Three dimensional format biochips

专利摘要:
A biochip formed of a plurality of optically transparent hydrogel cells attached to an upper surface of a solid substrate in the form of an array. Each cell is formed from a hydrogel of polyethylene glycol, polypropylene glycol or polymers thereof having active isocyanate groups. Non-hybridized binding entities are immobilized in these cells, which are effective for selective sequestration of target proteins or other comparable molecules. Different binding entities are immobilized on the cells to produce biochips that are used for analysis for multiple target biomolecules.
公开号:KR20020089315A
申请号:KR1020027008285
申请日:2001-10-26
公开日:2002-11-29
发明作者:파그나니로베르토；한순갑；동시아오팡；피르쉐토니；마쯔모토산드라；친버그파벨
申请人:바이오셉트 인코포레이티드；
IPC主号:

专利说明:

3D Format Biochips {THREE DIMENSIONAL FORMAT BIOCHIPS}
[5] Currently, there are many known methods used to study protein-ligand, protein-protein, protein-DNA interactions. These methods all have significant limitations in that they are cumbersome, expensive, require large amounts of protein, or are not suitable for rapid high throughput analysis of protein interactions.
[6] The initial method used to study protein interactions is protein-affinity columns. In this method, the capture protein is covalently immobilized to agarose beads and used to affinity-purify the target protein from a heterogeneous mixture containing a large number of contaminating proteins through the use of affinity chromatography. This method requires a relatively large amount of capture protein for proper fixation to agarose beads, which is not suitable for rapid high throughput screening of protein interactions.
[7] Another method used to study protein interactions is the yeast two-hybrid system. In this method, a target protein library is constructed in yeast. This system is designed to express these corresponding proteins, each of which is linked to a transcriptional activation region. DNA encoding a bait protein (or a protein tested for other possible interacting proteins) is fused to a DNA binding domain and also expressed within the same library. Also included are reporter genes that carry DNA sequences corresponding to the genetic code for the detection system, such as fluorescent proteins or proteins that are readily detectable for biological activity. When the target protein of interest binds to the bait protein, subsequent interaction between the two achieves the activation of the reporter gene and results in signal generation. Although this method is used relatively frequently, it is slow and cumbersome, requires considerable molecular biology expertise and does not provide rapid high throughput screening of economical protein-protein interactions.
[8] Another early method commonly adopted for studying protein interactions is to immunoprecipitate both capture and target proteins, and then analyze the resulting complexes using polyacrylamide gel electrophoresis. In this method, the capture protein is first incubated with a heterogeneous mixture of proteins to bind to the target. The resulting complex is then immunoprecipitated using antibodies positive for 1 protein in a pair, and the complex is separated by gel electrophoresis for analysis and followed by a detection step such as staining with a dye. This method is slow and cumbersome, requires significant biochemical expertise and similarly does not confer rapid high throughput analysis of protein interactions.
[9] Another method used to study protein interactions is phage display. In this method, the protein library is a flagella of specific filamentous phage proteins expressed on the surface of host bacteria, such as, for example, E. coli, to provide an affinity support for such "displayed" proteins. Expressed in the stomach. Phage libraries are then exposed to a number of potential target proteins. Binding of the displayed protein to the target protein allows for identification of the target. This method has a number of limitations, for example, high molecular weight proteins are difficult to display, and only a few phage filamentous proteins are suitable for this use. In addition, the structural compaction of the displayed protein is known to reduce its affinity and consequently affect its ability to bind to its natural ligands.
[10] The preparation of high-throughput-capable microarrays or biochips suitable for binding of entities such as proteins can be used for surface detection in such a way that they can later be used for detection by easily interacting with the molecules of interest, such as proteins or other substances often referred to as targets. It will generally require the use of methods of attaching proteins to them. For example, a protein can bind directly to a surface treated with divalent or trivalent metal ions such as Cu ²⁺ , Fe ^3+, and the protein will naturally bind according to varying degrees of affinity. If the target then binds to the probes, they can be detected and identified by SELDI ^™ (surface-enhanced laser desorption / ionization) in combination with a mass spectrometer, as described in US Pat. No. 5,719,060. In alternative methods described by G. MacBeath and S. Schreiber ( Science 289 : 1760, 2000), chemical bonds are also used to attach proteins to the substrate surface, while target ligands are labeled with fluorescent tags; Thus, any interaction between the probe and the labeled target can be detected using a fluorescence-based slide scanner.
[11] Since the protein fixation methods for providing binding entities generally employ direct chemical conjugation of the protein to the surface of the substrate, these methods may be responsible for protein function due to improper chemical conjugation at the active site or loss of native structure. It is embodied as a major limitation that results in loss. When this happens, only a small amount of immobilized protein remains active, resulting in difficulty in detection and low assay sensitivity. In addition, the lack of complexity and precision of these methods generally imposes inadequacies for use in the manufacture of high throughput applications high density microarrays.
[12] US Pat. No. 6,087,102 describes a method for utilizing polyacrylamide gels to create individual cells consisting of electrophoretic protein spots that can be subsequently crosslinked in situ into the gel to form biochips. Limitations of this method include the challenge of potential disruption effects on capture proteins during the fabrication and crosslinking of fine small cells on biochips. U. S. Patent No. 5,847, 019 describes another approach to fabricating biochips using photo-reactive free-radical chemistry to form patterned network layers by utilizing photopolymerizable polymers. This photoactivation approach used to anchor proteins to biochips appears to be limited to specific photoactivation chemicals, including acrylamide polymers, and also the use of free radical photochemistry capture proteins used in biochips manufactured in this way. May cause potential free radical damage.
[13] The use of isocyanate-capping liquid polyurethane prepolymers with proteins to directly fix proteins in polyurethane foams is described in US Pat. Nos. 4,098,645 and 3,672,955, which describe proteins through amino and hydroxyl moieties of isocyanate-functional hydrogel systems. Direct binding to an enzyme / antibody reaction based on enzyme reactors and affinity columns. While the described method may be suitable for this purpose, these methods do not form geometrically controlled optically transparent hydrogels suitable for biochip applications. In addition, using these methods without inhibiting potential conjugation to protein residues can very easily cause undesirable crosslinking of the protein to the polymer, and excessive crosslinking may reduce or destroy the protein's original structure. And thus reduce the biological activity of the protein, which would impose this method unsuitable for high precision binding assays in which biochips are used.
[14] Despite these technical hurdles, the importance of understanding interactions with protein-proteins and other comparable biomolecules is practical and suitable for the incorporation of many different non-hybridized binding entities, tools designed for various studies, and commercial applications in biological sciences. To achieve flexible format biochips. Briefly, there is a need for an efficient fixation or support system that provides a support for an entity in a manner that maintains maximum binding activity and thus allows fabrication of microarrays based on nonnucleic acid / hybridization.
[15] In the other methods mentioned, it is desirable to provide a method that allows binding entities such as proteins to be fixed or encapsulated in a manner that allows them to retain their original structure and function so that they can be free to sequester the target.
[16] Summary of the Invention
[17] By providing a suitable gel with the desired immobilization chemistry, microarrays or biochips can be provided that incorporate a number of different nonhybridized binding entities into an array in a three-dimensional format suitable for high throughput analysis and characterization of biomolecule interactions. This has been found in the present invention.
[18] The present invention provides a biochip in the form of an array of optically transparent PEG or PPG-based polymer microdroplets arranged on a solid substrate, providing formation capacity equivalent to 1,000 individual reaction cells per square centimeter. Each cell will typically contain at least one binding entity immobilized on or within the volume of the microdroplets. By altering the different binding entities in the cells in the array in a known manner, efficient screening of compounds or activities for biological samples or binding interactions can be performed and quantified. These cells are in the form of maximizing the amount of binding entity contained in each cell and thus maximizing detection sensitivity, preferably in three dimensions.
[19] The polymer microdroplets that form each cell of such a biochip are selected to provide an environment that contributes to maintaining the original structure of a fixed binding entity such as, for example, a protein or peptide. This resulting polymer is preferably a hydrogel that is physically and chemically stable to allow subsequent washing and other liquid processing steps and handling to be employed in the manufacture and use of biochips. Polyethylene glycol-based prepolymers having isocyanate-functional active groups are preferably utilized, and when polymerized, polyethylene glycol hydrogel networks are formed, reinforced and crosslinked by urethane bonds. After initiation of the polymerization reaction, the prepolymer is microspotted to form an array of three-dimensional reaction cells and to fully polymerize onto the biochip substrate. Specific polymer chemistries for cells are described in US Pat. No. 6,174,683 and International Patent Application Publication No. WO 00/65097, which describe biochips using nucleic acid oligomers as hybridization capture probes. As a result of technological advances, it has been found that polyethylene glycol, polypropylene glycol, or copolymers thereof can be efficiently employed in the manufacture of three-dimensional biochips suitable for containing a variety of non-hybridized binding entities, including proteins and peptides. The present invention has been found.
[20] In one particular aspect, the present invention provides a composition comprising (a) a solid substrate having a surface; (b) at least one optically transparent hydrogel cell formed from an isocyanate-acting polymer and attached to the surface of the substrate; (c) Provides an optically transparent hydrogel biochip comprising non-hybridized binding entities immobilized on or on the hydrogel cell, which are effective for selectively sequestering a target protein or other comparable molecule.
[21] In another particular aspect, the invention provides (a) an optically transparent hydrogel biochip having a substrate having a surface to which at least two hydrogel cells are bound, each cell having a thickness of at least about 30 μm and preferentially polyethylene glycol , Hydrogel cells comprising polyethylene glycol or copolymers thereof, each hydrogel cell comprising a different protein binding entity immobilized therein or on and (b) a hydrogel with an assay solution containing the target biomolecule under binding conditions Contacting the biochip; (c) washing the hydrogel biochip under conditions that remove non-selective and unbound target biomolecules; (d) providing a method of using a biochip for performing a biochemical analysis, comprising detecting a target biomolecule bound to one of the cells.
[22] In another particular aspect, the present invention provides an organic solvent solution of (a) an isocyanate-functional hydrogel prepolymer; (b) providing a solution of the hybridized binding entity; (c) the binding entity is covalently bound to the isocyanate-functional hydrogel prepolymer via reaction with up to 15% of the active isocyanate; (d) initiating polymerization of the isocyanate-functional hydrogel prepolymer under conditions that will produce an optically clear hydrogel; (e) dispensing the polymerized isocyanate-acting hydrogel prepolymer in the form of droplets onto a solid substrate, wherein the optically clear hydrogel prepolymer containing the binding entity is attached to the substrate, or immobilized on the biochip. And an optically clear isocyanate-acting hydrogel biochip having a non-hydrided binding entity that is efficient for selectively sequestering a target protein or target biomolecule.
[23] In another particular aspect, the present invention provides an organic solvent solution of (a) an isocyanate-functional hydrogel prepolymer; (b) providing a solution of the desired protein capture agent; (c) covalently coupling the mediating coupling agent for the protein with an isocyanate-functional hydrogel prepolymer; (d) initiates polymerization of said isocyanate-functional hydrogel prepolymer; (e) dispensing droplets of polymerized isocyanate-functional hydrogel prepolymer onto a solid substrate so that the polymer adheres to the substrate; (f) exposing each hydrogel droplet to one of the desired protein solutions to fix the protein capture agent inside or on a biochip via linkage with the coupling agent, thereby polymerizing the droplets with different protein capture agents. Provided is a method of making an isocyanate-acting hydrogel biochip having a protein selected to be immobilized within or on the biochip, which acts as a capture agent, comprising producing a biochip having a plurality of cells.
[24] In another particular aspect, the present invention provides an article of manufacture comprising: (a) a solid substrate having an upper surface; (b) a plurality of hydrogel cells comprising polyethylene glycol, polypropylene glycol, or copolymers thereof bonded to the upper surface of the substrate; (c) a mediator immobilized in or on the hydrogel of the cell; (d) interacting in such a way that the protein binding entity can exhibit its original structure, thereby providing a hydrogel biochip comprising binding different protein binding entities to at least several mediators within the hydrogel cell. .
[25] In another particular aspect, the present invention provides an organic solvent solution of (a) an isocyanate-functional hydrogel prepolymer; (b) initiate the polymerization of the isocyanate-functional hydrogel prepolymer; (c) distributing the polymerized isocyanate-functional hydrogel prepolymer into a solid substrate such that droplets can adhere to the substrate and form multiple cells; (d) physically immobilizing different proteins selected within or on the biochip, wherein at least two of each cell are selected to act as a binding agent to selectively sequester specific biomolecules. Eggplants provide a method of making an isocyanate-acting hydrogel biochip with multiple cells.
[1] Microarrays composed of immobilized nucleic acids such as DNA have demonstrated tremendous utility in high throughput analysis and characterization of biological samples. Through analysis of such samples using multiple combinations of biological probes on nucleic acid biochips, information regarding the nucleic acid components of the sample is obtained. Such biochips use simple flat plates, such as glass slides, or recessed or well formed plates. Typically, the various sequences, DNA, ie single stranded DNA, RNA or PNA of the nucleic acid oligomer are fixed in a form that can then hybridize to complementary sequences from a sample, which is described in U.S. See patent no. 6,242,246. Thanks to the specificity of this hybridization and the ability to quickly examine various combinations of nucleic acid sequences, the data obtained are useful for determining gene expression and sequence characteristics from a sample. These data may be key factors in determining the genetic basis of disease mechanisms and in identifying potential diagnostic and therapeutic targets.
[2] Nucleic acid microarray methods utilize available DNA synthesizers, PCR methods, and developing gene target information. Antibodies, for example, that can be used to allow other high throughput assays that utilize non-hydriding interactions, which can potentially provide a route towards new biological insights that are not easily deduced from the use of nucleic acid microarrays. Or promising interest exists in extending the use of such microarrays in the immobilization of other binding entities of biological interest such as other proteins.
[3] However, compared to nucleic acid biochips, it is anticipated that difficulties will be encountered in fabricating biochips capable of appropriately immobilizing many different binding entities such as proteins and peptides. Immobilization chemistries commonly used for immobilization of materials such as proteins frequently induce denaturation of these materials because they adhere or are in direct contact with the solid support surface. In addition, attachment chemistry may be limited because of multiple competitively active residues present on multiple binding entities, such as proteins, independent of nucleic acids, and the structure of many other binding entities, such as proteins, may be their biological activity. It is well known that it is key to preserving and can be easily destroyed as it is immobilized via molecular multiple moieties.
[4] This potential loss of activity becomes even more important in such situations where detection of the target protein will be inherently more difficult due to the ineffectiveness of DNA amplification methods such as polymerase chain reaction (PCR). That is, in many cases, only a very limited amount of binding entity, such as a protein, isolated from a tissue sample is available, and the lack of the ability to produce large quantities of these materials in an easy and convenient manner will interfere with such an assay, as in nucleic acids. .
[26] 1 is a schematic of hydrogel polymerization following reaction of a hydrogel prepolymer with a protein in an organic solvent, embodying various features of the invention as part of the process.
[27] 2 is a schematic of an alternative reaction of a hydrogel prepolymer with a protein in water during the polymerization of the hydrogel.
[28] 3A and 3B are schematic diagrams of another alternative reaction of the hydrogel prepolymer with a chelating agent, followed by chelating with metals and subsequent binding to proteins containing multiple histidines in the tail.
[29] 4A is a schematic diagram of the experiment described in Example 4. FIG.
[30] 4B is an illustration of two single stranded nucleic acids used in Example 4. FIG.
[31] 5 is a schematic of the experiments performed in Example 5. FIG.
[32] 6 is a schematic of the experiments performed in Example 9. FIG.
[33] The hydrogel preferably has a suitable pore size and high moisture content to allow diffusion of molecules into and out of the matrix, the ability to bind to the surface of the glass or analog, and to minimize any optical interference due to the fluorescent tag in the fully polymerized state. It is a class of polymers that can provide gel matrices with sufficient optical clarity, good structural integrity when fully polymerized, and suitable shelf life for common research and medical applications. Hydrogels are hydrophilic network polymers that are like glass in the dehydrated state and swell and form elastic gels when water is present. Isocyanate-acting hydrogels have a number of features that can serve as advantages in the immobilization of non-hybridized bond entities such as, for example, proteins. Isocyanate-acting hydrogels refer to organic polymers capped with isocyanate groups to effect the desired addition polymerization and also to covalently bond with proteins or analogs or in the case of protein attachment. For example, polyurethane polymers that are well known in the art and that can be formed by reaction between diisocyanates and polyether or polyester polyols can provide suitable hydrogels for this purpose.
[34] The prepolymer is preferably used as starting material in forming the biochip using this isocyanate-functional hydrogel, and also preferably the prepolymer is formulated and hydrated polyurethane, polyurea-urethane and / or polyurea polymer gel To provide. Hydrogel polymers have been prepared from a variety of prepolymers and are used in a wide variety of applications. Typically, hydrogels are formed by polymerizing hydrophilic monomers in an aqueous solution under conditions such that the lightly crosslinked prepolymer has a three-dimensional polymer network, which is a gel in concentrated form, and is initially formed. Polyurethane hydrogels are formed through the production of urea and urethane linkages by polymerizing isocyanate-terminated-capped prepolymers.
[35] Suitable isocyanate-functional prepolymers are often prepared from relatively high molecular weight polyoxyalkylene diols or polyols which react with bi- or multi-functional isocyanate compounds. Preferred prepolymers are prepared from polyoxyalkylene diols or polyols which may comprise a single polymer of ethylene oxide units or blocks or random copolymers containing ethylene oxide units and propylene oxide or butylene oxide units. In the case of block or random copolymers, preferably at least 75% of the units should be ethylene oxide units. Alternatively, although less preferred, homopolymers of polypropylene oxide may also be used. The polyoxyalkylene diol or polyol molecular weight is preferably 2,000 to 30,000, more preferably 5,000 to 30,000. Suitable prepolymers can be prepared, for example, by reacting selected polyoxyalkylene diols or polyols with polyisocyanates at an isocyanate to hydroxy ratio of from about 1.2 to about 2.2, where essentially all of the hydroxy groups are capped with polyisocyanates. have. The isocyanate-functional prepolymer should preferably contain an amount of active isocyanate of about 0.1 to about 1.2 meq / g, preferably about 0.2 to about 0.8 meq / g. In general, considerably lower molecular weight prepolymers, for example up to 3,000 MW, should preferably contain a relatively high isocyanate content (about 1 meq / g or more). The polymerization rate of such prepolymers should be controlled so as not to polymerize too quickly for efficient microspotting, and from this point of view, high molecular weight prepolymers containing relatively low isocyanate content are usually preferred.
[36] Such high molecular weight prepolymers (1) react a polyol (triol or higher) having a molecular weight of at least 2,000 with a polyisocyanate, such as isophorone diisocyanate, or (2) diol having a molecular weight of at least 2,000, Although often prepared in one of two general ways of reacting with a crosslinking agent such as glycerol, trimethylolpropane, trimethylolethane, triethanolamine or organic triamine, etc., other methods not known in the art may also be used.
[37] Aromatic, aliphatic or cycloaliphatic polyisocyanates can be used. High molecular weight aliphatic isocyanate-capped prepolymers typically gel in the hydrated polymer state within about 20 to 90 minutes, while prepolymers capped with aromatic polyisocyanates gel much more rapidly. Examples of suitable 2- or multi-functional isocyanates are as follows: toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, isophorone diisocyanate, ethylene diisocyanate, ethylidine diisocyanate, propylene- 1,2-diisocyanate, cyclobexylene-1,2-diisocyanate, cyclohexylene-1,4-diisocyanate, phenylene diisocyanate, 3,3 "-diphenyl-4,4" -biphenylene Diisocyanate, 1,6-hexamethylene diisocyanate, 1,4-tetramethylene diisocyanate, 1,10-decamethylene diisocyanate, cumene-2,4-diisocyanate, 1,5-naphthalene diisocyanate, methylene dicyclo Hexyl diisocyanate, 1,4-cyclohexylene diisocyanate, p-tetramethyl xylylene diisocyanate, p-phenylene diisocyanate, 4-methoxy-1,3-phenylene diisocyanate , 4-chloro-1,3-phenylene diisocyanate, 4-bromo-1,3-phenylene diisocyanate, 4-ethoxyl-1,3-phenylene diisocyanate, 2,4-dimethyl-1, 3-phenylene diisocyanate, 2,4-dimethyl-1,3-phenylene diisocyanate, 5,6-dimethyl-1,3-phenylene diisocyanate, 1,4-diisocyanatodiphenyl ether, 4, 4'-diisocyanatodi-phenylether, benzidine diisocyanate, 4,6-dimethyl-1,3-phenylene diisocyanate, 9,10-anthracene diisocyanate, 4,4'-diisocyanatodibenzyl, 3 , 3'-dimethyl-4,4'-diisocyanatodiphenylmethane, 1,6-dimethyl-4,4'-diisocyanatodiphenyl, 2,4-diisocyanatostibene, 3,3'- Dimethoxy-4,4'-diisocyanatodiphenyl, 1,4-anthracene diisocyanate, 2,5-fluoronediisocyanate, 1,8-naphthalene diisocyanate, 2,6-diisocyanatobenzulurane, 2 , 4,6-toluene triisocyanate, p, p ', p'-triphenylmethane triisocyanate, trifunctional trimer of isophorone diisocyanate (isocyanurate), trifunctional burette of hexamethylene diisocyanate, trifunctional trimer of hexamethylene diisocyanate (isocyanurate ), Polymer 4,4'-diphenylmethane diisocyanate, xylylene diisocyanate and m-tetramethyl xylene diisocyanate.
[38] Capping the selected diol or polyol with polyisocyanates to form the prepolymer can be accomplished using stoichiometry of the reactants. The ratio of isocyanate to hydroxy groups can vary as is known in the art, but should preferably be from about 1 to about 3, and more preferably from about 1.2 to about 2.2. The capping reaction can be carried out at about 20 to about 150 ° C. using any suitable conditions, such as under dry nitrogen, for about 2 hours to about 14 days, preferably in the absence of catalyst. Preferred temperatures are about 60 to 100 ° C. and the reaction ends when the isocyanate concentrations are close to theory.
[39] Preferred prepolymers include polyethylene glycol end-capped with toluene diisocyanate: copolymers of ethylene oxide and propylene oxide (optionally with trimethylolpropane) and toluene diisocyanate; Toluene diisocyanate-polyethylene glycol-trimethylolpropane, methylene diisocyanate-methylene homopolymer; Polymeric methylene diisocyanate-polyethylene glycol; Ethylene oxide-propylene oxide-trimethylolpropane and isophorone diisocyanate, and polymers of polyethylene glycol trilactate and toluene diisocyanate. Suitable prepolymers of this type are available as HYPOL PreMA ^R G-50, HYPOL ^R 2000, HYPOL ^R 3000, HYPOL ^R 4000 and HYPOL ^R 5000 from Hampshire Chemical Corp. of Lexington, Mass., And these formulations are generally polyethylene oxide And small amounts of copolymers of polypropylene oxide.
[40] The main chain of the hydrogel polymer in which all are considered preferably comprises polyethylene glycol, polypropylene glycol, or copolymers of polyethylene glycol and polypropylene glycol. Without being bound by any theoretical mechanism, the nonionic, hydrophilic character of polyethylene glycol and polypropylene glycol hydrogels is a good way to maintain low levels of nonspecific binding of analytes to hydrogels and to maintain the original structure and biological reactivity of immobilized biomolecules. Provide conformance. Isocyanate-functional hydrogels advantageously quickly absorb large amounts of liquid in a relatively constant manner such that the basic overall shape of the gel material is maintained. In addition, the moisture absorbed by this material remains in the absorbent material even under the applied pressure. Polyurethane based isocyanate-acting hydrogels of this general type are described in US Pat. Nos. 3,939,123 (Mathews, et al.), 4,110,286 (Vandegaer, et al) and 4,098,645 (Hartdegan, et al). Such polyurethane-based hydrogels have been widely used as surface coatings and also in flexible or rigid forms; It has also been used to form foam for enzyme reactor systems.
[41] In a preferred embodiment, the biochip is based on high molecular weight polyethylene oxide, diols or triols of polypropylene oxide, or copolymers of polyethylene oxide and polypropylene oxide, capped with water-active diisocyanate, and optionally with a lightly suitable crosslinker. It is made using an isocyanate-functional hydrogel that can be crosslinked. The amount of active isocyanate present in the prepolymer is for example predictable as an amount of about 0.1 to 1 meq / g, but not more than 0.8 meq / g. Generally preferred diisocyanates include aromatic diisocyanates such as toluene diisocyanate or methylene diphenyl-isocyanate and aliphatic diisocyanates such as isophorone diisocyanate. Preferably, about 15% to about 5% of the active isocyanate in the polymer is used to provide a site for fixing the binding entity, and more preferably 10% or less of the active isocyanate in the prepolymer anchors the binding entity. It is used to Polymerization of prepolymers for biochip fabrication, which may be preformulated in water-miscible organic solvents, takes place by the formation of urea bonds that simply occur with the addition of water.
[42] The binding entity may be immobilized directly or indirectly before, during, or after polymerization of the cell material in each cell or microdroplet. Indirect fixation envisages the adoption of a mediator that can be used as a secondary mediator that is primarily linked to and also to the hydrogel. For example, the primary mediator encapsulated in a hydrogel matrix can be an antibody against calmodulin. Once the calmodulin binds to the antibody, the calmodulin acts as a secondary mediator when used to sequester a calmodulin-binding-protein, such as calcium / chalmodulin dependent kinase II. This approach of attaching CaM kinase II (term) to hydrogels provides a good way to fix proteins via naturally occurring binding motifs, ie, calmodulin proteins. CaM kinase II is now liberated in probe assay solutions aimed at examining, for example, the regulatory events (phosphorylation, dephosphorylation) of CaM kinase II or investigating possible docking proteins or other intracellular trafficking proteins. Alternative methods of immobilizing CaM kinase II can lead to loss of function or other deleterious effects.
[43] The term binding entity is used to refer to a substance that can interact in a particular way with one or more target substances to physically sequester the target substance by a mechanism other than hybridization. These nonhybridized binding entities include receptors, peptides, enzymes, enzyme inhibitors, enzyme substrates, immunoglobulins such as antibodies, antigens, lectins, modified proteins, modified peptides, double-stranded DNA, biological origin amines and complex carbohydrates. Biological materials such as, but not limited to; It may also include, for example, drugs or synthetic ligands designed to have this type of specific binding activity. By “modified” protein or polypeptide is meant a protein or peptide having one or more amino acids in the substance modified by any combination of addition of new chemical residues, removal or removal and addition of existing chemical residues. This change may include both natural and artificial modifications. Natural modifications include, but are not limited to, phosphorylation, sulfation, saccharification, nucleic acid addition, and lipidation. Artificial alterations include, but are not limited to, chemical linkers to facilitate binding to hydrogels, and the addition of fluorescent dyes, microstructures, nanostructures such as quantum dots or other artificial materials. In addition, modifications may include removal of functional groups present, such as hydroxy, sulfhydryl or phenyl groups, or removal or alteration of the original side chain or polypeptide amide backbone. Complex hydrocarbons include natural and artificial straight and branched chain oligosaccharides, glycolipids, peptidoglycans, modified polysaccharides such as glycosaminoglycans or acetylated species, and heterologous oligosaccharides such as N-acetylglucosamine or sulfated species. Including but not limited to. Also included are their artificial modifications, such as the addition of molecules such as drugs, ligands, dyes or other agents useful for screening and quantitative purposes. Examples of naturally occurring complex hydrocarbons include hydrocarbon residues found on chitin, hyaluronic acid, keratan sulfate, chondroitan sulfate, heparin, cellulose and modified proteins such as albumin and IgG. Combinations of two or more of these beings will be fixed at some position on the microchip array, which combinations may be added as one mixture of the two beings or added sequentially.
[44] Depicting the interaction between an immobilized entity and a target in sequestration or dehybridization binding involves the use of two or more substances in certain selective ways, typically covalent or non-covalent (eg, van der Waals forces and / or ionic interactions). By means of action). Certain targets may be simple substances that may be present in a complex mixture of biological or synthetic materials. Sequestration or binding may be transient, such as during covalent modifications or extended natural reactions such as antigen-antibody reactions, or for example during phosphorylation events.
[45] DNA binding entities include, but are not limited to, synthetic and natural double-stranded polymers of deoxyribonucleotides, synthetic and natural polyribonucleotides, aptamers, synthetic polynucleotides having one or more modified or non-naturally occurring chemical entities It is not limited. This use of DNA as a binder / isolator is arranged in conventional nucleotide hybrid arrays that typically use a single strand of DNA (oligonucleotide or cDNA) to which the target DNA hybridizes. Double-stranded DNA can be used to interact with DNA binding proteins, suitable biomolecules such as transcription factors such as estrogen receptors, or synthetic agents or molecules for binding or sequestering biomolecules (as opposed to hybridization). For example, general transcription factors such as TBP or SP1, or specific transcription factors such as nuclear hormone receptors can be attracted and sequestered by helix, double stranded DNA. Aptamers are described in US Pat. No. 5,840,867 which appears to act like a monoclonal antibody.
[46] Alternatively, one aspect may use initial binding entities, such as selective antibodies physically copolymerized in the gel matrix or other selective binders such as aptamers, where one or more different antibodies will be immobilized to each cell of the array. . Subsequent application of a complex mixture of biological materials to such an array will inherent binding properties of these immobilized antibodies contained in each cell will create a new array that "self-classifies" and "self-assembles" this complex mixture. ; The new array will be complementary to the initial binding entity. For example, antibodies to specific antigens are immobilized on each gel microdroplet during polymerization; Thereafter, by exposing a mixture of protein or peptide antigens to such an array, specific protein or peptide antigens are provided to bind to each cognate antibody. One example of the use of such mediated antibody arrays is to self-classify a complex mixture of proteins from cell extracts without the need for separate isolation of each protein. The array thus formed can then be used to assess what effect it has on each exposure site, to protein kinases or other protein-modified residues added later. This concept can be extended to examine whether such modifying activity can be affected by drugs or other added chemical compounds.
[47] As a further alternative, other binding entities may be located or immobilized in each cell of the biochip array after polymerization through the use of an agent to be initially immobilized. For example, a suitable mediator, such as Protein A, is immobilized during polymerization, after which the desired immunoglobulin capture agent is bound to immobilized Protein A through controlled exposure to immunoglobulins in solution.
[48] In other embodiments, the initially immobilized binding entity can be modified later. Such modifications include (a) biological modifications, such as, for example, phosphorylation, glycosylation, acetylation, methylation, ubiquitination, lipid modification, and ADP-ribosylation, or (b) eg, dye modifications, biotinylation, alkylation and abnormalities. Non-biological modifications such as residue incorporation and conjugation with proteins or enzymes that produce a modified final form of the array. In another embodiment, the double-stranded nucleic acid oligonucleotide (or polymer) is immobilized during the polymerization; The protein of interest is then bound to this nucleic acid by nucleic acid sequence-specific protein interactions, producing a self-assembled protein-nucleic acid complex array.
[49] By first reacting the prepolymer with the binding entity in an aprotic solvent, the binding entity is effectively immobilized on the prepolymer, and this process can have a number of advantages in the preparation of the hydrogel. This may help later generation of a homogeneous solution of the prepolymer in water and also contribute to lowering the generation of carbon dioxide during the polymerization step by lowering the viscosity of the polymerization mixture to allow the CO ₂ to slow and foam. In the polymerization step of the polyurethane-based hydrogel, for example, some gaseous carbon dioxide is produced by the reaction of isocyanate groups with water of the hydrogel prepolymer. This reaction is illustrated in FIGS. 1 and 2, which may be advantageous for controlling the generation of carbon dioxide gas and its escape from the gel when biochips are made from the prepolymer. If the polymerization takes place too quickly in a high viscosity mixture, the resulting carbon dioxide cannot escape and become trapped in the gel. This results in an irregular foam matrix that can be a problem in the continuity of the gel matrix, and can also interfere with optical clarity. In biochip designs, the greater the optical clarity, the more accurate the detection of fluorescence, which may be an indicator of successful target binding. One way to control carbon dioxide generation is to maintain the pH at about 8.5 or below to control the reaction rate and thus the diffusion of carbon dioxide in the polymerization solution.
[50] A further advantage of derivatizing the hydrogel in an aprotic solvent is to minimize the precipitation of the prepolymer to improve the optical or transparency of the hydrogel. Therefore another method of achieving slow polymerization of the gel and slow production of CO ₂ to provide a continuous and transparent gel matrix provides a ratio of aprotic solvent to water of at least about 0.25 to 1 and more preferably more, eg For 0.3-0.35 to 1. Derivatization and polymerization of the hydrogels are generally completed in about 30 minutes at this ratio. The amount of binding entity bound to the prepolymer of any cell can be adjusted by simply varying the amount of binding entity added to the reaction (eg, from about 10 fmol to about 1 pmol of protein for each microdroplet, so that each hydro Allows for precise control of the amount of bound binding entity in the gel microdroplets.
[51] The ease of diffusion that allows promising target molecules or other secondary binding entities through the gel to react with immobilized mediators or primary binding entities in the gel matrix will in some cases be determined by the percentage of hydrogel prepolymer in the solution used. Can be. The '683 patent describes the adoption of a 5% solution of prepolymer to formulate hydrogel droplets; However, at the 5% level, diffusion of larger materials, such as proteins, into polymerized hydrogels is slower than desired. It has been found that the lower the percentage of the prepolymer, for example 3.5% is preferred to facilitate the passage of large material into the hydrogel. However, in up to about 3% prepolymer solution, the resulting gel may lack sufficient structural integrity and / or suitable polymerization to be useful. Therefore, for various applications using antibodies as visualization tools, the preferred range of polymer is considered to be about 3% to 5%. Other applications or uses for testing substances of a smaller size than typical antibodies, such as, for example, IgG (or larger when the gel encloses or immobilizes the microspheres, for example), have a high or low percentage of polymer in solution, respectively. Can be adopted.
[52] When the hydrogel is firstly derivatized with protein and deposited with a solid substrate, after the start and before completion of the polymerization, the transport can be carried out by any convenient method; For example, a conventional microspotting machine may be used that deposits a gel to form an array of microdroplets. Such gels are inherently noncovalently attached to some substrates, while the substrate surface is generally derivatized to achieve firm attachment of the gel to the substrate prior to addition of the hydrogel. For example, in one preferred embodiment where glass is used as the substrate, the glass is derivatized with an amine prior to the deposition of the polymerized hydrogel. Polymerized derivatized hydrogels then bind strongly to the substrate through reaction of some of the active isocyanate groups with amines present on the surface of the glass when deposited onto the derivatized glass substrate. This provides covalent attachment of the hydrogel to the substrate, preferably about 5% or less of the original active isocyanate group in the prepolymer is used for this action.
[53] In certain embodiments, partial initial blocking of the binding entity may be desirable to maximize the efficient fixation of the binding entity. Reactivity of certain binding entities, which are certain chemical moieties that may include, for example, primary amines with isocyanate prepolymers, can lead to excessive crosslinking between the binding entity and the polymer, and can also lead to denaturation of the binding entity or to target compounds. The binding affinity of the binding entity for can be impaired. This can be avoided or limited by protecting at least some of these residues during polymerization; Deprotection after polymerization will restore the functionality and utility of the binding entity in the array. That is, post-polymerization de-blocking allows the binding entity to have its original structure. Such blocking / de-blocking may be performed by shared or non-shared means. For example, when an antibody is used as the binding entity, the antigen recognition site that is sensitive to crosslinking with the polymer is incubated with an uncrosslinked peptide (or other epitope analog) prior to mixing with the prepolymer. Such peptide or epitope analogs will protect the antigen recognition site from conjugation with active isocyanate groups during the polymerization process. After polymerization, such peptides will be released from the antibody by brief exposure to acid, for example pH 3.0, to re-expose the antigen recognition site of the antibody. Similar techniques can be used to protect selected sulfhydryl residues or amines on binding entities; It may utilize well known reversible chemical derivatization to protect these functional groups during the polymerization process.
[54] It should be noted in the '683 patent that polyethylene glycol can be added as a thickener to facilitate further linear expansion during polymerization. It has been found that other compounds can be added during polymerization to maintain the stability and inherent activity of binding entities such as proteins. Unbound additives may optionally be included in the prepolymer mixture to stabilize the binding entity. These additives include, but are not limited to, glycerol, Ficoll and ethylene glycol and sugars such as mannitol, sucrose and trehalose. Other bulking agents, including nonspecific (non-binding) proteins, such as bovine serum albumin, can also be used to aid the activity of an entity such as a protein when trying to limit the degree of crosslinking to the hydrogel.
[55] Another optional use of the additive is to use materials that produce regions or domains in the polymerization hydrogel. Upon completion of the polymerization, these materials dissolve or diffuse in aqueous solution, leaving large pores, vesicles or channels in the hydrogel polymer that would not be present in the absence of these materials. The presence of such large pores creates a large surface area within or on the hydrogel and provides increased capacity for binding biomolecules and the like, which may be too large to readily diffuse through the hydrogel matrix.
[56] Hydrogel polymers are also suitable for other binding entities including, but not limited to, synthetic molecules, drugs, non-peptide receptor ligands, mixed organic / inorganic species such as metal porphyrins, inorganic materials such as zeolites, and the like. . In one preferred embodiment, this entity is used to isolate the compound from solution based on specific interactions between the binding entity and the analytical species. In another preferred embodiment, this binding entity temporarily interacts with the species in solution. This is the case when the binding entity acts as an optional substrate for reactive processes such as phosphorylation, methylation, cleavage or other forms of modification. In another aspect of the invention, the incorporated material may be involved in a catalytic reaction. Such catalytic materials are useful in biological reactors. Alternatively, arrays of different catalyst presences can be used to screen the most efficient catalyst presences. Generally, these hydrogels are formulated such that the binding entity is useful for a variety of tasks, including but not limited to biological analysis, material screening, and sensors.
[57] Alternatively, non-biological compounds such as tridentate or tetradentate metal chelating agents, for example iminodiacetic acid or nitrilotriacetic acid with suitable linkers of amine-derivatized C ₄ -C ₈ are schematically shown in FIG. 3A. It may be immobilized with a hydrogel as an intermediary binder before or during polymerization as depicted. The desired binding entity, such as a protein, is preferably synthesized or modified to have multiple histidine-containing sequences, for example as the tail or head end of the protein, which is immobilized chelate, as depicted schematically in FIG. 3B. Each cell of the biochip by exposure with divalent or trivalent metal ions such as Cu ²⁺ or Fe ³⁺ to allow chelation with these terminal residues to physically fix the protein in the hydrogel by linking with a topical agent Can be fixed to. By exposing each cell to a specific binding entity such as, for example, a different protein trapping agent, protein chips that are stable for analytical applications are formed. One advantage of using mediators to produce such polymeric microdroplets is excellent confidence in avoiding potential denaturation of particularly sensitive proteins, so that the structure and arrangement of the last binding entity protein remains unchanged. In addition, preparation can be simplified by using the same chelating agent to generate each microdrop or cell in a particular protein microarray and subsequently linking the binder thereafter.
[58] Biochip substrates can be composed of a variety of materials and formats that contribute to automated processing during binding assays and post detection of target molecules binding to each cell. Although solid flat plates such as glass slides are suitable, plates with depressions or wells formed to hold each cell may also be used. Optically transparent substrates, such as glass or transparent polystyrene, will allow transmission light detection through the cell and are convenient for detection modalities using fluorescence or optical absorption. Due to the high binding capacity of three-dimensional hydrogel cells, reflective optical methods are also possible and allow the use of opaque substrates. The use of a rigid substrate allows for the precision of the alignment in the detection run of the assay using the biochip, but this is unnecessary if the proper arrangement is incorporated into the cell to facilitate detection. Flexible formats such as, for example, tapes or filaments may be precisely detected by a scanning scheme similar to the use of magnetic tapes. Although optical methods and suitable substrates are preferred due to their simplicity, other biochemical detection methods, such as the detection of radioactive agents, may alternatively be used. In general, any number of cells, for example 1 to 1000, can be provided on the biochip. To aid in automated processing, multiplexes of 96 cells can often be used; For example, 384 cells may be provided in an array on a 3 inch (7.6 cm) x 5 inch (12.7 cm) plate. Preferably multiple cells are used, but in certain circumstances a biochip using only a single cell may be satisfactory.
[59] In certain embodiments, it may be desirable to load the binding entity into the hydrogel cell after polymerization of the hydrogel cell. Simple diffusion can be an inefficient tool to achieve this. In later course of use, small molecules that rapidly diffuse into the hydrogel diffuse rapidly out of the hydrogel, thus causing a loss of this binding entity. It is therefore desirable to have mechanisms for covalently conjugating these agents to the polymer matrix after diffusion into the matrix for such readily diffusing agents such as small substances and peptides. One preferred means to achieve this is to utilize moieties suitable for carrying out crosslinking, such as photoactive or chemical crosslinking reactants, which are contained within the polymer or diffused into the polymer as part of the component.
[60] In contrast, larger binding entities, such as large fragments of proteins or DNA, may be inefficient to migrate to the hydrogel matrix through passive diffusion. To facilitate diffusion into a large class of matrices, an electric field is applied in a solution that has a pH value different from the isoelectric point of the protein, in a way that causes a controlled transfer of the class with a full charge, such as a protein. This method is called "electrophoresis". When the hydrogel cell is in the path of movement of the charged kind, the charged kind undergoes the passive force as well as the additional force provided by the applied electric field, thus accelerating insertion into the hydrogel cell. The advantage of this electric field-promoting diffusion is that larger binding entities do not readily and passively diffuse out of the hydrogel matrix during subsequent analysis steps.
[61] Subsequent to polymerization of the hydrogel cell, the substrate surface not occupied by the hydrogel cell may be treated with an agent or substance to reduce subsequent nonspecific or unwanted adhesion of the analyte, target material or other substance. This is particularly useful in applications where the analyte reactant potentially nonspecifically binds to the surface and thus can substantially reduce the effective concentration of the analyte or target material in solution. Alternatively, this treatment can be employed to reduce the amount of background signal observed from the surface and thus increase the efficiency of the hydrogel cell for analytical purposes.
[62] Treatment of the exposed surface area involves the application of the reactants to react with the primary amine present as an initial layer or coating on the substrate surface. The reactants include activated polyethylene glycol polymers having at least one terminus containing active moieties such as isocyanates to covalently bond to the primary amine; And nonpolymeric molecules functionalized with small nucleophilic-reactive moieties such as succinyl esters. Examples of standard blocking reactants, such as silanization of free or bovine serum albumin, which are customarily employed to reduce background signals, well known to those skilled in molecular biology, may alternatively be used.
[63] Advantageously, all reactions involved in this system, i.e. (1) derivatize the hydrogel prepolymer either directly or with a media probe, (2) polymerization of the hydrogel, (3) onto the substrate surface of the derivatized hydrogel Bonds involve the formation of strong urea or urethane (carbamate) bonds. This combination gives the microdrop arrays mechanical integrity and significantly increases the half-life of the biochip.
[64] In certain preferred embodiments to be described below, the hydrogel microdroplets after polymerization on a substrate are preferably at least about 30 μm thick, more preferably at least about 50 μm thick and most preferably about 50 to 100 μm thick. In addition, the microdroplets are generally elliptical, as opposed to the rectangular cells previously used in some systems. The larger size of the entirety of the gel microdroplets (or cells) allows to significantly increase the amount of binding entity immobilized there, reducing the low detection limit of the biochip and facilitating its use. By appropriately modifying the distribution mechanism to be used for reducing the viscosity of the polymer solution and microspotting onto the biochip substrate, even smaller cells can produce very dense biochip arrays. If a substrate with wells is used, microdroplets should be deposited onto the bottom of the wells.
[65] The following examples illustrate some applications for protein chips. Representative biochips suitable for protein-protein interaction studies are described by binding calmodulin to calcineurin in a calcium-dependent manner. Biochips suitable for protein-DNA interactions are described by binding lambda refresher proteins to DNA. This biochip will be a pathway to understand suitable biochips for antigen-antibody interactions and interactions as mentioned previously, which may not be described in detail in the Examples.
[66] Example 1 Use of Additives (Glycerol / Trehalose) to Enhance Biological Activity
[67] The following examples show that inactive proteins, simple carbohydrates and diluents enhance overall signal and assay performance and have a protective effect on the antibody activity immobilized on the hydrogel of this biochip.
[68] Panel A- Trehalose. In this experiment, an aliquot of trehalose stock solution, 50% w / v D (+) trehalose dihydrate in 50 mM sodium borate buffer, pH 8, is added to 50 μl volume of the final hydrogel formulation. The formulation also contains pre-contained 3.5 wt% HYPOL PreMA ^R G-50 hydrogel prepolymer (HYPOL, acetonitrile, N-methyl-2-pyrrolidinone in a w / w / w ratio of 1: 3: 3 respectively). Mixed stock solution), anti-transferrin (4 mg / ml phosphate saline 1 × (PBS), 2 μl bovine IgG (50 / mg / ml in PBS and 1.25% glycerol) The amount of trehalose is 0, 1% Varying from 0 to 10 μl, corresponding to the final w / v% of 2%, 5% and 10% trehalose, blank hydrogel spots containing no protein are also included. Spotted at 2 spots per pin on glass slides coated with amine The encapsulated test protein is anti-transferrin and the system is a transferrin (Amersham, 1% bovine serum albumin (BSA), 0.1% Triton) labeled with a Cy3 fluorescent dye About 0.1 μg / ml in PBS containing X100 (PBST) and shake at 45 ° C. After incubation, slides are washed twice in PBST for 10 minutes and then imaged using ScanArray Lite slide scanner Blank hydrogel spots have no detection signal and 0% trehalose have a weak signal 1% And 2% trehalose have a slightly strong, 5% strong signal and 10% the strongest signal, indicating that addition of trehalose has a positive effect on the biological activity of the test antibody in the hydrogel.
[69] Panel B-Glycerol. Glycerol dissolved as 20% stock in pH 8.0 sodium borate buffer had a final concentration of 0%, 0.5 in the hydrogel formulation containing 3.5% final HYPOL PreMA ^R G-50, anti-transferrin, bovine IgG, 5% trehalose. For example, 0, 1.25 μl and 2.5 μl of stock glycerol are added to be% and 1%. As in the assay, a transferrin system labeled with a Cy3 fluorescent dye is used for the assay. In panel B, where glycerol is increased by 50% each, a positive effect on antibody activity is demonstrated and signal intensity is increased.
[70] Example 2. Diffusion (% 3% Hydrogel vs. 5%)
[71] The following examples show the effect of% of hydrogel on the diffusion of binding entities into the hydrogel matrix.
[72] Using the methodology described in Example 1, mouse IgG is immobilized on 3%, 4% and 5% hydrogels, respectively. BSA is included as a differential spot as a nonspecific binding control. Following curing of the polymer, the array is incubated for 1 hour with a rabbit anti-mouse antibody solution labeled with rhodamine, and then washed. Rabbit anti-mouse antibodies bind to mouse IgG antibodies and the extent of binding is measured by fluorescence at each location using the ScanArray Lite slide scanner. Under the same binding conditions and binding times, low% hydrogel spots show strong binding signals; This is an indication of the rapid diffusion rate of rhodamine-rabbit-anti-mouse IgG into the hydrogel matrix at this low percentage.
[73] Example 3 Use of Coatings for Low Background of Nonspecific Binding / Slides
[74] N-hydroxysuccinimidyl active ester (NHS), activated by polyethylene glycol (PEG) polymer, mPEG-SPA-NHS 5K (Shearwater Corporation) is 0.05 at pH 8.25 buffered to a final PEG concentration of 50 mg / ml. M is dissolved in sodium bicarbonate. Corning aminosilane slides are used for surface grafting of polymers. Grace-Biolabs hybridization chamber (SA500-3LCLR) is used as the reaction chamber. To coat the surface, 3 slides are treated on a shaker for 3 hours at room temperature (NSH room temperature) with PEG solution, 3 slides are treated for 3 hours at 45 ° C. (NSH 45) and an additional slide is 45 ° C. as a control. Treated in deionized water for 3 hours at
[75] After PEG treatment, the hybridization chamber is removed and the slides are washed in PBS for 10 minutes, then for 10 minutes with distilled water and air dried. Glyal-derived neurotropic factors labeled with Cy3 are dissolved in PBST. Incubate the slides for 1 hour at room temperature. It is then washed in PBS for 10 minutes and in distilled water for 10 minutes. The slides are subsequently scanned using the ScanArray Lite slide scanner. A 5-10-fold lower background intensity signal following a series of conditions treatment demonstrates the efficacy of the PEG coating in reducing the nonspecific absorption of fluorescent material to the surface.
[76] Example 4 Protein-DNA Interactions on Biochips
[77] In the next example, the single stranded DNA is primaryly linked to the hydrogel and then hybridized to create a double-stranded binding entity that is effective to sequester the target protein as outlined in FIG. 4A.
[78] 5'amino-modified single-strand bacterial λ-repressor binding sequence O _R 2 O _R 1 (wt) and its muts that carry a single base mutation at the binding site (sequence is indicated by the binding site underlined at 4b Visible) is printed on amino-silanated slides at 130 μM in 3.75% HYPOL ^™ . Printed spots are enclosed in individual hybridization chambers and allowed to hybridize with corresponding complementary sequences at 1 μM of 3 × SSC, 0.1% Triton X-100, 5 mM MgCl ₂ for 18 hours at 45 ° C. The resulting double-stranded DNA was then bound to binding buffer (50 mM Tris.HCl, pH 7.6), 100 mM NaCl, 1 mM CaCl ₂ , 0.1 mM EDTA, 0.1 mg / ml BSA, 2.5 μg / ml poly (dA-dT) , 0.05% Tween 20, 1 mM DTT) and incubated for 2 hours at room temperature with bacterial phage lambda refresher λCI labeled with 1.5 μg / ml Cy3. Cy3-labeled [lambda] CI is removed at the end of the binding reaction and the slides are simply washed with binding buffer and then deionized with H ₂ O (dH ₂ O) and imaged by a GSI laser scanner. In separate slides, double-stranded DNA is stained with SYBR Gold (Molecular Probe) according to the manufacturer's protocol and visualized with a GSI scanner for its total DNA content.
[79] Binding of the λ repressor labeled Cy3 to the native operon dsDNA sequence is shown by acquisition of a fluorescence signal at the corresponding spot. The absence of strong fluorescence in the mutant spots indicates that the interaction is sequence-specific. Comparison of Cy3 fluorescence from lambda refresher, SYBR Gold (double-stranded DNA strain) stained with fluorescence of printed slides, for heterogeneous printed DNA producing Cy3 signals related to wild type O _R 2 O _R 1 sequences It confirms that it is a sequence-specific lambda repressor-lambda operon interaction rather than any nonspecific protein binding. The 100-fold difference in signal strength between wild-type sequence linkages relative to mutant sequences confirms the specificity of the response to double-stranded DNA immobilized on the hydrogel matrix.
[80] Example 5. Protein-DNA Interactions on Biochips
[81] In this experiment, double stranded DNA is hybridized before polymerization and fixation, after which target protein binding occurs.
[82] Double-strand (ds) DNA biochips can also be made by directly printing 5 ′ amino-modified Pre-hybridized dsDNA. This method contrasts with the previous example where single-stranded DNA is printed and cognate oligonucleotides are subsequently hybridized to the printed oligonucleotides to form binding entities.
[83] In this example, the 53 kD protein, estrogen receptor (ER), binds to its consensus estrogen response factor (ERE) as a homodimer. Wild-type ERE sequences differ from mutant sequences by 4 nucleotides in a region known to be important for binding by the receptor. The wild type sequence is a 32-sequence oligomer consisting of the sequence 5'-tttacggtagaggtcactgtgacctctacccg-3 '. The mutant sequence has the sequence 5'-tttacggtagaggtcactgt atgg tctacccg-3 'and differs in 4 oligonucleotides (underlined). To produce dsDNA for printing, 5 μl and complementary nucleotides of each 650 μM stock of amine-linked oligonucleotides of interest were added in 40 μl DNA hybridization buffer (3 × SSC, 5 mM MgCl ₂ ) at pH 8. Dilute to 1: 650 (65 μM final concentration) so that the final reactant volume is 50 μl. The reaction product is incubated at 95 ° C. for 10 minutes and then cooled on ice for 3 minutes. 10 μl of this double-stranded DNA is printed on 450 μM hydrogel spots using a solution consisting of 3.75% polymer, 0.5% glycerol and 50 mM sodium borate buffer at pH 8.0. After blocking for 1 hour with 1% BSA in PBST solution, 1 μl of transcription factor in the form of an ER concentration of 1.153 μM was added to the appropriate binding buffer (10% glycerol, 10 mM HEPES, 30 mM KCl, 0.1 mM EDTA, 0.25 mM DTT). , 1 mM Na ₂ HPO ₄ , pH 7.9) and allowed to bind to dsDNA for 1 hour at room temperature; It is then washed with PBST for 10 minutes. The ER-ERE complex is subsequently incubated with a 1: 100 dilution of rabbit anti-ERβ antibody for 1 hour at room temperature and washed for 30 minutes with PBST. It is then incubated for 1 hour at room temperature with a 1: 1000 dilution of goat anti-rabbit IgG-Cy3 conjugate and washed for 30 minutes with PBST. The entire experiment is depicted approximately in FIG. 5. The slides are cleaned with dH ₂ O and air dried before imaging with the ScanArray Lite scanner. Signal analysis is performed using ArrayPro 4.0 software. The increased signal observed from the spot containing the wild type sequence relative to the mutant sequence signal is similar to the control and indicates the maintenance of binding specificity by the estrogen receptor for the target sequence in the hydrogel matrix.
[84] Example 6. Antigen-Biochip
[85] This experiment shows that the hydrogel platform can be used as a matrix to fix other binding entities such as antigens. Antibody-antigen reactions are commonly used in a variety of biological assays, and the ability to immobilize components (antibodies or antigens) is a desirable feature in supports. In this experiment the antigen is immobilized on a hydrogel matrix.
[86] Using the methodology described in Example 1, the protein antigen, human transferrin (0.2 mg / ml) was directly onto amine-coated glass slides at different dilution concentrations in 3.3% hydrogel with 5% trehalose, 2 mg / ml BSA. It is fixed. After blocking with 5% skim dry milk powder, the slides are incubated with mouse ascites fluid containing anti-human transferrin at various concentrations for 1 hour. After incubation, the slides are washed three times for 10 minutes with PBST. Bound mouse anti-transferrin antibodies are visualized by laser scanner imaging after incubating slides with Cy3-labeled donkey anti-mouse IgG. A linear dosing reaction is observed over three dilutions, that is, from 0.1 to 0.001. This input-response relationship represents the function of the antigen immobilized on the hydrogel matrix and the permeability of the hydrogel matrix to support sequential diffusion of the antibody into the matrix as part of the overall analysis methodology.
[87] Example 7. Antibody-Biochips
[88] As mentioned in the previous examples, antigen-antibody responses are commonly employed in biological assays. In this example, the antibody is immobilized on the hydrogel matrix as opposed to the immobilization of the antigen in Example 6.
[89] Anti-human transferrin, anti-BSA and anti-PSA antibodies (0.4-0.8 mg / ml) were treated with 5% trehalose, 2 mg / ml bovine IgG and 0.5% on amino-silanated glass slides according to the methodology of Example 1 In the presence of glycerol is fixed to 3.3% hydrogel. Slides are then incubated overnight at room temperature with Cy3 labeled individual antigens at a concentration of 1 mg / ml in PBST containing 1% BSA. Bound proteins are visualized by laser scanner imaging after vigorous washing with PBST. The presence of labeled target protein at the corresponding antibody site on the microarray indicates maintenance of antibody function in the hydrogel matrix.
[90] Example 8. Multilayer ELISA Analysis
[91] The ability to support complex binding interactions may also be a desirable feature for hydrogel matrices. In this example, a hydrogel is used to immobilize the antibody as the primary binding entity. Subsequent specific positioning of its antigen is caused by further binding events for visualization purposes, which shows the biocompatibility of the hydrogel with respect to multiple binding events by firm maintenance of protein and protein function.
[92] Rat anti-mouse IL-2 monoclonal capture antibody (BD, Pharmingen) was subjected to 3.3% hydrogel with 5% trehalose, 2 mg / ml bovine IgG on amino-silanated glass slides by the methodology outlined in Example 1 Is fixed directly to Slides are incubated with moderate mixing for 1 hour at room temperature with culture medium diluted from phytohemagglutinin-stimulated mouse LBRM-33 4A1 cells or unstimulated cells. After two 15-minute washes with PBST washes, the slides are incubated with biotinylated rat monoclonal anti-mouse IL-2 detection antibody (BD, Pharmingen) for 1 hour at room temperature. Free antibody is removed by three PBST washes for 15 minutes each. Horseradish peroxidase-conjugated streptavidin is added to the slides while incubating for another hour at room temperature. Cy3-tyramide substrate from the TSA reactant system is added to the slide to fully cover all printed spots after a strong wash of streptavidin-HRP according to the recommended protocol. After washing the unreacted substrate, the slides are analyzed by laser scanner imaging. An 8-fold increase in fluorescence signal indicates the presence of bound antigen by immobilizing the antibody on the hydrogel.
[93] Example 9. Multilayer Small Molecule Mediated (CaM / Calcineurin)
[94] Complex interactions between different proteins are often difficult to achieve on the support surface; However, the following example is illustrated schematically in FIG. 6 and demonstrates the use of multiple protein interactions mediated by small molecules.
[95] Mouse anti-bovine brain calcineurin monoclonal antibody (0.4 mg / ml, Sigma), both anti-bovine calmodulin antibody (0.2 mg / ml, Chemicon) and control bovine IgG (0.4 mg / ml), respectively. The methodology of 1 is directly immobilized on a 3.3% hydrogel with 5% trehalose and 2 mg / ml bovine IgG onto an amine-coated glass slide. Slides are incubated overnight with 0.1 mg / ml bovine calcineurin in 20 mM HEPES (pH 7.6), 130 mM KCl, 0.1% Triton X-100, 10 μg / ml polyglutamic acid after 5% dry milk blocking. Cy3-labeled chicken calmodulin is allowed to bind to calcineurin-treated slides in the presence of 1 mM CaCl ₂ or 5 mM EGTA in PBST, 1% BSA for 1 hour at room temperature. Bound calmodulin is visualized at Cy3 excitation and emission wavelengths using laser scanner imaging. A six-fold increase in signal intensity was observed in the presence of calcium compared to the absence of calcium (ie, in the presence of EGTA) hydrogels to support complex biomolecular interactions involving both proteins and small molecules at the anticalcineurin antibody site. Indicates the ability of the matrix.
[96] Example 10 Specific Detection of Tyrosine Phosphorylated Peptides on Biochips
[97] Hydrogel matrices are useful in a variety of binding entities and assay formats. In this example, the use of phosphorylated amino acids in peptide binding entities is shown.
[98] Each peptide is printed on the slide as 2 quadruple pairs next to 40 μM concentration; The peptide is immobilized on 3.5% HYPOL ^™ containing 0.5% glycerol by the methodology of Example 1. The peptides which are then printed on the slides are described in Table 1.
[99] Table 1
[100]
[101] Idiomatic abbreviations were used with pTry = phosphotyrosine and pSer = phosphoserine.
[102] In all subsequent incubation steps, the glass slide is incubated on a rocker in a glass slide-dye dish. Peptide biochips were blocked for 60 minutes at room temperature with 1% BSA in PBS containing 0.1% Triton X-100 and at 1: 2000 dilution in biotinylated anti-phosphotyrosine antibody in PBST containing 1% BSA. Incubate overnight at ° C. After washing twice with PBST for 10 minutes at room temperature, the slides are incubated with Cy3-labeled streptavidin at 1: 2000 dilution in PBST containing 1% BSA. The slides are then washed three times for 15 minutes in PBST. After a brief rinse with distilled water, the slides are dried in air and scanned using a GSI Lumonics scanner. The results show the presence of a fluorescence signal comprising phosphoserine at or without phosphotyrosine, indicating that the phosphopeptide maintains the proper native structure to be recognized by the antibody despite isocyanate binding to the hydrogel. Indicates.
[103] Example 11 Dephosphorylation of Tyrosine Phosphorylated Proteins on Biochips by Tyrosine Phosphatase
[104] The previous examples demonstrated the use of hydrogel matrices to support binding interactions of extended nature (minutes or hours). The examples below show that the matrix also supports transient binding interactions including enzymatic activity and the like. In this example, the phosphopeptide substrate is immobilized on a hydrogel matrix, which then serves as a substrate for the enzyme to remove phosphate groups. Remaining phosphoric acid is then detected using the methodology in Example 10.
[105] Using the same experimental method as described in Example 10, the printed slides were prepared in reaction buffer (1 × LAR buffer: 25 mM Tris-HCl, 50 mM NaCl, 2 mM Na ₂ EDTA, 5 mM dithiothreitol, 0.01%). Brij-35, pH 7.0 at 25 ° C .; 1 × YOB buffer: 50 mM Tris-HCl, 100 mM NaCl, 2 mM Na ₂ EDTA, 5 mM dithiothreitol, 0.01% Brij-35, pH 7.0 at 25 ° C.) Incubate for 10 minutes at room temperature with 6 units leukocyte antigen related (LAR) protein tyrosine phosphatase or 6 units Yersinia enterocolitica (YOP) protein tyrosine phosphatase in this supplied 430 μl chamber. Thereafter, the chamber is removed and the glass slide is transferred to a glass slide-dye dish. The reaction is stopped by washing the slide twice for 10 minutes at room temperature with 1 mM sodium pervanadate (universal tyrosine phosphatase inhibitor) in PBST. The slide is then blocked with 1% BSA, incubated with biotinylated anti-phosphotyrosine antibody followed by Cy3-streptavidin binding as described in Example 10. Loss of the fluorescence signal already observed in Example 10 suggests the ability of the phosphorylase enzyme to enter the hydrogel, maintain biological activity and temporarily interact with one or more substrates, ie immobilized phosphopeptides. More specifically, the results show that LAR-PTPase selectively completely removes phosphate groups from peptide No. 1 and low levels of remaining peptides containing pTyr residues. YOB-PTase enzyme substantially completely removes phosphate groups from peptides No. 1, 3, 6, 8 and 13; It exhibits a greater degree of removal of phosphate groups from peptides Nos. 2, 7, 10, 14 and 15, that is, greater than LAR-PTPase in removing the same peptide. Therefore, the results of fluorescence with various phosphopeptides show a favorable specificity for the portion of the 2 phosphorylase enzyme directed towards a portion of the phosphopeptide sequence.
[106] Example 12. Metal Chelates
[107] The binding entity need not be a biological entity in the origin, but various synthetic molecules can be employed. In this example metal chelators are used to fix metal ions in the hydrogel matrix that contribute to binding to multiple histidine residues present in the protein molecule.
[108] Ni ⁺⁺ or Cu ⁺⁺ NTA hydrogels are produced by mixing HYPOL ^™ with various amounts of nitrilotriacetic acid and spotted on glass slides. The polymerized gel spot was washed with 50 mM acetic acid in dH ₂ O and charged with 50 mM Cu (NO ₃ ) ₂ or Ni (NO ₃ ) ₂ ; It is washed with 50 mM acetic acid in dH ₂ O containing 0.1 M KNO ₃ (pH 4.0) and finally washed with dH ₂ O. In PBST containing 1% BSA, 10 μg / ml of 6 X His tagged green fluorescent protein is added to the slide, which is grooved under appropriate excitation and emission filters after removal of free unbound 6 X His-GFP. Imaging in PBS using a built-in CCD camera. An increased fluorescence signal and corresponding increased fluorescence signal is observed, suggesting that the hydrogel matrix supports the use of small molecules as intermediary binders.
[109] Example 13. Alpha-2-Macroglobulin-Trypsin Interaction on Biochip (Electric Field Base Loading)
[110] Alpha-2-macroglobulin is a mechanism that protects the body from excessive protease activity, essentially preventing the body from "digesting" itself, specifically binding to and invalidating proteases in the blood. It is a large plasma protein (molecular weight 800,000) that circulates to make. The relationship between proteases such as alpha-2-macroglobulin and trypsin is very close, and alpha-2-macroglobulins immobilized on agarose beads are used to affinity-purify trypsin and other proteases.
[111] Three sets of hydrogel microdroplets are spotted with amine-derivatized glass. Glass slides are first treated to block nonspecific binding sites for 2 hours at room temperature with 1% BSA solution in 10 mM sodium phosphate buffer at pH 7.4 and 150 mM NaCl (PBS). Failure to do this results in some fluorescently-labeled proteins that bind nonspecifically and increase the signal-to-noise ratio. The hydrogel consists of a prepolymer comprising an isocyanate-functional HYPOL ^™ . The polymerization is initiated with an aqueous solution and the polymerization kinetic is controlled by pH and temperature. Each hydrogel microdroplet forms one cell of the microarray, causing polymerization at a controlled rate to prevent opacity due to carbon dioxide gas production. The first set of such hydrogels is loaded with alpha-2-macroglobulin using 50 μl solution at 5 mg / ml PBS concentration. The high molecular weight of alpha-2-macroglobulin limits the rapid diffusion into the hydrogel droplets, and the diffusion rate can be increased using mild current (2.5-5 mV) carried by the small electrode system. Once alpha-2-macroglobulin diffuses into the hydrogel droplets, its high molecular weight significantly prevents subsequent diffusion from the droplets. Ferritin serves as a negative control as is known that trypsin does not bind. Ferritin is similarly diffused into the second set of hydrogel droplets using a mild current under the same electrode system and under the same conditions. The third set of droplets is not treated with any protein but serves as an additional negative control. All three sets of droplets are then exposed to FITC-labeled trypsin for about 15 minutes and washed with 1% BSA-PBS at pH 7.4 for about 5-20 minutes. Fluorescence intensity is measured with a CCD camera and the results are shown in Table 2.
[112] Table 2. Specific binding of FITC-trypsin to alpha-2-macroglobulin
[113] Immobilized proteinFluorescence intensity (au) Alpha-2-macroglobulin800 Ferritin20 No protein10
[114] The results suggest that FITC-labeled trypsin specifically binds its natural ligand, alpha-2-macroglobulin, in hydrogel droplets, and there is little detectable binding activity for negative control protein ferritin or hydrogels.
[115] Although the present invention has been described in terms of a number of different embodiments, including the best mode contemplated by the present inventors, it is contemplated that changes or modifications apparent to those skilled in the art may occur without departing from the scope of the invention as set forth in the appended claims. do. For example, although certain fluoropores such as FITC and Cy3 are used, other fluoropores or other reporters may alternatively also be used. Although the use of biochips having multiple cells to transfer different nonhybridized binding entities is advantageous, single-cell biochips may be suitable in certain cases.
[116] Certain aspects of the invention are highlighted in the following claims.

权利要求:
Claims (37)
[1" claim-type="Currently amended] a) a solid substrate having a surface;
b) at least one optically transparent hydrogel cell attached to the surface of the substrate, formed from an isocyanate-functional polymer; And
c) An optically clear hydrogel biochip comprising a non-hybridized binding entity immobilized in or on a hydrogel cell effective for selective sequestration of a target protein or other comparable molecule.
[2" claim-type="Currently amended] The biochip of claim 1, wherein the hydrogel comprises a polymer having a urethane bond.
[3" claim-type="Currently amended] The biochip of claim 1, wherein the hydrogel comprises polyethylene glycol, polypropylene glycol, or polymers thereof.
[4" claim-type="Currently amended] The biochip of claim 1, wherein the hydrogel cell is at least 30 μm thick.
[5" claim-type="Currently amended] The biochip of claim 4, wherein the hydrogel cell is about 30 to about 100 μm thick.
[6" claim-type="Currently amended] The biochip of claim 1, wherein the binding entity is covalently bound onto and into the hydrogel cell through reaction with an isocyanate group.
[7" claim-type="Currently amended] 6. The biochip of claim 5, wherein up to about 15% of the active isocyanates in the polymer of the cell react with the binding entity.
[8" claim-type="Currently amended] The biochip of claim 6, wherein up to about 10% of the active isocyanates in the polymer of the cell react with the binding entity.
[9" claim-type="Currently amended] The biochip of claim 1, wherein the binding entity comprises an immunoglobulin, an enzyme, a receptor, an enzyme inhibitor, an enzyme substrate, or a peptide.
[10" claim-type="Currently amended] The biochip of claim 1, wherein the binding entity is immobilized inside the hydrogel through reaction with a mediator.
[11" claim-type="Currently amended] 10. The biochip of claim 9, wherein the binding entity is a protein that binds to the metal chelate immobilized on the hydrogel and also constitutes a mediator.
[12" claim-type="Currently amended] The biochip of claim 10, wherein the protein is bound to the metal chelate via histidine-containing polypeptide at one end thereof.
[13" claim-type="Currently amended] The biochip of claim 10, wherein the binding entity is immobilized via a primary mediator linked to the hydrogel and a secondary mediator linked to the primary mediator.
[14" claim-type="Currently amended] The biochip of claim 13, wherein the primary mediator is an antibody and the secondary mediator is a protein.
[15" claim-type="Currently amended] The biochip of claim 1, wherein the substrate has a plurality of hydrogel cells attached to a surface, wherein different binding entities are immobilized on different hydrogel cells.
[16" claim-type="Currently amended] The biochip according to any one of claims 1 to 15, wherein the substrate is optically transparent.
[17" claim-type="Currently amended] 17. The biochip of any one of claims 1 to 16, wherein the substrate has an active substance which allows the hydrogel to covalently bond to the upper surface.
[18" claim-type="Currently amended] The biochip of claim 17, wherein the hydrogel cell is covalently bonded to the substrate via some isocyanate groups of the polymer.
[19" claim-type="Currently amended] a) a solid substrate having an upper surface;
b) a plurality of hydrogel cells comprising polyethylene glycol, polypropylene glycol, or copolymers thereof bonded to the upper surface of the substrate;
c) mediators immobilized on or in the hydrogel cell; And
d) a hydrogel biochip comprising different protein binding entities bound to a mediator in at least several hydrogel cells, interacting in such a way as to reveal the original structure of the protein.
[20" claim-type="Currently amended] (a) providing an optically transparent hydrogel biochip having a substrate having a surface to which at least two hydrogel cells are bound, each cell having at least about 30 μm thickness and consisting mainly of polyethylene glycol, polyethylene glycol or copolymers thereof Each hydrogel cell comprises a different protein binding entity immobilized therein or on it;
(b) contacting the hydrogel biochip with an analyte solution containing the target biomolecule under binding conditions;
(c) washing the hydrogel biochip under conditions that remove non-selective and unbound target biomolecules;
(d) detecting the target biomolecule bound to the one cell, using the biochip to perform biochemical analysis.
[21" claim-type="Currently amended] The method of claim 20, wherein the target biomolecule results in a compositional change of the binding entity.
[22" claim-type="Currently amended] The method of claim 21, wherein the compositional change is a phosphorylation event.
[23" claim-type="Currently amended] The method of claim 22, wherein the compositional change is a dephosphorylation event.
[24" claim-type="Currently amended] 24. The method of any of claims 21 to 23, wherein the binding entity that undergoes a compositional change is bound to the hydrogel by one or more mediators.
[25" claim-type="Currently amended] (a) providing an organic solvent solution of isocyanate-functional hydrogel prepolymer;
(b) providing a solution of the hybridized binding entity;
(c) the binding entity is covalently bound to the isocyanate-functional hydrogel polypolymer via reaction with up to 15% of the active isocyanate;
(d) initiate polymerization of the isocyanate-functional hydrogel prepolymer under conditions such that an optically clear hydrogel is produced;
(e) dispensing the polymerized isocyanate-acting hydrogel prepolymer onto a solid substrate in the form of droplets, whereby an optically clear hydrogel polymer containing a binding entity is attached to the substrate, the target being on or onto the biochip. A method of making an optically clear isocyanate-acting hydrogel biochip having a non-hybridized binding entity effective for selective sequestration of a protein or target biomolecule.
[26" claim-type="Currently amended] The method of claim 25, wherein the binding of the entity is carried out simultaneously with the polymerization.
[27" claim-type="Currently amended] 27. The method of any one of claims 25 or 26, wherein the viscosity and pH are selected to control the generation of carbon dioxide to ensure transparency of the resulting hydrogel.
[28" claim-type="Currently amended] 28. The method of any one of claims 25-27, wherein the substrate is treated to provide an active moiety of the top surface to which the polymeric hydrogel will covalently bond to the substrate.
[29" claim-type="Currently amended] a) providing an organic solvent solution of isocyanate-functional hydrogel polypolymer;
b) providing a solution of the desired protein capture agent;
c) the intermediate coupling agent for the protein is covalently bonded to the isocyanate-functional hydrogel prepolymer;
d) initiate the polymerization of the isocyanate-functional hydrogel prepolymer;
e) dispensing droplets of the polymerized isocyanate-functional hydrogel prepolymer onto a solid substrate, allowing the polymer to adhere to the substrate;
f) Exposing each hydrogel droplet to one desired protein solution to immobilize the protein trapping agent via linkage in or phase with a coupling agent such that the droplet is polymerized to produce a biochip having multiple cells with different protein trapping agents. A method of making an isocyanate-acting hydrogel biochip comprising a protein selected to act as a capture agent and having a protein immobilized on or in the biochip.
[30" claim-type="Currently amended] The method of claim 29, wherein the linking of the protein to the coupling agent is performed concurrently with the polymerization.
[31" claim-type="Currently amended] The method of claim 29, wherein the linking of the protein to the coupling agent is performed after polymerization.
[32" claim-type="Currently amended] 32. The method of any one of claims 29-31, wherein the coupling agent is a chelating agent.
[33" claim-type="Currently amended] 33. The method of claim 32, wherein the proteins each comprise a histidine-containing terminal peptide sequence.
[34" claim-type="Currently amended] 34. The method of any one of claims 29 to 33, wherein the reaction conditions during the polymerization are adjusted to slow the rate of carbon dioxide evolution to ensure the optical clarity of the resulting hydrogel.
[35" claim-type="Currently amended] 35. The method of any one of claims 29-34, wherein the substrate is treated to provide an active moiety on the top surface to which the hydrogel will covalently bind to the substrate.
[36" claim-type="Currently amended] a) providing an organic solvent solution of isocyanate-functional hydrogel prepolymer;
b) initiate polymerization of the isocyanate-functional hydrogel prepolymer;
c) dispensing the droplets of polymerized isocyanate-functional hydrogel prepolymer into the solid substrate such that the droplets bind to the substrate and form a plurality of cells;
d) physically immobilizing different proteins selected to act as binding entities to selectively sequester specific biomolecules within or on each cell. A method for producing an isocyanate-functional hydrogel biochip.
[37" claim-type="Currently amended] The method of claim 36, wherein the fixing step comprises an entrapment of the protein having a molecular weight of about 100,000 or greater through the use of a current to cause the protein to diffuse into the cell.

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同族专利:

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公开号 | 公开日

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WO2002059372A3|2002-09-19|

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AT421694T|2009-02-15|

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EP1328810B1|2009-01-21|

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EP1328810A2|2003-07-23|

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DE60137523D1|2009-03-12|

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JP2004518138A|2004-06-17|

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WO2002059372A2|2002-08-01|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-10-26|Priority to US24369900P

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2000-10-26|Priority to US60/243,699

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2001-10-26|Application filed by 바이오셉트 인코포레이티드

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2001-10-26|Priority to PCT/US2001/051265

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2002-11-29|Publication of KR20020089315A

优先权:

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申请号 | 申请日 | 专利标题

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US24369900P| true| 2000-10-26|2000-10-26|

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US60/243,699|2000-10-26|

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PCT/US2001/051265|WO2002059372A2|2000-10-26|2001-10-26|Three dimensional biochip|