 0.2 M Tris, pH 8.5 for 30 minutes at 50 C. The silylated slides were incubated in a humidified incubator for 4 hours and then reduced with sodium borohydride as suggested by the manufacturer.
The polylysine slides were UV crosslinked and then blocked with succinic anhydride as described in the literature'. All the processed slides were hybridized with fmole/ 1 of complementary detection oligonucleotide ID 9 in 4X SSC, 2 mg/ml tRNA, 0.1% lauroylsarcosine at 45 C overnight. The slides were washed and hybridization signals were scanned as described in Example 25.
The results are shown in the following Table 11. There was no difference in signals between amine-modified versus un-modified DNA on silylated and polylysine slides. Only SurModics coatings demonstrated that specific attachment was due to having a 5'-amine on the PCR products. This provides evidence of end-point attachment of DNA up to 1 kb with SurModics coatings. Polylysine slides had the highest background probably due to ionic and/or non-specific binding of the DNA
onto the surfaces.
Table 11: Hybridization Signals With Immobilized 1 Kb DNA and a Complementary Detection Oligonucleotide ID 9 on Coated Glass Slides. Comparison of Compound IX-A/Compound X-A Coated Slides and Commercial Glass Slides.
Coating Amine-primer Non-modified Background PCR product primer PCR product Compound IX-A and 26,580 3,219 946 185 88 Compound X-A Mixture Silylated 5,611 2,063 7,050 2,211 114 Polylysine 4,3674 2,832 4,3206 4,743 3,075 Example 28 Immobilization and Hybridization of PCR Products with cDNA Detection Probe on Photo-Polymeric NOS Coated Glass Slides.
Two sets of slides were prepared as described in Example 26. Three PCR
product sequences (designated F 11, XEF, daf) containing an amine on both, the forward, the reverse or neither strand (provided by Axys Pharmaceuticals, La Jolla, California) were dissolved in printing buffer (80ng/111), heated at 100 C, cooled on ice, and printed on the slides using a Generation II Arrayer (Molecular Dynamics, Sunnyvale, California). After incubation overnight as described in Example 25, the slides were placed in a boiling water bath for 2 minutes, washed twice with PBS/0.05% tween-20, rinsed twice with water, and put in blocking buffer for 30 minutes at 50 C. The slides were than rinsed with water and spun dry. Slides were prehybridized as described in Example 26 and hybridized to a mixture of fluorescently (Cy3) labeled cDNA (provided by Axys Pharmaceuticals) in 50% formamide, 5X
SSC, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA at 42 C overnight. This mixture contained complementary probes to the forward strand of all three PCR product targets. The Fll probe was spiked at a 1 to 50,000 mass ratio relative to the other two sequences. After hybridization, the slides were washed and scanned as described in Example 25. The average intensities of the spots are shown in Table 12. Slides which were hybridized to a cDNA probe mixture which did not contain the Fll probe showed no signal in these spots. The results show that both coating types give comparable hybridization results. The coating containing compound X-A had much higher background. This was especially true in the area near where the PCR
product was printed.
Table 12: Immobilization of PCR Products and Hybridization to Fluorescently Labeled cDNA on Glass Microscope Slides. Numbers are Fluorescent Signal'.
coated with amine on compound IX-A both strands forward reverse strand neither strand strand 0.85 Kb XEF 2664.5 6125.5 759.5 3590.5 1 Kb daf 42921.5 14294 1 Kb F 1 1 588 1859.5 123.5 891.5 background= 80 coated with mixture amine on compounds IX-A & both strands forward reverse strand neither strand X-A strand 0.85 Kb XEF 3001 12896 779 4119 1 Kb daf 44132.5 13269.5 1 Kb F 1 1 535 1687.5 133 860.5 background= varies from 100 to 2500 1 Laser power set at 80% and photomultiplier tube set at 80%
Table 13: Compounds.

el CI

COMPOUND I
H2N- (CH2)3- NH ____________________________ Sr_ COMPOUND II
= 0 II 0111 NH- (CH2)3-- NH ______ /0 '/-- CH3 COMPOUND III

II )\-----I N - (CH2)5- C - 0 - N
--------\< )7----COMPOUND IV

)\-----\N - (CH2)5- C - 0 - N
)r---cH3 0 0 COMPOUND V

1101 1101 CH2Br COMPOUND VI

OH

ili C-N-(CH2)2-SH

0 Oil CH2-0 COMPOUND VII

1101 NH- (CH2)10- 8N)\---)7----COMPOUND VIII

H CH H H
i I 3 I I
___________________ CH2 C ___ CH2 _______________ C C _________ C=0 C=0 I I ONLO

I (CH2)5 (CH2)3 NH C=0 I I
C=0 0 I

___ ___ ___ ___. _ -COMPOUND IX
_ _ ___ _____________ CH2 C __________ CH2 C __________ CH2 C __________ I I I
C=0 C=0 C=0 I
I
(CH2)3 (CH2)3 NH I

C=0 + N(CH3)3 CI-S

_ _ _ _ COMPOUND X

¨ ¨¨ ¨
I

I
______ CH2 C __________ CH2 C _________________ CH2 C ___________ CH2 C __ I I I I
C= 0 C= 0 C= 0 C= 0 i I I I

(CH2)3 (CH2)5 (CH2)3 i NH I I
I C= 0 +
C= 0 I
N(CH3)3 so CI"

N

¨ ¨ ¨ ___. _ ____ __. _ COMPOUND XI

CH2-0 _ _ _ I 1 _____ 41, ,¨(cH2)2 s ______ cH2 C __ c c C=O

(CH2)5 I
C=0 I
N
0 ..0 - _ COMPOUND XII

____ _ ___ _ _______________________ CH2 _________________ CH2 C __ I
C=0 C=0 I I
NH NH
I
I
(CH2)3 (CH2)3 NH I
I
C=0 N(CH3)2 - - ___ COMPOUND XIII

I II II )\-----H2C=CH-CHiN-C-CHiCHiCHiC-0-N
)1----COMPOUND XIV

I I I
_______ CH2 C _____________ CH C _____ CH2 C ________ C = 0 N
Nr. 0 CH

I I
NH NH
I I
(CH 2) 3 - C = 0 I I
NH 1( CH2 ) 3 I
1101C = 0 I

I
N

40 C*0 - -Compound XV

SEQUENCE LISTING
<110> SURMODICS, INC.
<120> TARGET MOLECULE ATTACHMENT TO SURFACES
<130> 13181-25 WKS
<140> 2,360,000 <141> 2000-01-10 <150> 09/227,913 <151> 1999-01-08 <160> 9 <170> PatentIn Ver. 2.0 <210> 1 <211> 50 <212> DNA
<213> Homo sapiens <400> 1 gtctgagtcg gagccagggc ggccgccaac agcaggagca gcgtgcacgg 50 <210> 2 <211> 30 <212> DNA
<213> Homo sapiens <400> 2 gtctgagtcg gagccagggc ggccgccaac 30 <210> 3 <211> 50 <212> DNA
<213> Homo sapiens <400> 3 ccgtgcacgc tgctcctgct gttggcggcc gccctggctc cgactcagac 50 <210> 4 <211> 50 <212> DNA
<213> Homo sapiens <400> 4 cggtggatgg agcaggaggg gcccgagtat tgggagcggg agacacagaa 50 <210> 5 <211> 30 <212> DNA
<213> Homo sapiens < 4 00> 5 ttctgtgtct cccgctccca atactcgggc 30 <210> 6 <211> 30 <212> DNA
<213> Homo sapiens <400> 6 ttctgtgtct cccgctccca atactcgggc 30 <210> 7 <211> 30 <212> DNA
<213> Homo sapiens <400> 7 gtctgagtcg gagccagggc ggccgccaac 30 <210> 8 <211> 50 <212> DNA
<213> Homo sapiens <400> 8 cggtggatgg agcaggaggg gcccgagtat tgggagcggg agacacagaa 50 <210> 9 <211> 20 <212> DNA
<213> Homo sapiens <400> 9 acgccgagtt aacgccatca 20

Claims (30)

The embodiments of the present invention in which an exclusive property or privilege is claimed are defined as follows:
1. A method of attaching a target molecule to a substrate, the method comprising:
a) providing a reagent composition comprising a polymeric backbone, and more than two thermochemically reactive groups attached to the polymeric backbone, wherein the thermochemically reactive groups are configured and arranged to form covalent bonds with functional groups on the target molecule;
b) coating and immobilizing the reagent composition on the substrate to form a bound composition;
c) providing a solution comprising target molecule having one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition;
d) applying one or more discrete spots of up to about 1 nanoliter of the solution to the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without use of attracting groups to attract the target molecule to the bound reagent.
2. The method according to claim 1 wherein the target molecule comprises nucleic acid and the substrate comprises plastic, silicon hydride, or an organosilane-pretreated glass or silicone slide.
3. The method according to claim 2 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
4. The method according to claim 1 wherein the step of providing a solution comprising target molecule having one or more functional groups reactive with thermochemically reactive groups provided by the reagent composition comprises providing the solution in an amount of up to about 20 nanoliters.
5. The method according to claim 1 wherein the reagent composition further comprises one or more photoreactive groups for attaching the reagent composition to the substrate upon application of energy from a suitable source.
6. The method according to claim 5 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones and the photoreactive groups are photoreactive aryl ketones.
7. The method according to claim 6 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
8. The method according to claim 1 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the pendent thermochemically reactive groups are selected from the group consisting of activated esters, epoxides, azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and a-iodoacetamide, and the backbone further comprises one or more pendent aryl ketone photoreactive groups.
9. The method according to claim 1 wherein the method is used to prepare one or more microarrays of nucleic acids upon a substrate comprising plastic, silicon hydride, or an organosilane-pretreated glass or silicone slide, each microarray including discrete regions of nucleic acids in amounts of 0.1 femtomoles to 10 nanomoles.
10. An activated slide for binding target molecule in a sample, the slide comprising:
a) a substrate; and b) a bound composition attached to the substrate, wherein the bound composition comprises a polymeric backbone having more than two thermochemically reactive groups attached thereto, and wherein the bound composition is configured and arranged to form covalent bonds with functional groups on the target molecule without use of attracting groups to attract the target molecule to the bound composition.
11. The activated slide according to claim 10, wherein the activated slide is configured and arranged to receive a sample comprising target molecule in an amount of up to about 20 nanoliters.
12. The activated slide according to claim 10, wherein the activated slide is adapted for fabricating a microarray, and wherein the target molecule comprises a nucleic acid and the substrate comprises plastic, silicon hydride, or an organosilane-pretreated glass or silicone slide.
13. The activated slide according to claim 12, wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
14. The activated slide according to claim 10, wherein the bound composition comprises a reaction product of a reagent composition with the substrate, wherein the reagent composition comprises one or more photoreactive groups for attaching the reagent composition to the substrate upon application of energy from a suitable source, wherein the photoreactive groups immobilize the reagent composition onto the substrate to form the bound composition.
15. The activated slide according to claim 14, wherein the reagent composition comprises thermochemically reactive groups and photoreactive groups, and wherein the themochemically reactive groups and photoreactive groups are pendent from one or more hydrophilic polymeric backbones and the photoreactive groups are photoreactive aryl ketones.
16. The activated slide according to claim 15, wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
17. The activated slide according to claim 10, wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the thermochemically reactive groups are selected from the goup consisting of activated esters. epoxides, azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and .alpha.-iodoacetamide, and the backbone further comprises one or more pendent aryl ketone photoreactive groups.
18. A microarray comprising a substrate, a bound composition disposed on the substrate, and target molecule covalently coupled with the bound composition, wherein the microarray is prepared by a method comprising:
a) providing a reagent composition comprising a polymeric backbone, and more than two thermochemically reactive groups attached to the polymeric backbone;
b) coating and immobilizing the reagent composition on the substrate to form a bound composition;
c) providing a solution comprising target molecule, wherein the target molecule comprises one or more functional groups reactive with the thermochemically reactive groups provided by the bound composition;
d) applying one or more discrete spots of up to about 1 nanoliter of the solution on the substrate; and e) allowing the thermochemically reactive groups provided by the bound composition to form covalent bonds with the functional groups provided by the target molecule in order to attach the target molecule to the substrate without the use of attracting groups.
19. The microarray according to claim 18 wherein the target molecule comprises nucleic acid and the substrate comprises the plastic, silicon hydride, or an organosilane-pretreated glass or silicone slide.
20. The microarray according to claim 19 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulthydryl groups.
21. The microarray according to claim 20 wherein the microarray is configured and arranged to receive the solution comprising target molecule in volumes of nanoliters or less.
22. The microarray according to claim 18 wherein the reagent composition further comprises one or more photoreactive groups for attaching the reagent composition to the surface upon application of energy from a suitable source.
23. The microarray according to claim 22 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones and the photoreactive groups are photoreactive aryl ketones.
24. The microarray according to claim 23 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
25. The microarray according to claim 18 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the thermochemically reactive groups are selected from the group consisting of activated esters, epoxides, azlactones, activated hydroxyls, aldehydes, isocyanates, thioisocyanates, carboxylic acid chlorides, alkyl halides, maleimide, and .alpha.-iodoacetamide, and the backbone further comprises one or more pendent aryl ketone photoreactive groups.
26. The microarray according to claim 18 wherein the microarray comprises a surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide, and wherein the microarray provides at least about 100/cm2 distinct nucleic acids having a length of at least 10 nucleotides, the nucleic acids each being spotted in discrete regions and defined amounts of 0.1 femtomoles to 10 nanomoles.
27. The microarray according to claim 26 wherein the regions are generally circular in shape, having a diameter of between about 10 µm and about 500 µm and separated from other regions in the array by center to center spacing of 20 µm to 1000 µm.
28. The method according to claim 1, wherein the more than two thermochemically reactive groups comprise amine-reactive or sulfhydryl-reactive groups.
29. The activated slide according to claim 10, wherein the more than two thermochemically reactive groups comprise amine-reactive or sulfhydryl-reactive groups.
30. The microarray according to claim 18, wherein the more than two thermochemically reactive groups comprise amine-reactive or sulfhydryl-reactive groups.
CA2360000A 1999-01-08 2000-01-10 Target molecule attachment to surfaces Expired - Lifetime CA2360000C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/227,913 US6465178B2 (en) 1997-09-30 1999-01-08 Target molecule attachment to surfaces
US09/227,913 1999-01-08
PCT/US2000/000535 WO2000040593A2 (en) 1999-01-08 2000-01-10 Target molecule attachment to surfaces

Publications (2)

Publication Number Publication Date
CA2360000A1 CA2360000A1 (en) 2000-07-13
CA2360000C true CA2360000C (en) 2014-03-25

Family

ID=22854960

Family Applications (1)

Application Number Title Priority Date Filing Date
CA2360000A Expired - Lifetime CA2360000C (en) 1999-01-08 2000-01-10 Target molecule attachment to surfaces

Country Status (6)

Country Link
US (3) US6465178B2 (en)
EP (1) EP1141385B1 (en)
JP (1) JP4768130B2 (en)
AU (1) AU778265B2 (en)
CA (1) CA2360000C (en)
WO (1) WO2000040593A2 (en)

Families Citing this family (233)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6762019B2 (en) * 1997-09-30 2004-07-13 Surmodics, Inc. Epoxide polymer surfaces
US6465178B2 (en) 1997-09-30 2002-10-15 Surmodics, Inc. Target molecule attachment to surfaces
WO2000038838A1 (en) * 1998-12-23 2000-07-06 American Registry Of Pathology Apparatus and methods for efficient processing of biological samples on slides
US7091033B2 (en) * 2000-07-21 2006-08-15 Phase-1 Molecular Toxicology, Inc. Array of toxicologically relevant canine genes and uses thereof
US20050266494A1 (en) * 2000-09-06 2005-12-01 Hodge Timothy A System and method for computer network ordering of biological testing
US7494817B2 (en) * 2000-09-06 2009-02-24 Transnet Yx, Inc. Methods for genotype screening using magnetic particles
US20050239125A1 (en) * 2000-09-06 2005-10-27 Hodge Timothy A Methods for genotype screening
EP1978110B1 (en) * 2000-09-06 2010-05-26 Transnetyx, Inc. Computer-based method and system for screening genomic DNA
JP2004508057A (en) * 2000-09-06 2004-03-18 ホッジ ティモシー エイ System, method and apparatus for screening for transgenic and target specific mutations
US20050272085A1 (en) * 2000-09-06 2005-12-08 Hodge Timothy A Methods for forensic and congenic screening
EP2465943A3 (en) * 2001-03-16 2012-10-03 Kalim Mir Linear polymer display
US20020146684A1 (en) * 2001-04-09 2002-10-10 Meldal Morten Peter One dimensional unichemo protection (UCP) in organic synthesis
US7501157B2 (en) * 2001-06-26 2009-03-10 Accelr8 Technology Corporation Hydroxyl functional surface coating
US6844028B2 (en) * 2001-06-26 2005-01-18 Accelr8 Technology Corporation Functional surface coating
US10272409B2 (en) 2001-07-11 2019-04-30 Michael E. Hogan Methods and devices based upon a novel form of nucleic acid duplex on a surface
DE60231408D1 (en) * 2001-07-11 2009-04-16 Baylor College Medicine METHOD AND DEVICES BASED ON A NEW FORM OF A NUCLEIC ACID ELEMENT ON A SURFACE
US7297553B2 (en) * 2002-05-28 2007-11-20 Nanosphere, Inc. Method for attachment of silylated molecules to glass surfaces
AU2002322458A1 (en) 2001-07-13 2003-01-29 Nanosphere, Inc. Method for immobilizing molecules onto surfaces
WO2005108625A2 (en) * 2001-07-13 2005-11-17 Nanosphere, Inc. Method for preparing substrates having immobilized molecules and substrates
US6444318B1 (en) 2001-07-17 2002-09-03 Surmodics, Inc. Self assembling monolayer compositions
US6902894B2 (en) * 2001-09-07 2005-06-07 Genetel Pharmaceuticals Ltd. Mutation detection on RNA polmerase beta subunit gene having rifampin resistance
CA2457564C (en) * 2001-10-05 2009-04-07 Surmodics, Inc. Particle immobilized coatings and uses thereof
US7439346B2 (en) * 2001-10-12 2008-10-21 Perkinelmer Las Inc. Nucleic acids arrays and methods of use therefor
US7348055B2 (en) 2001-12-21 2008-03-25 Surmodics, Inc. Reagent and method for providing coatings on surfaces
NL1020066C2 (en) * 2002-02-26 2003-08-27 Dutch Space B V Device for manufacturing a DNA test array.
US7226771B2 (en) 2002-04-19 2007-06-05 Diversa Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
EP1497418B1 (en) 2002-04-19 2012-10-17 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
US20040142864A1 (en) * 2002-09-16 2004-07-22 Plexxikon, Inc. Crystal structure of PIM-1 kinase
US20070082019A1 (en) * 2003-02-21 2007-04-12 Ciphergen Biosystems Inc. Photocrosslinked hydrogel surface coatings
JP2007524374A (en) * 2003-02-28 2007-08-30 プレキシコン,インコーポレーテッド PYK2 crystal structure and use
ES2713024T3 (en) 2003-03-06 2019-05-17 Basf Enzymes Llc Amylases, nucleic acids that encode them and methods for their manufacture and use
EP1601332A4 (en) 2003-03-07 2012-05-02 Verenium Corp Hydrolases, nucleic acids encoding them and mehods for making and using them
EP1615034A4 (en) * 2003-03-31 2010-11-24 Rikagaku Kenkyusho Fixing agents, method of fixing substance with the same, and substrate having substance fixed thereto with the same
ES2545639T3 (en) 2003-04-04 2015-09-14 Basf Enzymes Llc Pectate liases, nucleic acids that encode them and methods for their preparation and use
WO2004092250A1 (en) * 2003-04-15 2004-10-28 Biogenon Ltd. Biocompatible material
US7635734B2 (en) * 2004-02-17 2009-12-22 The Children's Hospital Of Philadelphia Photochemical activation of surfaces for attaching biomaterial
AU2004231992B2 (en) * 2003-04-16 2011-04-21 Drexel University Magnetically controllable drug and gene delivery stents
US9694103B2 (en) 2003-04-16 2017-07-04 The Children's Hospital Of Philadelphia Photochemical activation of surfaces for attaching biomaterial
US20040234964A1 (en) * 2003-05-19 2004-11-25 Affymetrix, Inc. Methods for reducing background on polynucleotide arrays
DE10325098B3 (en) * 2003-06-03 2004-12-02 IPK-Institut für Pflanzengenetik und Kulturpflanzenforschung Procedure for SNP analysis on biochips with oligonucleotide areas
JP2005017155A (en) * 2003-06-27 2005-01-20 Toyobo Co Ltd Method for manufacturing array on metal substrate
US7960148B2 (en) 2003-07-02 2011-06-14 Verenium Corporation Glucanases, nucleic acids encoding them and methods for making and using them
US20050079548A1 (en) * 2003-07-07 2005-04-14 Plexxikon, Inc. Ligand development using PDE4B crystal structures
CA2535526C (en) 2003-08-11 2015-09-29 Diversa Corporation Laccases, nucleic acids encoding them and methods for making and using them
US20050164300A1 (en) * 2003-09-15 2005-07-28 Plexxikon, Inc. Molecular scaffolds for kinase ligand development
US7309593B2 (en) * 2003-10-01 2007-12-18 Surmodics, Inc. Attachment of molecules to surfaces
US20050074406A1 (en) * 2003-10-03 2005-04-07 Scimed Life Systems, Inc. Ultrasound coating for enhancing visualization of medical device in ultrasound images
WO2005037338A1 (en) * 2003-10-14 2005-04-28 Cook Incorporated Hydrophilic coated medical device
EP1696920B8 (en) 2003-12-19 2015-05-06 Plexxikon Inc. Compounds and methods for development of ret modulators
US20070066641A1 (en) * 2003-12-19 2007-03-22 Prabha Ibrahim Compounds and methods for development of RET modulators
EP3673986A1 (en) 2004-01-07 2020-07-01 Illumina Cambridge Limited Improvements in or relating to molecular arrays
US20050220853A1 (en) * 2004-04-02 2005-10-06 Kinh-Luan Dao Controlled delivery of therapeutic agents from medical articles
US20050238542A1 (en) * 2004-04-22 2005-10-27 Applera Corporation Pins for spotting nucleic acids
CA2565965A1 (en) 2004-05-06 2006-07-27 Plexxikon, Inc. Pde4b inhibitors and uses therefor
WO2005113834A2 (en) * 2004-05-20 2005-12-01 Quest Diagnostics Investments Incorporated Single label comparative hybridization
US20050281857A1 (en) * 2004-05-25 2005-12-22 Heyer Toni M Methods and reagents for preparing biomolecule-containing coatings
EA013993B1 (en) 2004-06-16 2010-08-30 Верениум Корпорейшн Compositions and methods for enzymatic de-colorization of chlorophyll
EP1755597A2 (en) * 2004-06-17 2007-02-28 Plexxikon, Inc. Azaindoles modulating c-kit activity and uses therefor
US7498342B2 (en) 2004-06-17 2009-03-03 Plexxikon, Inc. Compounds modulating c-kit activity
US8536661B1 (en) 2004-06-25 2013-09-17 University Of Hawaii Biosensor chip sensor protection methods
AU2005279795A1 (en) * 2004-09-03 2006-03-09 Plexxikon, Inc. Bicyclic heteroaryl PDE4B inhibitors
WO2007008246A2 (en) 2004-11-12 2007-01-18 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for dna and other molecules
US20060134656A1 (en) * 2004-12-16 2006-06-22 Hamers Robert J Reduction of non-specific adsorption of biological agents on surfaces
DE602006018861D1 (en) * 2005-01-27 2011-01-27 Quest Diagnostics Invest Inc FAST COMPARATIVE GENOM HYBRIDIZATION
WO2006099207A2 (en) 2005-03-10 2006-09-21 Diversa Corporation Lyase enzymes, nucleic acids encoding them and methods for making and using them
CA2861310A1 (en) 2005-03-15 2006-09-28 Bp Corporation North America Inc. Cellulases, nucleic acids encoding them and methods for making and using them
WO2007013896A2 (en) * 2005-05-17 2007-02-01 Plexxikon, Inc. Pyrrol (2,3-b) pyridine derivatives protein kinase inhibitors
HUE027370T2 (en) * 2005-06-22 2016-10-28 Plexxikon Inc Pyrrolo [2,3-b]pyridine derivatives as protein kinase inhibitors
CA2615717A1 (en) * 2005-07-20 2007-01-25 Surmodics, Inc. Polymer coated nanofibrillar structures and methods for cell maintenance and differentiation
US8162555B2 (en) * 2005-07-21 2012-04-24 The Regents Of The University Of California Printing pins having selective wettability and method of making same
US8076074B2 (en) 2005-11-29 2011-12-13 Quest Diagnostics Investments Incorporated Balanced translocation in comparative hybridization
EP1987142A4 (en) 2006-02-02 2009-07-15 Verenium Corp Esterases and related nucleic acids and methods
MY160772A (en) 2006-02-10 2017-03-15 Verenium Corp Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them
BRPI0707784B1 (en) 2006-02-14 2018-05-22 Verenium Corporation ISOLATED, SYNTHETIC OR RECOMBINANT NUCLEIC ACID, EXPRESSION CASSETTE, CLONING VECTOR OR VEHICLE, TRANSFORMED ISOLATED HOST CELL, AND METHOD FOR PRODUCTION OF A RECOMBINANT POLYPEPTIDE
ATE548450T1 (en) 2006-03-07 2012-03-15 Verenium Corp ALDOLASES, NUCLEIC ACIDS FOR ENCODING THEM AND METHODS FOR THEIR PRODUCTION AND USE
US8043837B2 (en) 2006-03-07 2011-10-25 Cargill, Incorporated Aldolases, nucleic acids encoding them and methods for making and using them
US20070232556A1 (en) * 2006-03-31 2007-10-04 Montine Thomas J Methods and compositions for the treatment of neurological diseases and disorders
US20090215098A1 (en) * 2006-04-28 2009-08-27 Ucl Business Plc. Quantification of enzyme activity by mass spectrometry
CN101528766A (en) 2006-08-04 2009-09-09 维莱尼姆公司 Glucanases, nucleic acids encoding them and methods for making and using them
CA2663001A1 (en) 2006-09-21 2008-03-27 Verenium Corporation Phospholipases, nucleic acids encoding them and methods for making and using them
EP2617729B1 (en) 2006-09-21 2016-03-16 BASF Enzymes LLC Phytases, nucleic acids encoding them and methods for making and using them
US8618248B2 (en) 2006-10-31 2013-12-31 President And Fellows Of Harvard College Phosphopeptide compositions and anti-phosphopeptide antibody compositions and methods of detecting phosphorylated peptides
WO2008063888A2 (en) 2006-11-22 2008-05-29 Plexxikon, Inc. Compounds modulating c-fms and/or c-kit activity and uses therefor
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
GB2457851B (en) 2006-12-14 2011-01-05 Ion Torrent Systems Inc Methods and apparatus for measuring analytes using large scale fet arrays
PE20121126A1 (en) * 2006-12-21 2012-08-24 Plexxikon Inc PIRROLO [2,3-B] PYRIDINES COMPOUNDS AS KINASE MODULATORS
WO2008079909A1 (en) * 2006-12-21 2008-07-03 Plexxikon, Inc. Pyrrolo [2,3-b] pyridines as kinase modulators
RU2009122670A (en) 2006-12-21 2011-01-27 Плекссикон, Инк. (Us) COMPOUNDS AND METHODS FOR MODULATION OF KINASES AND INDICATIONS FOR THEIR USE
DK3101128T3 (en) 2006-12-21 2019-07-08 Basf Enzymes Llc AMYLASES AND GLUCOAMYLASES, NUCLEAR ACIDS CODING FOR THESE, AND METHODS OF MANUFACTURE AND USE THEREOF
WO2008095033A2 (en) 2007-01-30 2008-08-07 Verenium Corporation Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
EP2147100B1 (en) 2007-04-27 2017-06-07 The Regents of The University of California Plant co2 sensors, nucleic acids encoding them, and methods for making and using them
NZ582772A (en) 2007-07-17 2012-06-29 Plexxikon Inc Compounds and methods for kinase modulation, and indications therefor
US7507539B2 (en) * 2007-07-30 2009-03-24 Quest Diagnostics Investments Incorporated Substractive single label comparative hybridization
US8486680B2 (en) 2007-10-03 2013-07-16 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
CN104651381A (en) 2008-01-03 2015-05-27 巴斯夫酶有限责任公司 Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them
EP2982437B1 (en) 2008-06-25 2017-12-06 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale fet arrays
US8357503B2 (en) 2008-08-29 2013-01-22 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US8198062B2 (en) 2008-08-29 2012-06-12 Dsm Ip Assets B.V. Hydrolases, nucleic acids encoding them and methods for making and using them
US8153391B2 (en) 2008-08-29 2012-04-10 Bunge Oils, Inc. Hydrolases, nucleic acids encoding them and methods for making and using them
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US8993068B2 (en) 2008-11-04 2015-03-31 The Trustees Of Columbia University In The City Of New York Heterobifunctional polymers and methods for layer-by-layer construction of multilayer films
EP3282021A1 (en) 2009-03-09 2018-02-14 Bioatla, LLC Mirac proteins
TWI404719B (en) * 2009-04-03 2013-08-11 Hoffmann La Roche Compositions and uses thereof
CN102427834A (en) 2009-04-28 2012-04-25 苏尔莫迪克斯公司 Devices and methods for delivery of bioactive agents
BRPI1011160A8 (en) 2009-05-21 2018-01-02 Verenium Corp PHYTASES, PROTEIN PREPARATION INCLUDING THEM, AND THEIR USES
US20120261274A1 (en) 2009-05-29 2012-10-18 Life Technologies Corporation Methods and apparatus for measuring analytes
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US8574835B2 (en) 2009-05-29 2013-11-05 Life Technologies Corporation Scaffolded nucleic acid polymer particles and methods of making and using
US8329724B2 (en) 2009-08-03 2012-12-11 Hoffmann-La Roche Inc. Process for the manufacture of pharmaceutically active compounds
CN102630250A (en) * 2009-09-25 2012-08-08 基因诊断测试公司 Multiplex (+/-) stranded arrays and assays for detecting chromosomal abnormalities associated with cancer and other diseases
US20110073412A1 (en) 2009-09-28 2011-03-31 Tlt-Babcock, Inc. Axial fan compact bearing viscous pump
UA109884C2 (en) 2009-10-16 2015-10-26 A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD
UA111708C2 (en) 2009-10-16 2016-06-10 Бандж Ойлз, Інк. METHOD OF OIL REFINING
CN106220623A (en) 2009-11-06 2016-12-14 普莱希科公司 Compounds and methods for and indication thereof for kinases regulation
US20110144373A1 (en) * 2009-12-10 2011-06-16 Surmodics, Inc. Water-soluble degradable photo-crosslinker
EP2516567B1 (en) 2009-12-21 2016-08-03 Innovative Surface Technologies, Inc. Coating agents and coated articles
TWI539172B (en) 2010-06-30 2016-06-21 生命技術公司 Methods and apparatus for testing isfet arrays
TWI547688B (en) 2010-06-30 2016-09-01 生命技術公司 Ion-sensing charge-accumulation circuits and methods
US8823380B2 (en) 2010-06-30 2014-09-02 Life Technologies Corporation Capacitive charge pump
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
CN103168341B (en) 2010-07-03 2016-10-05 生命科技公司 There is the chemosensitive sensor of lightly doped discharger
US9618475B2 (en) 2010-09-15 2017-04-11 Life Technologies Corporation Methods and apparatus for measuring analytes
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
BR112013008347A2 (en) 2010-10-06 2016-06-14 Bp Corp North America Inc cbh variant polypeptides i
US10315987B2 (en) 2010-12-13 2019-06-11 Surmodics, Inc. Photo-crosslinker
WO2012109075A1 (en) 2011-02-07 2012-08-16 Plexxikon Inc. Compounds and methods for kinase modulation, and indications therefor
AR085279A1 (en) 2011-02-21 2013-09-18 Plexxikon Inc SOLID FORMS OF {3- [5- (4-CHLORINE-PHENYL) -1H-PIRROLO [2,3-B] PIRIDINA-3-CARBONIL] -2,4-DIFLUOR-PHENIL} -AMIDE OF PROPANE ACID-1- SULFONIC
US9006345B2 (en) 2011-03-25 2015-04-14 The Trustees Of Columbia University In The City Of New York Heterotrifunctional molecules and methods for the synthesis of dendrimeric materials
WO2012158957A2 (en) 2011-05-17 2012-11-22 Plexxikon Inc. Kinase modulation and indications therefor
US9861727B2 (en) 2011-05-20 2018-01-09 Surmodics, Inc. Delivery of hydrophobic active agent particles
US10213529B2 (en) 2011-05-20 2019-02-26 Surmodics, Inc. Delivery of coated hydrophobic active agent particles
US9757497B2 (en) 2011-05-20 2017-09-12 Surmodics, Inc. Delivery of coated hydrophobic active agent particles
EP2718465B1 (en) 2011-06-09 2022-04-13 Illumina, Inc. Method of making an analyte array
CN102503849B (en) * 2011-10-08 2013-08-07 武汉志邦化学技术有限公司 Synthetic method of N-(3-amino propyl)methacrylamide hydrochloride
EP2771103B1 (en) 2011-10-28 2017-08-16 Illumina, Inc. Microarray fabrication system and method
WO2013070627A2 (en) 2011-11-07 2013-05-16 Illumina, Inc. Integrated sequencing apparatuses and methods of use
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
CN103373937A (en) * 2012-04-23 2013-10-30 佟太锋 Method for synthesizing N-(3-aminopropyl)methacrylate
US8786331B2 (en) 2012-05-29 2014-07-22 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9150570B2 (en) 2012-05-31 2015-10-06 Plexxikon Inc. Synthesis of heterocyclic compounds
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
US11246963B2 (en) 2012-11-05 2022-02-15 Surmodics, Inc. Compositions and methods for delivery of hydrophobic active agents
JP6438406B2 (en) 2012-11-05 2018-12-12 サーモディクス,インコーポレイテッド Compositions and methods for delivering hydrophobic bioactive agents
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
US9267171B2 (en) 2013-02-28 2016-02-23 New York University DNA photolithography with cinnamate crosslinkers
ES2620427T3 (en) 2013-03-08 2017-06-28 Illumina Cambridge Limited Rhodamine compounds and their use as fluorescent markers
DK2964612T3 (en) 2013-03-08 2017-04-03 Illumina Cambridge Ltd POLYMETHIN COMPOUNDS AND USE THEREOF AS FLUORESCING LABELS
US8841217B1 (en) 2013-03-13 2014-09-23 Life Technologies Corporation Chemical sensor with protruded sensor surface
US8963216B2 (en) 2013-03-13 2015-02-24 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
EP2972281B1 (en) 2013-03-15 2023-07-26 Life Technologies Corporation Chemical device with thin conductive element
US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface
CN105283758B (en) 2013-03-15 2018-06-05 生命科技公司 Chemical sensor with consistent sensor surface area
EP2972280B1 (en) 2013-03-15 2021-09-29 Life Technologies Corporation Chemical sensor with consistent sensor surface areas
CN103204784B (en) * 2013-04-03 2015-06-10 甘肃科瑞生物科技有限公司 Synthesis method for N-(3-aminopropyl) methacrylamide hydrochloride
US20140336063A1 (en) 2013-05-09 2014-11-13 Life Technologies Corporation Windowed Sequencing
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
KR102266002B1 (en) 2013-07-01 2021-06-16 일루미나, 인코포레이티드 Catalyst-free surface functionalization and polymer grafting
EP3726213A1 (en) 2013-08-19 2020-10-21 Singular Bio Inc. Assays for single molecule detection and use thereof
CN105874385B (en) 2013-12-19 2021-08-20 Illumina公司 Substrate comprising a nanopatterned surface and method for the production thereof
WO2015103225A1 (en) 2013-12-31 2015-07-09 Illumina, Inc. Addressable flow cell using patterned electrodes
GB201408077D0 (en) 2014-05-07 2014-06-18 Illumina Cambridge Ltd Polymethine compounds and their use as fluorescent labels
ES2913205T3 (en) 2014-05-13 2022-06-01 Bioatla Inc Conditionally active biological proteins
US11111288B2 (en) 2014-08-28 2021-09-07 Bioatla, Inc. Conditionally active chimeric antigen receptors for modified t-cells
KR20230172625A (en) 2014-08-28 2023-12-22 바이오아트라, 인코퍼레이티드 Conditionally active chimeric antigen receptors for modified t-cells
WO2016036916A1 (en) 2014-09-03 2016-03-10 Bioatla, Llc Discovering and producing conditionally active biologic proteins in the same eukaryotic cell production hosts
WO2016100486A1 (en) 2014-12-18 2016-06-23 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
CN111505087A (en) 2014-12-18 2020-08-07 生命科技公司 Method and apparatus for measuring analytes using large scale FET arrays
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
WO2016123480A1 (en) 2015-01-29 2016-08-04 Surmodics, Inc. Delivery of hydrophobic active agent particles
CN113637729B (en) 2015-02-18 2024-02-23 因威塔公司 Assay for single molecule detection and uses thereof
EP3262217A4 (en) 2015-02-24 2018-07-18 BioAtla LLC Conditionally active biological proteins
GB201508858D0 (en) 2015-05-22 2015-07-01 Illumina Cambridge Ltd Polymethine compounds with long stokes shifts and their use as fluorescent labels
ES2945607T3 (en) 2015-07-17 2023-07-04 Illumina Inc Polymer sheets for sequencing applications
WO2017019804A2 (en) 2015-07-28 2017-02-02 Plexxikon Inc. Compounds and methods for kinase modulation, and indications therefor
US10478546B2 (en) 2015-09-15 2019-11-19 Surmodics, Inc. Hemodialysis catheter sleeve
GB201516987D0 (en) 2015-09-25 2015-11-11 Illumina Cambridge Ltd Polymethine compounds and their use as fluorescent labels
BR112018011475A2 (en) 2015-12-07 2018-12-04 Plexxikon Inc compounds and methods for kinase modulation and indication for kinase
JP6813247B2 (en) * 2016-03-23 2021-01-13 株式会社ダイセル Stationary phase for chromatography
US10806904B2 (en) 2016-03-31 2020-10-20 Surmodics, Inc. Two-part insertion tool and methods
US10918835B2 (en) 2016-03-31 2021-02-16 Surmodics, Inc. Delivery system for active agent coated balloon
US20170281914A1 (en) 2016-03-31 2017-10-05 Surmodics, Inc. Localized treatment of tissues through transcatheter delivery of active agents
SI3455261T1 (en) 2016-05-13 2023-01-31 Bioatla, Inc. Anti-ror2 antibodies, antibody fragments, their immunoconjugates and uses thereof
EP3458913B1 (en) 2016-05-18 2020-12-23 Illumina, Inc. Self assembled patterning using patterned hydrophobic surfaces
US10391292B2 (en) 2016-06-15 2019-08-27 Surmodics, Inc. Hemostasis sealing device with constriction ring
CN110198756B (en) 2016-09-16 2022-07-12 舒尔默迪克斯公司 Lubricious insertion tool for medical devices and methods of using same
TW201815766A (en) 2016-09-22 2018-05-01 美商普雷辛肯公司 Compounds and methods for IDO and TDO modulation, and indications therefor
US10385214B2 (en) 2016-09-30 2019-08-20 Illumina Cambridge Limited Fluorescent dyes and their uses as biomarkers
ES2937782T3 (en) 2016-10-03 2023-03-31 Illumina Inc Fluorescent detection of amines and hydrazines and methods of testing the same
US10758719B2 (en) 2016-12-15 2020-09-01 Surmodics, Inc. Low-friction sealing devices
US10898446B2 (en) 2016-12-20 2021-01-26 Surmodics, Inc. Delivery of hydrophobic active agents from hydrophilic polyether block amide copolymer surfaces
CN109476674B (en) 2016-12-22 2021-12-10 伊鲁米纳剑桥有限公司 Coumarin compound and application thereof as fluorescent marker
AU2017395023B2 (en) 2016-12-23 2022-04-07 Plexxikon Inc. Compounds and methods for CDK8 modulation and indications therefor
US10428067B2 (en) 2017-06-07 2019-10-01 Plexxikon Inc. Compounds and methods for kinase modulation
GB201711219D0 (en) 2017-07-12 2017-08-23 Illumina Cambridge Ltd Short pendant arm linkers for nucleotides in sequencing applications
GB201716931D0 (en) 2017-10-16 2017-11-29 Illumina Cambridge Ltd New fluorescent compounds and their use as biomarkers
DE202019005610U1 (en) 2018-12-07 2021-06-10 Element Biosciences, Inc. Flow cell device and its use
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
WO2020178231A1 (en) 2019-03-01 2020-09-10 Illumina, Inc. Multiplexed fluorescent detection of analytes
KR20210134210A (en) 2019-03-01 2021-11-09 일루미나 케임브리지 리미티드 Tertiary amine substituted coumarin compounds and their use as fluorescent labels
SG11202012555UA (en) 2019-03-01 2021-01-28 Illumina Cambridge Ltd Exocyclic amine-substituted coumarin compounds and their uses as fluorescent labels
NL2023327B1 (en) 2019-03-01 2020-09-17 Illumina Inc Multiplexed fluorescent detection of analytes
US11421271B2 (en) 2019-03-28 2022-08-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing using photoswitchable labels
KR20220107231A (en) 2019-11-27 2022-08-02 일루미나 케임브리지 리미티드 Dyes and compositions containing cyclooctatetraene
CN115867560A (en) 2020-06-22 2023-03-28 伊鲁米纳剑桥有限公司 Nucleosides and nucleotides having 3' acetal capping groups
US20220033900A1 (en) 2020-07-28 2022-02-03 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
US20220195516A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Methods, systems and compositions for nucleic acid sequencing
US20220195196A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Alkylpyridinium coumarin dyes and uses in sequencing applications
US20220195517A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Long stokes shift chromenoquinoline dyes and uses in sequencing applications
US20220195518A1 (en) 2020-12-22 2022-06-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing
EP4334327A1 (en) 2021-05-05 2024-03-13 Illumina Cambridge Limited Fluorescent dyes containing bis-boron fused heterocycles and uses in sequencing
CA3216735A1 (en) 2021-05-20 2022-11-24 Patrizia IAVICOLI Compositions and methods for sequencing by synthesis
CA3222829A1 (en) 2021-12-16 2023-06-22 Gabriele CANZI Methods for metal directed cleavage of surface-bound polynucleotides
CA3222842A1 (en) 2021-12-20 2023-06-29 Oliver MILLER Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides
CA3223274A1 (en) 2021-12-20 2023-06-29 Illumina, Inc. Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides
WO2023186815A1 (en) 2022-03-28 2023-10-05 Illumina Cambridge Limited Labeled avidin and methods for sequencing
AU2023246772A1 (en) 2022-03-29 2024-01-18 Illumina Inc. Chromenoquinoline dyes and uses in sequencing
AU2023246691A1 (en) 2022-03-30 2024-01-18 Illumina, Inc. Methods for chemical cleavage of surface-bound polynucleotides
WO2023186982A1 (en) 2022-03-31 2023-10-05 Illumina, Inc. Compositions and methods for improving sequencing signals
WO2023192900A1 (en) 2022-03-31 2023-10-05 Illumina Singapore Pte. Ltd. Nucleosides and nucleotides with 3' vinyl blocking group useful in sequencing by synthesis
US20230338623A1 (en) 2022-04-25 2023-10-26 Surmodics, Inc. Medical device coatings with microcrystalline active agents
WO2023232829A1 (en) 2022-05-31 2023-12-07 Illumina, Inc Compositions and methods for nucleic acid sequencing
US20230416279A1 (en) 2022-06-28 2023-12-28 Illumina Cambridge Limited Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing
WO2024039516A1 (en) 2022-08-19 2024-02-22 Illumina, Inc. Third dna base pair site-specific dna detection

Family Cites Families (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4056208A (en) 1976-08-11 1977-11-01 George Wyatt Prejean Caustic-resistant polymer coatings for glass
SE7712058L (en) 1976-11-12 1978-05-13 Ceskoslovenska Akademie Ved THREE DIMENSIONAL CARRIERS OF INORGANIC, POROST MATERIAL AND A REACTIVE POLYMER AND A PROCEDURE FOR THE MANUFACTURE OF IT
US4139577A (en) * 1976-12-07 1979-02-13 Ici Americas Inc. Polymerizable composition comprising a tetracarboxylic ester monomer useful for imparting flame retardance to resin polymers
US4363634A (en) 1980-07-18 1982-12-14 Akzona Incorporated Glass support coated with synthetic polymer for bioprocess
US4357142A (en) 1980-07-18 1982-11-02 Akzona Incorporated Glass support coated with synthetic polymer for bioprocess
US4452102A (en) * 1981-04-23 1984-06-05 New Age Industries, Inc. Epicycloidal gearing
US5073484A (en) 1982-03-09 1991-12-17 Bio-Metric Systems, Inc. Quantitative analysis apparatus and method
US4973493A (en) 1982-09-29 1990-11-27 Bio-Metric Systems, Inc. Method of improving the biocompatibility of solid surfaces
US4722906A (en) 1982-09-29 1988-02-02 Bio-Metric Systems, Inc. Binding reagents and methods
US5217492A (en) 1982-09-29 1993-06-08 Bio-Metric Systems, Inc. Biomolecule attachment to hydrophobic surfaces
US5512329A (en) 1982-09-29 1996-04-30 Bsi Corporation Substrate surface preparation
US5002582A (en) 1982-09-29 1991-03-26 Bio-Metric Systems, Inc. Preparation of polymeric surfaces via covalently attaching polymers
US4994373A (en) 1983-01-27 1991-02-19 Enzo Biochem, Inc. Method and structures employing chemically-labelled polynucleotide probes
US4542102A (en) 1983-07-05 1985-09-17 Molecular Diagnostics, Inc. Coupling of nucleic acids to solid support by photochemical methods
US4582860A (en) 1983-12-15 1986-04-15 Rohm And Haas Company Oxirane resins for enzyme immobilization
US4826759A (en) 1984-10-04 1989-05-02 Bio-Metric Systems, Inc. Field assay for ligands
US4979959A (en) 1986-10-17 1990-12-25 Bio-Metric Systems, Inc. Biocompatible coating for solid surfaces
CA1335721C (en) * 1987-12-24 1995-05-30 Patrick E. Guire Biomolecule attached to a solid surface by means of a spacer and methods of attaching biomolecules to surfaces
US6309822B1 (en) 1989-06-07 2001-10-30 Affymetrix, Inc. Method for comparing copy number of nucleic acid sequences
ATE155524T1 (en) 1990-04-12 1997-08-15 Hans Dr Sigrist METHOD FOR THE LIGHT-INDUCED IMMOBILIZATION OF BIOMOLECULES ON CHEMICALLY INERT SURFACES
KR920009328B1 (en) * 1990-08-18 1992-10-15 삼성전관 주식회사 Method of manufacturing cathode
US5663318A (en) * 1991-03-01 1997-09-02 Pegg; Randall Kevin Assay preparation containing capture and detection polynucleotides covalently bound to substrates with a heterobifunctional crosslinking agent
FR2679255B1 (en) 1991-07-17 1993-10-22 Bio Merieux METHOD OF IMMOBILIZING A NUCLEIC FRAGMENT BY PASSIVE FIXING ON A SOLID SUPPORT, SOLID SUPPORT THUS OBTAINED AND ITS USE.
EP0585436B1 (en) 1992-02-13 2000-05-03 SurModics, Inc. Immobilization of chemical species in crosslinked matrices
US5414075A (en) 1992-11-06 1995-05-09 Bsi Corporation Restrained multifunctional reagent for surface modification
JPH08509751A (en) 1993-01-21 1996-10-15 オレゴン州 Chemical functionalization of the surface
US5610287A (en) 1993-12-06 1997-03-11 Molecular Tool, Inc. Method for immobilizing nucleic acid molecules
US5807522A (en) 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
US5641658A (en) 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
US5643580A (en) 1994-10-17 1997-07-01 Surface Genesis, Inc. Biocompatible coating, medical device using the same and methods
US5707818A (en) 1994-12-13 1998-01-13 Bsi Corporation Device and method for simultaneously performing multiple competitive immunoassays
US6239273B1 (en) 1995-02-27 2001-05-29 Affymetrix, Inc. Printing molecular library arrays
US6033784A (en) 1995-04-07 2000-03-07 Jacobsen; Mogens Havsteen Method of photochemical immobilization of ligands using quinones
DE69634013T2 (en) 1995-05-26 2005-12-15 SurModics, Inc., Eden Prairie PROCESS AND IMPLANTABLE OBJECT FOR PROMOTING ENDOTHELIALIZATION
US5783502A (en) 1995-06-07 1998-07-21 Bsi Corporation Virus inactivating coatings
DE19533682A1 (en) 1995-09-12 1997-03-13 Biotronik Mess & Therapieg Process for depositing and immobilizing heparin on inorganic substrate surfaces of cardiovascular implants
US5714360A (en) 1995-11-03 1998-02-03 Bsi Corporation Photoactivatable water soluble cross-linking agents containing an onium group
US5942555A (en) 1996-03-21 1999-08-24 Surmodics, Inc. Photoactivatable chain transfer agents and semi-telechelic photoactivatable polymers prepared therefrom
AU3568897A (en) 1996-06-07 1998-01-05 Eos Biotechnology, Inc. Immobilised linear oligonucleotide arrays
WO1998020020A2 (en) 1996-11-06 1998-05-14 Sequenom, Inc. High density immobilization of nucleic acids
EP0843019A3 (en) * 1996-11-08 2002-10-09 Kyowa Medex Co., Ltd. Method for determination of specific nucleic acid sequence, and a reagent therefor
US5919626A (en) 1997-06-06 1999-07-06 Orchid Bio Computer, Inc. Attachment of unmodified nucleic acids to silanized solid phase surfaces
JP4313861B2 (en) 1997-08-01 2009-08-12 キヤノン株式会社 Manufacturing method of probe array
US6121027A (en) * 1997-08-15 2000-09-19 Surmodics, Inc. Polybifunctional reagent having a polymeric backbone and photoreactive moieties and bioactive groups
US5858653A (en) 1997-09-30 1999-01-12 Surmodics, Inc. Reagent and method for attaching target molecules to a surface
US6762019B2 (en) * 1997-09-30 2004-07-13 Surmodics, Inc. Epoxide polymer surfaces
US6465178B2 (en) 1997-09-30 2002-10-15 Surmodics, Inc. Target molecule attachment to surfaces
DE19804518C2 (en) 1998-02-05 2000-10-05 Roehm Gmbh Process for the production of pearl-shaped copolymers based on acrylate, carrier polymer materials produced thereafter and their use
US6410044B1 (en) * 1998-03-19 2002-06-25 Surmodics, Inc. Crosslinkable macromers
AU3628999A (en) 1998-05-08 1999-11-29 Kyowa Medex Co., Ltd. Method for quantitating specific gene sequence and quantitating reagent
US6551557B1 (en) 1998-07-07 2003-04-22 Cartesian Technologies, Inc. Tip design and random access array for microfluidic transfer
US6391937B1 (en) 1998-11-25 2002-05-21 Motorola, Inc. Polyacrylamide hydrogels and hydrogel arrays made from polyacrylamide reactive prepolymers
CA2335377C (en) 2001-03-05 2010-12-14 Bioneer Corporation Bio-molecular microchip and manufacturing process thereof
US6444318B1 (en) * 2001-07-17 2002-09-03 Surmodics, Inc. Self assembling monolayer compositions

Also Published As

Publication number Publication date
US6465178B2 (en) 2002-10-15
AU2497900A (en) 2000-07-24
AU778265B2 (en) 2004-11-25
EP1141385B1 (en) 2017-07-05
US20010014448A1 (en) 2001-08-16
WO2000040593A2 (en) 2000-07-13
US7691787B2 (en) 2010-04-06
US20030148308A1 (en) 2003-08-07
US20050170427A1 (en) 2005-08-04
EP1141385A2 (en) 2001-10-10
WO2000040593A3 (en) 2000-12-28
JP4768130B2 (en) 2011-09-07
JP2002534663A (en) 2002-10-15
CA2360000A1 (en) 2000-07-13

Similar Documents

Publication Publication Date Title
CA2360000C (en) Target molecule attachment to surfaces
EP1019424B1 (en) Reagent and method for attaching target molecules to a surface
US7309593B2 (en) Attachment of molecules to surfaces
JP5005869B2 (en) Epoxide polymer surface
MXPA01006935A (en) Target molecule attachment to surfaces
MXPA00003045A (en) Reagent and method for attaching target molecules to a surface

Legal Events

Date Code Title Description
EEER Examination request
MKEX Expiry

Effective date: 20200110