CN101052672A - Photocrosslinked hydrogel blend surface coatings - Google Patents

Abstract

Hydrogel polymer blends including precursors and crosslinked forms of the compositions. The blends provide an improved approach to achieve high quality, uniform coatings with better commercial viability than other approaches including copolymerization. Applications include mass spectral analysis of biomolecular analytes such as proteins. Dextran and acrylamide systems are preferred. Benzophenone groups can be used as photocrosslinking groups. Photoinitiators are not needed. Functionalities which can selectively bind to biomolecular analytes are included.

Description

The hydrogel blend top coat of photo-crosslinking

Background of invention

Term " hydrogel " ordinary representation swelling and contain the hydrophilic cross-linking organic polymer material (hydrophilic polymer network just) (for example referring to WO00/66265 and the US6 of Ciphergen Biosystems, 613,234 (Voute)) of water in water.Hydrogel has multiple commercial applications, for example is used for that contact lens, transmitter, tissue adhesive, medicine are sent, dressing and top coat.For example, referring to the US 6,017,577 of Hostettler etc.Especially, the hydrogel surface coating is used for bio-medical instrument, and as catheter, catheter ball and support, as the US 5,601 of Demm, 538 is described.Hydrogel can be used as successive layers or zone of dispersion pattern and is administered to the surface and goes up (example gel " fragment " or " liner ").

Hydrogel in water degree of depth swelling, form Stability Analysis of Structures but the special performance of the structure compatible with liquid comes from their lightly crosslinked characteristics, this characteristic comes from its preparation method again.A kind of this type of preparation method is respectively by the preparation of photopolymerization and photo-crosslinking, and it is disclosed in US 5,567, in 435 and 6,156,478.But ' 478 patent has been described based on azlactone functional monomer's the Photocrosslinkable and the hydrogel composition of light patternization.These hydrogels can be by photomask or laser induced thermal imaging and on matrix patterning, and azlactone functional group can be used for biomolecules is incorporated into this matrix.According to ' 478 patent, described hydrogel composition can be used for preparation " microchip ", as low-or height-density D NA chip or contain the gel pad microarray of enzyme.

The other method of preparation hydrogel material is, monomer solution is deposited on the stromal surface, and uses thermal initiator or light trigger in-situ polymerization and cross-linking monomer mixture, and it is disclosed among the PCT application WO 00/66265.Change the consumption of monomer and linking agent, can influence the thickness and the aperture of the hydrogel layer that obtains.

The US 5,512,329 and 5,002,582 of Guire etc. discloses the polymkeric substance with the potential reaction functional group that is used to be covalently bonded to stromal surface.When by outside stimulus such as actinic radiation the potential reactive group being intensified, these polymkeric substance are covalently bonded to stromal surface.But these polymkeric substance are typically designed to and repel protein rather than absorb protein, perhaps the specific control by functional group's chemistry make its optionally with protein interaction or conjugated protein.And these polymkeric substance are not to prepare by controlled copolymerization process, and controlled copolymerization process can form the suitable Chemical bond selectivity of suitable hydrogel structure and acquisition and protein and other biomolecules.

Although in these documents, described multifunctionality and suitability, but in the biochip based method of protein detection, especially in the substance assistant laser desorpted/ionization (MALDI) and surface-enhanced laser desorb/ionization (SELDI) mass spectrum that mass-spectrometric technique for example becomes more and more popular in the protein analysis, do not develop the potentiality of hydrogel yet fully.And the common method that is used to prepare hydrogel can not obtain to help MALDI or the mass spectral all even homogeneous coating of SEDLI usually.For example, adopt the in-situ polymerization monomer mixture that controlled polymerization process is not provided usually.Polymerization and surface attachment take place on each aspect usually simultaneously, and the independent reactor of each some representative.The hydrogel material that obtains can incur loss because of the variation of point-to-point and chip to chip.Method commonly used does not provide the three dimensional polymeric structure with enough surface-area, controlled porosity and ligand density of being used to capture wide molecular weight scope protein and biomolecules usually yet.Hydrogel with enough surface-area can be given high binding capacity of probe and susceptibility, and it is very little at the sample size that can be used for analyzing to be attractive with having in limited time.Hydrogel with controllable bore diameter and ligand density can be given desired selectivity of probe and binding capacity, to satisfy the needs that particular organisms is used.And method commonly used does not provide usually and should help MALDI or mass spectral coating uniformity of SEDLI and uniformity.For example, the more accurate flight time of sample (time of flight) that evenly can provide of hydrogel surface coating is analyzed, this be because all to be absorbed in the energy source of analyte on the detecting probe surface and gas phase ion spectrometer equidistant.And the existing prescription of probe material may be incompatible with desired treatment process (spin coating, dip-coating, light patternization or its combination that is suitable for).The existence of low molecular weight compositions can cause problem.For example, referring to PCT application WO00/66265 and US patent application 20020060290A1.Other document comprises US 6,579,719 (Hutchens), 6,610,630 (Schwarz) and 6,675,104 (Paulse).

The needs that exist are, improve detection of biological molecule such as proteinic biochip method, comprise probe material by using more high uniformity and structural stability feature, the MALDI, the SELDI that change of control coating thickness, hydrogel porosity and point and the detection of other mass spectroscopy better.Advantage provided by the invention comprises the hydrogel surface value maximization that is used in SELDI and maldi analysis, include, but are not limited to following factors: density, (6) of the stability of hydrogel, (5) control selective binding functional group prepare hydrogel easily and consistently and do not have the lower-molecular-weight component branch basically with (7) on the homogeneity of the control of the covering fully of (1) hydrogel, (2) hydrogel thickness and swelling capacity, (3) hydrogel coating, (4) surface, and it can spread apart and disturb this analysis by producing signal noise.

Hydrogel blend is known, comprises being described in US 6,586 blend among 493 (Massia), 6,211,296 (Frate) and 4,693,887 (Shah).But hydrogel blend does not develop usually and is used for mass spectrometry method for example SELDI and MALDI.The needs that exist are that improvement comprises improved processibility, synthetic diversity, economy and convenience to the control of aquogel system.But have challenge because sometimes in the aquogel system a kind of existence of useful functional group can disturb use and synthesis strategy to another functional group.

Summary of the invention

In this part the general introduction of the present invention several infinite aspect.Confirm among the present invention: for aquogel system, blend provides a kind of useful means, with the method for independently controlling of the required synthetic chemistry reaction of the useful hydrogel blend of preparation that provides a kind of other method such as copolyreaction more to be difficult to provide.The blend preparation allows that the stock polymer of use limited quantity prepares multiple material, and it has shortened the time of research and development, and required synthetic work amount.And, can realize better control to the polymkeric substance energy.

In the first embodiment, the invention provides a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, this precursor composition comprises: (a) first polymkeric substance, it comprises Photocrosslinkable functional group, the optional functional group of selective binding biomolecule analysis thing and (b) second polymkeric substance of also comprising of first polymkeric substance wherein, it comprises the functional group of selective binding biomolecule analysis thing, wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, and wherein the amount of the functional group of the amount of first and second polymkeric substance and Photocrosslinkable functional group and selective binding biomolecule analysis thing is given performance that this hydrogel precursor polymer blend compositions photo-crosslinking becomes hydrogel and the performance that makes hydrogel selective binding biomolecule analysis thing respectively.In preferred embodiments, there is the functional group of selective binding biomolecule analysis thing in first polymkeric substance, and can be identical with the functional group of selective binding biomolecule analysis thing in second polymkeric substance.In preferred embodiments, first polymkeric substance can comprise the linear polymer main chain with side group, and this side group comprises Photocrosslinkable functional group; And second polymkeric substance comprises the linear polymer main chain with side group, and this side group comprises the functional group of selective binding biomolecule analysis thing.In preferred embodiments, selective binding functional group can be effectively and biomolecule analysis thing covalent attachment or non-covalent the combination.In addition, selective binding functional group can be stratographic or biologic specificity in conjunction with functional group.As required, this hydrogel composition can further comprise energy absorbing portion or one or more fluorophor.Preferred this hydrogel composition does not contain light trigger substantially.

In second embodiment, the present invention also provides a kind of aquogel polymer blend composition, it is prepared as follows: (A) cross-linked hydrogel polymer blend precursor composition, said composition comprises: (i) first polymkeric substance, it comprises Photocrosslinkable functional group, the optional functional group of selective binding biomolecule analysis thing and (ii) second polymkeric substance of also comprising of first polymkeric substance wherein, it can carry out synthesis modification to comprise the functional group of selective binding biomolecule analysis thing, forms crosslinked hydrogel thus; (B) this cross-linked hydrogel of synthesis modification, make its functional group that comprises selective binding biomolecule analysis thing, wherein the amount of first and second polymkeric substance is given this hydrogel precursor blend polymer photo-crosslinking performance that becomes hydrogel and the performance that makes hydrogel selective binding biomolecule analysis thing with the amount of Photocrosslinkable functional group and selective binding functional group.

The 3rd embodiment is a kind of matrix, it comprises stromal surface and the aquogel polymer blend composition on stromal surface, wherein said composition comprises that (i) comprises first polymkeric substance of crosslinked functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, second polymkeric substance that (ii) comprises the functional group of selective binding biomolecule analysis thing, the functional group of the selective binding biomolecule analysis thing of this functional group and first polymkeric substance can be identical or different.

A kind of method of using hydrogel composition to make functionalisation of surfaces also is provided in the 4th embodiment, this method comprises: (A) provide (i) to have the matrix on surface, (ii) according to the hydrogel blend precursor composition of above-mentioned first and second embodiments in this overview section, (B) contact is according to the hydrogel blend precursor composition of above-mentioned first and second embodiments in this overview section, to form the layer of said composition from the teeth outwards, (C) make lip-deep at least some compositions crosslinked, to form the hydrogel that contacts with the surface.

Other embodiment comprises the method for a kind of preparation according to the hydrogel blend precursor composition of above-mentioned first and second embodiments in this general introduction, and it comprises that mixing first and second polymkeric substance are to form the step of this hydrogel precursor blend polymer.

In addition, the invention provides a kind of particle, it comprises the hydrogel according to the cross-linked form hydrogel composition of above-mentioned first and second embodiments in this overview section.

Further, the invention provides a kind of method that is used for detection of biological analysis of molecules thing, it comprises: the matrix according to above-mentioned the 3rd embodiment in this overview section is contacted with the sample that contains the biomolecule analysis thing, and (ii) detect this biomolecule analysis thing by biomolecule analysis thing and combining of selective binding functional group subsequently

Another embodiment is a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, this precursor composition comprises: (a) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second polymkeric substance, it comprises energy absorption functional group, and wherein amount and the amount of Photocrosslinkable functional group and energy absorption functional group of first and second polymkeric substance are given performance and the hydrogel that this hydrogel precursor polymer blend compositions photo-crosslinking becomes hydrogel respectively and promoted analyte of interest desorption when being subjected to high energy gamma source such as laser bombardment to enter the performance of gas phase.

Another embodiment is a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, this precursor composition comprises: (a) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second polymkeric substance, and wherein this hydrogel also comprises the reactive group that can form covalent linkage with biomolecules, and wherein the amount of first and second polymkeric substance is given the performance that this hydrogel precursor polymer blend compositions photo-crosslinking becomes hydrogel with the amount of Photocrosslinkable functional group and energy absorption functional group.

The present invention also provides a kind of aquogel polymer blend composition, it comprises (a) first polymkeric substance, it comprises Photocrosslinkable functional group, (b) second polymkeric substance, it comprises that (i) is one or more by the functional group of non-covalent combination selective binding biomolecule analysis thing, (ii) one or more functional group, (iii) one or more energy absorbing portion, or its combination by covalent attachment mode selective binding biomolecule analysis thing.In preferred embodiments, second polymkeric substance comprises (i) one or more functional groups by non-covalent combination selective binding biomolecule analysis thing.In another preferred embodiment, second polymkeric substance comprises (ii) one or more functional groups by covalent attachment mode selective binding biomolecule analysis thing.In another preferred embodiment, second polymkeric substance comprises (iii) one or more energy absorbing portion.No matter covalently or non-covalently in this embodiment, second polymkeric substance needn't have the functional group of selective binding biomolecule analysis thing, combination.In this embodiment, as required, first polymkeric substance also can comprise covalently or non-covalently bonded selective binding functional group or energy absorbing portion.For specific application, can control amount and the functional group of selective binding and the amount of energy absorbing portion of first and second polymkeric substance, the feasible hydrogel that is formed with usefulness.

The present invention also provides a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, this precursor composition comprises: (a) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second polymkeric substance, it comprises energy absorption functional group, and wherein amount and the amount of Photocrosslinkable functional group and energy absorption functional group of first and second polymkeric substance are given performance and the hydrogel that this hydrogel precursor polymer blend compositions photo-crosslinking becomes hydrogel respectively and promoted analyte of interest desorption when being subjected to high energy gamma source such as laser bombardment to enter the performance of gas phase.

The present invention also provides a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, this precursor composition comprises: (a) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second polymkeric substance, and wherein this hydrogel also comprises the reactive group that can form covalent linkage with biomolecules, and wherein the amount of first and second polymkeric substance is given the performance that this hydrogel precursor polymer blend compositions photo-crosslinking becomes hydrogel with the amount of Photocrosslinkable functional group and energy absorption functional group.

A kind of hydrogel coating external member also is provided, it comprises: (a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second composition, its bag is second polymkeric substance also, this second polymkeric substance comprises the functional group of (i) selective binding biomolecule analysis thing, wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, or (ii) one or more energy absorbing portion.

Another kind of hydrogel coating external member comprises: (a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second composition, it comprises second polymkeric substance, and this second polymkeric substance can carry out synthesis modification to comprise the functional group of selective binding biomolecule analysis thing.

The fundamental sum new feature indefiniteness ground of other indefiniteness of the present invention comprises: light trigger does not need as independent composition.And, even polymer molecule can not have the group of Photocrosslinkable, also crosslinking reaction can take place in the polymer molecule.For example, the benzophenone group can shift a plurality of c h bonds.Many crosslinking reactions require by the unsaturated group that connects along main polymer chain for example acrylate or methacrylate based group radical polymerization or react by photodimerization.

Description of drawings

Fig. 1: the synoptic diagram of polymer blend method.

Fig. 2: [2-(methacryloyl oxygen base) ethyl] (4-benzoyl phenmethyl) dimethyl brometo de amonio is monomeric synthetic.

Fig. 3: the preparation of SAX monomer and the monomeric multipolymer of Photocrosslinkable.

Fig. 4: the serum of the SAX chip by blend preparation detects figure.

Fig. 5: the reaction of dextran and 4-benzoyl phenylformic acid.

Synthesizing of Fig. 6: 4-(glycidyl ether oxygen base) benzophenone.

Fig. 7: the reaction of dextran and 4-(glycidyl ether oxygen base) benzophenone.

Fig. 8: (a) the emission FTIR spectrum of CDI activatory dextran hydrogel on dextran hydrogel, (b) Al chip.

Fig. 9: the SELDI spectrum that is used for the CDI-dextran chip of antibody-antigen recognition research.

Figure 10: the serum of the deae dextran chip of blend preparation detects figure.

Figure 11: with the painted MEP of Ponceau S.

Figure 12: selective binding/washing of the last IgG of MEP.

Figure 13: remove albuminous serum detection figure.

Figure 14: remove albuminous serum detection figure.

Figure 15: remove albuminous serum detection figure.

Describe in detail

I. foreword

In this application, specifically described improved hydrogel material and method and the application in analysis of protein (proteomic) and mass spectral analysis, wherein used following term:

" surface-enhanced laser desorb/ionization " or " SELDI " refer to a kind of desorb/ionization gas phase ion spectrometry method (for example mass spectrum), wherein analyte is captured on the surface of SELDI probe, and this SELDI probe engages gas phase The probe interface of ionic light spectrometer. In " SELDI MS ", gas phase ion spectrometer is mass spectrograph. SELDI Technical description is at for example US 5,719,060 (Hutchens and Yip) and US 6,225,047 (Hutchens and Yip) in. " surface strengthen affinity capture " (" SEAC ") or " affinity gas phase ion spectrometry method " (" affinity for example Mass spectrography ") be the SEDLI method version of a kind of use probe (" SEAC probe ") of containing absorption surface. " inhale The subordinate list face " refer to that there is the probe that is attached with adsorbent (being also referred to as " capture reagent " or " affinity reagent ") in sample The surface. Adsorbent be arbitrarily can bound analyte (for example target polypeptides or nucleic acid) material. " chromatographic adsorbent " Refer to normally used material in the chromatography. Chromatographic adsorbent comprises, for example, and ion exchange material, metal chelating Mixture (for example nitrilo-acetic acid or iminodiacetic acid), fixing metal chelate (for example Cu, Fe, Ni, Zn, gallium), hydrophobicity interaction adsorbent, hydrophily interaction adsorbent, dyestuff, simple biomolecules (example Such as nucleotides, amino acid, monose and aliphatic acid) and (for example hydrophobicity attraction/Coulomb repulsion of mixed mode adsorbent Adsorbent). " biologic specificity adsorbent " refer to comprise biomolecule (for example nucleic acid molecules (for example aptamer), The conjugate of polypeptide, polysaccharide, lipid, steroids or these molecules (for example glycoprotein, lipoprotein, glycolipid (example Such as DNA)-protein conjugate)) adsorbent. In some cases, the biologic specificity adsorbent can be big branch Minor structure is such as polyprotein matter complex compound, biomembrane or virus. The example of biologic specificity adsorbent is antibody, is subjected to Body protein and nucleic acid. The biologic specificity adsorbent has higher spy than chromatogram adsorbent to target analytes usually The opposite sex. If the affinity of adsorbent bound analyte is at least 10^-8M、10 ^-9M、10 ^-10M、10 ^-11M or 10^-12During M, then this adsorbent is " biological selectivity " to analyte. Other of adsorbent that is used for SEDLI Example can be at US 6,225,047 (Hutchens and Yip, " Use of retentate chromatography to Generate difference maps, " May1,2001) in find.

In some embodiments, the SEAC probe is provided as pre-activating surface, can change this surface The property to provide adsorbent to select. For example, some probe is provided have can be by the covalent bonds biomolecule Reactive part. Epoxides and carbonization diimidazole (carbodiimidizole) are to inhale for the covalent bond biologic specificity The attached dose of useful reactive part of antibody or cell receptor for example.

In preferred embodiments, the affinity mass spectrography comprises that the liquor sample that will contain analyte is applied to SELDI The surface of probe. The analyte such as the polypeptide that adsorbent are had affinity are combined on the detecting probe surface. Usually, subsequently Clean surface removing unconjugated molecule, and stays the molecule of reservation. The degree that analyte keeps is adopted with institute With the stringency of cleaning relevant. Subsequently, energy absorbing material (for example matrix) is applied to adsorbent surface. Detect subsequently the molecule of reservation by laser desorption/ionization mass spectrometry.

SELDI is applicable to protein detection, wherein detects examination with one or several different SELDI surface Protein in the sample. On the contrary, protein detection is applicable to diference mapping, wherein with the protein detection of various sample Figure compares, with the difference of protein expression between the test samples.

" surface strengthens clean desorption " or " SEND " are a kind of SELDI type, and it comprises using to comprise and adheres to Probe (" SEND probe ") in the energy absorption molecular layer of detecting probe surface. Adhere to and for example to pass through covalency or non-The chemical bond of covalency is realized. Different from traditional MALDI, the analyte among the SEND do not need to be collected in for Within the crystalline matrix of the energy absorption molecule of desorb/ionization. " energy absorption molecule (" EAM ") " referring to can The desorb of the analyte molecule that absorbed energy and subsequently promotion are in contact with it from laser desorption/ionization source and ionization Molecule. This phrase comprises the molecule for MALDI, usually is called " matrix ", and comprises clearly Chinese cassia tree Acid derivative, sinapic acid (" SPA "), cyano group-hydroxyl-cinnamic acid (" CHCA ") and two hydroxybenzoic acid, asafoetide Acid, hydroxyacetophenone derivative etc. It also comprises the EAM for SELDI. In certain embodiments, will The energy absorption molecule is attached in linearity or the cross-linked polymer (for example polymethacrylates). For example, composition It can be the copolymer of alpha-cyano-4-methacryloxy cinnamic acid and acrylate. In another embodiment, Composition is alpha-cyano-4-methacryloxy cinnamic acid, acrylate and 3-(trimethoxy) silicyl propyl group The copolymer of methacrylate. In another embodiment, composition is for comprising alpha-cyano-4-methacryloxypropyl The copolymer (" C18 SEND ") of base cinnamic acid and methacrylic acid octadecane ester. It is special that SEND also is described in the U.S. Sharp US 5,719,060 and WO03/64594 (Kitagawa, " Monomers And Polymers Having Energy Absorbing Moieties Of Use In Desorption/Ionization Of Analytes ", August 7,2003) in.

SEAC/SEND is one type SELDI, wherein simultaneously with capture reagent and energy absorption molecule attached On the surface that sample exists. Therefore, the SEAC/SEND probe can be captured with desorption by affinity and be captured branch Analyse thing, and do not need to use outer matrix. The C18SEND biochip is one type SEAC/SEND, its The C18 part of capturing the agent effect and the CHCA part that plays the energy absorbing portion effect have been comprised.

" photo-labile that the surface strengthens adheres to and discharges " or " SEPAR " are one type SELDI, its bag Draw together and use the probe with the part that is attached to the surface, this surface can the covalent bond analyte, and passes through subsequently For example interrupt after the laser in this part the key of photo-labile and discharge analyte being exposed to light. SEPAR advances One step was described in US 5,719, in 060.

" absorption " refers to analyte and adsorbent or captures detectable non-covalent combination between the agent.

" elutriant " or " washing lotion " refers to the reagent that is generally solution, its be used for impact or change analyte with The absorption of adsorbent surface, and/or remove unconjugated material from the surface. The elution characteristic of elutriant can depend on In for example pH, ionic strength, hydrophobicity, unordered (chaotropism) degree, detergent concentration and temperature.

" analyte " refers to any component branch of the detected sample of expectation. This term can refer to single in the sample Component or a plurality of component.

" biochip " refers to has the solid matrix that is generally plane surface that adheres to capture reagent (adsorbent). Usually, the surface of biochip comprises a plurality of addressable positions, and each position has in conjunction with the agent of capturing on it. Can make biochip be applicable to the engages probe interface, and play thus probe.

Explanation about hydrogel blend

The present invention relates generally to hydrogel blend, and hydrogel and blend are known in polymer arts. For example referring to (1) Contemporary Polymer Chemistry, Allcock and Lamp, Prentice Hall, 1981, (2) Textbook of Polymer Science, 3^rd Ed.，Billmeyer，Wiley-Interscience，1984。

In the present invention, can be by two or more mixed with polymers be prepared blend polymer together, Comprise binary and tertiary blending. Sometimes, can use more low-molecular-weight polymer or oligomer, but usually make Standby blend is the polymer of more HMW, film forming, self-supporting preferably. Can prepare in the present invention Blend is to obtain high-quality film or layer. Polymer can be various forms of, for example comprise homopolymers, Copolymer, cross-linked polymer, network polymer, short chain or long-chain branched polymer, interpenetrating polymer networks and poly-Known other type hybrid system in compound field. This blend polymer can swelling when being exposed to aqueous environments, And form the hydrogel state that is characterised in that aperture and high water content. The hydrogel that is used for the mass spectrum application is described in example In the U.S. Patent application document 2003/0218130 that equals to submit on April 14th, 2003 such as Boschetii, its tool Volume description a series of based on polysaccharide, especially based on the material of glucan, comprise interpenetrating networks, and require 2002 The provisional application No.60/376 that submit on May 2, in, 837 priority, these two pieces of documents all are introduced as at this Reference.

A kind of polymer blend comprises the functional group of Photocrosslinkable, and this functional group can be used for this polymkeric substance and other polymkeric substance crosslinked with himself.Another polymkeric substance comprises the functional group that can fall into one of several kinds.One class is in conjunction with functional group.Can comprise the group that is used for non-covalent bound analyte in conjunction with functional group, as chromatographic adsorbent or biologic specificity sorbent material.Also can have the method for covalent attachment molecule in conjunction with functional group, described molecule again can bound analyte.For example, epoxide or imidazoles can conjugated proteins, and protein again can be in conjunction with another protein or small molecules such as medicine.Another kind of is energy absorption functional group.Energy absorption functional group has preferential absorptivity at the wavelength place of the energy source that is used for laser desorption/ionization method.So, they promote desorb and the ionization of analyte to gas phase in the laser desorption method.In some embodiments, polymer blend can comprise combination and energy absorption functional group simultaneously in one or more polymkeric substance of blend.

More specifically, the present invention has investigated following crosslinked hydrogel.In one embodiment, this hydrogel be comprise first polymkeric substance of the photoreactivity part that can carry out crosslinking reaction as benzophenone and comprise be used for and analyte non covalent bond bonded partly second polymkeric substance such as chromatographic adsorbent or biologic specificity sorbent material between crosslinked product.In another embodiment, this hydrogel is to contain first polymkeric substance of photoreactivity part and contain crosslinked product between second polymkeric substance of energy absorbing portion, this energy absorbing portion can absorb the light of high energy gamma source such as laser, and the analyte molecule that links to each other with hydrogel of promotion is to the desorption of gas phase.In another embodiment, this hydrogel is to contain first polymkeric substance of photoreactivity part and do not contain crosslinked product between second polymkeric substance of photoreactive group, wherein after crosslinked, can utilize one or more can the covalent coupling biomolecules such as self can be used for absorption of proteins functional group, energy absorption functional group or the reactive functional groups of analyte binding molecule such as epoxide or imidazoles and come this hydrogel of derivatize.In another embodiment, these hydrogels are coated at least a portion matrix such as the biochip surface.

Fig. 1 provides the summary of blend strategy provided by the present invention.Fig. 1 has described the preferred embodiment of using based on the material of dextran, but the present invention is not limited to the dextran material.In Fig. 1, " dextran of light linking agent modification " is the embodiment of first polymkeric substance, below is described further based on the functional group of Photocrosslinkable; " dextran that part is functionalized " is the embodiment of second polymkeric substance, below is described further based on functional group that can selective binding biomolecule analysis thing.Fig. 1 also illustrates other component, and it can be unmodified polymkeric substance, and the polymkeric substance of for example unmodified dextran and mark for example can be fluorescently-labeled dextran.This unmodified polymkeric substance can be used for the thinner of functionalized dextran of part for example and/or photocrosslinking agent modified glucan, makes to control part and/or linking agent density.Fluorescently-labeled dextran can be used for for example double check quality control.

Blend of the present invention can comprise the monomer subunits that contains Photocrosslinkable functional group, it has been described among the PCT application No.PCT/US04/04887 (attny docket no.035394-0252) of submission on 02 20th, 2004, and it all is incorporated herein by reference at this.This blend also can comprise the monomer subunits that contains selective binding functional group, it also has been described among the PCT application No.PCT/US04/04887 (attny docket no.035394-0252) of submission on 02 20th, 2004, and it all is incorporated herein by reference at this.

The embodiment of hydrogel blend also is described on 09 12nd, 2003 U.S. Patent applications of submitting to formerly 10/660,738 (" Preparation and Use of Mixed Mode Solid Substrates forChromatography Adsorbents and Biochip Arrays "; Attny docket no.035394-0245) in, its whole disclosures are hereby incorporated by and depend on its disclosure.In the patent application of ' 738, a kind of solid carrier has been described at this, can by the covalent linkage coating for example silicomethane basic unit come its modification, its again can covalent linkage ground in conjunction with crosslinked polysaccharide.As shown in the embodiment 10 of ' 738 patent application, this polysaccharide is preferably the blend of two kinds of polysaccharide, comprises first polysaccharide and second polysaccharide that is replaced by one or more crosslinked groups.Preferably, according to ' 738 patent application, comprise dextran, Natvosol, starch, amylase and agarose, and most preferably be dextran at this polysaccharide.But suitable linking agent comprises and is not limited to benzophenone.When crosslinked, according to ' 738 patent application, the crosslinked while of polysaccharide also is covalently bonded in carrier.In this manual, hydrogel blend further describes to comprising first and second mixture of polymers.

Secondary embodiment A

A secondary embodiment (embodiment A) comprises the theme of claims, comprises its Equivalent, and its concrete disclosure with respect to ' 738 patent application is new.In this pair embodiment A, got rid of the particular topic of ' 738 patent application, it comprises based on the use of the description of the hydrogel blend of polysaccharide, benzophenone, embodiment 10 (35-37 page or leaf) and claim 68.

II. first polymkeric substance: the polymkeric substance of Photocrosslinkable

The invention provides first polymkeric substance that comprises Photocrosslinkable functional group, wherein the also optional functional group that comprises selective binding biomolecule analysis thing of this first polymkeric substance.First polymkeric substance can prepare by homopolymerization or one or more monomers of copolymerization.For example, first polymkeric substance can prepare with the multipolymer that formation comprises the functional group of Photocrosslinkable functional group and selective binding biomolecule analysis thing by making monomer copolymerization.For example, first polymkeric substance can be the Photocrosslinkable polymkeric substance that for example is described in PCT application (specifying the U.S.) sequence number No.PCT/US04/04887 (on 02 20th, 2004, all be incorporated herein by reference at this).Quantity and the density that adopts copolyreaction to control Photocrosslinkable group in multipolymer and the final hydrogel precursor has been described in this PCT application, and it controls the cross-linking density in the final hydrogel.It has also described the various biomolecules analyte.But in the present invention, single polymkeric substance or multipolymer can use with second combination of polymers of various consumptions, with the quantity of Photocrosslinkable group in the control hydrogel precursor and the cross-linking density in density and the final hydrogel.

First polymkeric substance also can be as PCT applies for described in the PCT/US04/04887, prepare by the existing polymkeric substance of modification, for example, by on the side group that the Photocrosslinkable group is coupled to polymer chain to introduce the functional group of Photocrosslinkable, make the first pre-functionalized polymeric have the photo-crosslinking group thus.This embodiment is interested especially when the hydrogel that is used to prepare the nonionic polysaccharide base comprises the dextran hydrogel.

First polymkeric substance can comprise the also functional group of binding biomolecules analyte, but this point is not necessary.On the contrary, second polymkeric substance can provide in conjunction with functional group.When having the functional group of selective binding in first polymkeric substance, they can be identical or different with the selective binding functional group of second polymkeric substance.Can use the operation of mixed mode.

Generally include the structure type of polyalcohol hydrogel precursor of first and second polymkeric substance and the type of main polymer chain and be not specially limited, but can for example be simple linear polymer, branched polymer or even be branch-shape polymer.Be preferably line style.Usually be not crosslinked before being converted into hydrogel under the crosslinked condition though comprise the polyalcohol hydrogel precursor of first and second polymkeric substance, it still can only be water-swellable and non-water-soluble in some cases.In other words, the polyalcohol hydrogel precursor that comprises first and/or second polymkeric substance can be water miscible or water-swellable.As known in the art is to use monomer and prepolymer that hydrogel is provided.Preferably, the polyalcohol hydrogel precursor comprises the simple linear polymer main chain of being made up of carbon, and this main chain has first side group and optional second side group with selective binding functional group that has with Photocrosslinkable functional group.

The polyalcohol hydrogel precursor that comprises first polymkeric substance can prepare by the synthetic method of various non-special restrictions.When synthetic, the polyalcohol hydrogel precursor can be that to have main polymer chain and at least two kinds of covalent bonds be the polymer chain of Photocrosslinkable functional group and alternative bonded functional group in the functional group of main polymer chain.Selective binding functional group can come preferential select target based on chemical interaction as known in the art (comprising for example covalent bonds, non covalent bond combination, static combination and other pattern that further describes herein).These functional groups can be regular ground or randomly distribute along main polymer chain.Random distribution can help to improve the homogeneity of polymer materials, and this can help the accuracy of mass spectrometry applications such as SELDI.When cross-linked hydrogel, selective binding functional group can be optionally and protein and other biomolecule analysis thing and goal response, comprises covalent linkage or non covalent bond association reaction.

In preferred embodiments, the polyalcohol hydrogel precursor that comprises first and second polymkeric substance is polysaccharide (for example dextran) or polyolefin compositions.In preferred embodiments, the polyalcohol hydrogel precursor that comprises first and second polymkeric substance comprises the line style carbon backbone chain of being represented by following monomer subunits:

-(CH ₂-CHR ₁)-and-(CH ₂-CHR ₂)-,-[CH ₂-C (CH ₃) R ₁]-and-[CH ₂-C (CH ₃) R ₂]-; R wherein ₁And R ₂The group that comprises functional group respectively with Photocrosslinkable and selective binding.Usually monomer or Photocrosslinkable functional group is provided or selective binding functional group is provided.But, in this polyalcohol hydrogel precursor, also can have other monomer.Can use these monomers of existence to come density desired in the controlled polymerization compositions in conjunction with functional group.Select monomer suitably, can make the resulting polymers hydrogel precursor have improved water-soluble, biocompatibility and have the non-specific adsorption of reduction.Preferred monomer provides the best of breed of these performances.

In the preferred embodiment of first polymkeric substance, the polyalcohol hydrogel precursor is made up of the line style copolymerization main chain with side group basically, this side group comprises the functional group of Photocrosslinkable and the functional group of selective binding, wherein other structural unit in this multipolymer can not influence by the performance of photo-crosslinking and when crosslinked under aqueous conditions with the performance of protein selective reaction.Usually, the structure of multipolymer is designed to selective binding protein, and does not repel protein.

First monomer subunits that comprises Photocrosslinkable functional group is not specially limited.These first monomer subunits can play light trigger in the Photocrosslinkable polymer composition.In other words, because these monomer subunits make and must and preferably do not add the light trigger that is used for photo-crosslinking in said compositions.For example, the functional group of Photocrosslinkable can be the curable functional group of UV-.The functional group of Photocrosslinkable is enough responsive to photon, and it is exposed to photo-crosslinking condition following time and becomes hyperergy, and making does not need light trigger to produce photo-crosslinking.For example, the functional group of this Photocrosslinkable can carry out hydrogen abstraction reaction being exposed to photo-crosslinking condition following time.The example of Photocrosslinkable functional group comprises benzophenone, diazo ester, aromatic yl azide and diazomethane (diazirine), comprises its derivative such as benzophenone derivates.The hydrogen abstraction reaction of benzophenone type compound for example is disclosed among the US 5,856,066.Other Photocrosslinkable functional group or so-called " potential reactive group " for example are described among the US 5,002,582.

In preferred embodiments, the functional group of Photocrosslinkable is ketone or organic carbonyl functional group, for example comprises aromatic ketone functional group, as benzophenone and its derivative that replaces.Carbonyl carbon can have the replacement or the unsubstituted aromatic ring of at least one and its bonding.In the preferred embodiment, can use the vinyl monomer of Photocrosslinkable.Preferred embodiment is acrylate, methacrylic ester, acrylamide and methacryloyl amine system.For example can using, the traditional linked reaction between the hydroxyl and carboxylic acid or amino and carboxyl forms the monomer with Photocrosslinkable group and described other group (comprising selective binding functional group and energy absorbing portion) herein.

Preferred benzophenone and its derivative because they have multiple advantage, comprising: chemical stability; Activate as under 350～360nm at the wavelength of avoiding protein damage; The c h bond reaction of preferential and anergy, even have water and also be like this under the nucleophilic reagents in a large number.This compounds also can with its oneself main polymer chain or with a plurality of position reactions of other polymer chain on chain, and just on another Photocrosslinkable group, do not react.

First polymkeric substance preferably comes crosslinked by photo-crosslinking.Can use other cross-linking method, for example heat cross-linking or chemically crosslinked.For example, can be with the multiple polymers blend together that reacts to each other.Can control this reaction, make before the reaction of bringing out between the polymkeric substance, to form high-quality film at the very start.And, can be with the polymeric film swelling of blend, and with linking agent introduce carry out in the swollen matrix crosslinked.

III. second polymkeric substance: functional polymer

The present invention also provides a kind of second polymkeric substance that is used for first polymer blending.In preferred embodiments, second polymkeric substance can comprise the functional group that is used for selective binding biomolecule analysis thing, and wherein the functional group of selective binding biomolecule analysis thing can be identical or different in first polymkeric substance and second polymkeric substance.For example, second polymkeric substance can with the coupling of selective binding functional group, and selective binding functional group can be the part of second monomeric unit.This coupling can be carried out before or after with second polymkeric substance and first mixed with polymers.Usually, second polymkeric substance needn't have the functional group of Photocrosslinkable, although it should have the functional group as c h bond or other reactive group that distributes along its polymer chain, this functional group can with the Photocrosslinkable functional group reactions of first polymkeric substance.Selective binding functional group as useful chromatographic adsorbent also can be used for this Chinese described coating biology chip application.

Comprise that being used for the particularly proteinic selective binding of binding biomolecules analyte functional group second monomer subunits limits especially, and some examples have been described when relating to first polymkeric substance in the above.Selective binding and reaction can be based on covalently or non-covalently interacting, and bound fraction can be covalent bonds part or non covalent bond bound fraction.For example, multiple choices described in conjunction with functional group among U.S. Patent application No.2002/0060290A1 that quotes and the WO00/66265 in the above.Thereby ' 290 patent documentation is since the 70th section sorbent material that discloses the multiple choices bound analytes.These sorbent materials comprise the sorbent material (the 84th section) based on the promoted interactional sorbent material of salt (the 73rd section), the interactional sorbent material of wetting ability (the 80th section), electrostatic interaction, the sorbent material (the 100th section) of the sorbent material of coordination covalent interaction (the 93rd section), the interactional sorbent material of enzyme active sites (the 98th section), reversible covalent interaction, the interactional sorbent material of glycoprotein (the 102nd section) and the interactional sorbent material of biologic specificity (the 104th section).Other interaction comprises hydrophobic interaction.Can use these interactional combinations.

In addition, WO 00/66265 discloses serial selective binding functional group or in conjunction with functional group, is included in listed those in the 13rd～15 page.

Scheme as an alternative, second polymkeric substance can comprise the reactive functional groups that is used to adhere to from as the biomolecules of selective binding functional group.

Scheme as an alternative, second polymkeric substance can comprise energy absorbing portion, and this part helps in laser desorption/ionization method analyte to the desorb/ionization of gas phase.

Functional group

Further describe in conjunction with functional group and EAM functional group.

1. in conjunction with functional group

Usually can be divided into the broad variety that comprises following two classes in conjunction with functional group: the reactive functional groups and the adsorption functional group that can form non covalent bond that can form covalent linkage with target with target.

A. reactive functional groups

Reactive functional groups is applicable to other molecule attached in hydrogel.For example, can for example polypeptide, nucleic acid, carbohydrate or lipid be attached to hydrogel with biomolecules.Exemplary reactive functional groups comprises:

(a) carboxy derivatives is as N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, acyl halide, acylimidazole, thioesters, p-nitrophenyl ester, alkyl, thiazolinyl, alkynyl and aromatic ester;

(b) haloalkyl, wherein halogenide subsequently can be by for example bromo ethanoyl replacement of nucleophilic group;

(c) aldehydes or ketones base makes it possible to by forming carbonyl derivative for example imines, hydrazone, semicarbazone or oxime or carry out subsequently derivatization by the mechanism as Grignard addition or lithium alkylide addition;

(d) alkylsulfonyl halide group is used for reacting with amine subsequently, for example forms sulphonamide;

(e) reactive thiol group, it can react with the disulphide on the protein, comprises 2-mercaptopyridine and adjacent pyridyl disulfide;

(f) sulfydryl, it can for example be acylations or alkylating;

(g) thiazolinyl, it can for example carry out (as maleimides) such as Michael additions;

(h) epoxide, its can with for example amine and oxy-compound reaction of nucleophilic reagent;

(i) diazanyl, itself and carbohydrate and glycoprotein reaction;

(j) vinyl sulphone;

(k) activation carbonyl.

Can selective reaction functional group, making them not participate in or not influencing them does not need to participate in wherein reaction.Scheme can protect this reactive group to avoid participating in the reaction by the existence of blocking group as an alternative.It will be appreciated by those skilled in the art that how to go to protect specific functional group and do not influence selected reaction conditions setting.For the example of useful blocking group, referring to the Protective Groups In OrganicSynthesis of Greene etc., John Wiley ﹠amp; Sons, New York, 1991.

It will be appreciated by those skilled in the art that reactive functional groups discussed herein just represented the sub-fraction of functional group that is applicable to assembling chip of the present invention.

Exemplary reactive functional monomer is imidazoles, phenyl carboxyl ethanol, N-hydroxy-succinamide, N-hydroxyl maleimide, cystamine/DTT, Racemic glycidol, right-nitrophenyl methylol carbonic ether, benzotriazole base methylol carbonic ether, MeSCH ₂CH ₂OH, Ellman ' s reagent (4-nitro-3-carboxylic acid) disulphide and O-pyridyl-disulphide.

B. adsorption functional group

Be applicable in conjunction with functional group's (it also can adhere to by reactive functional groups) and from sample, capture analyte, be used for further analysis.In conjunction with functional group can be divided into two classes-biologic specificity in conjunction with functional group and chromatogram in conjunction with functional group.

In conjunction with functional group can be stratographic or biologic specificity.Chromatogram interacts by electric charge-electric charge, wetting ability-wetting ability, hydrophobicity-hydrophobicity, Van der Waals in conjunction with functional group and it makes up binding substance.

Biologic specificity comprises the complementary three-dimensional structure usually in conjunction with functional group, relates to one or more above-mentioned interactions.The example of combination comprises and being not limited to but biologic specificity interacts: antigen and corresponding antibodies molecule, nucleotide sequence and its complementary sequence, effect body molecule and acceptor molecule, enzyme and inhibitor, the compound that contains sugar chain and Sugar receptors, antibody molecule and have combinations such as specific another antibody molecule, acceptor molecule and corresponding antibody molecule for the former antibody.Other example of specificity junction mixture matter comprises combinations such as the antibody molecule of the antibody molecule of the plain modification of chemical-biological or polynucleotide and avidin, bonding avidin and vitamin H.As mentioned above, biologic specificity functional group partly prepares by reactive part settled organism specificity usually.

In exemplary, in conjunction with the monomer comprise be selected from following in conjunction with functional group: the part of positively charged, electronegative part, anionresin part, cationic exchange part, complexing of metal ion part, metal complex, polarity part, hydrophobic parts.Other exemplary combination functional group comprises amino acid, dyestuff, carbohydrate, nucleic acid, polypeptide, lipid (for example phosphatide base choline) and carbohydrate.

Partly be for example diethylamino ethyl, triethylamine, sulfonate, tetraalkylammonium salt and carboxylate salt as the ion-exchange in conjunction with functional group in the polymkeric substance of the present invention.

In exemplary, in conjunction with functional group is the polyaminocarboxylate sequestrant, as ethylenediamine tetraacetic acid (EDTA) (EDTA) or. diethylene triaminepentaacetic acid(DTPA) (DTPA), dicarboxylic anhydride (the Aldrich ChemicalCo. that is purchased by use, Milwaukee WI) is attached to it amine on matrix or the spacerarm.When with complexing of metal ion, this metal chelate is incorporated into the material of mark, and as the protein of poly-histidyl-(polyhistidyl) mark, it can be used for identification and combining target material.Scheme as an alternative, the material of metal ion self or complexation of metal ions can be target compound.

But complexing of metal ion partly comprises and being not limited to: N-Oxyethylethylenediaminetriacetic acid (NTA), N, two (the carboxymethyl)-L-Methionins of N-, iminodiethanoic acid, amino-iso hydroximic acid, salicylic aldehyde, oxine, N, N, N '-three (carboxyl trimethylammonium) thanomin and N-(2-pyridylmethyl) aminoacetate.The complexing of metal ion agent can complexing any useful metal ion, for example copper, iron, nickel, cobalt, gallium and zinc.

Organo-functional group can be a kind of composition of organic molecule with ability of specific recognition analyte molecule.But exemplary organic molecule comprises and being not limited to: amino acid, heparin, vitamin H, avidin, streptavidin, carbohydrate, gsh, Nucleotide and nucleic acid.

In another exemplary, be biomolecules in conjunction with functional group, for example natural or synthetic peptide, antibody, nucleic acid, carbohydrate, lectin, receptor/ligand are in conjunction with right member, antigen, cell or its combination.Like this, in exemplary, in conjunction with functional group at target compound or at the antibody of the material that is similar to target compound on the structure.In another exemplary, be the avidin or derivatives thereof in conjunction with functional group, its analogue with the biotinylation (biotinylated) of target compound combines.In another exemplary, be nucleic acid in conjunction with functional group, it is bonded to sub-thread or bifilar target set nucleic acid thing, and this target compound has and the sequence complementary sequence that combines functional group.

In another exemplary, chip of the present invention is the oligonucleotide array, each addressable locational nucleic acid with specific nucleotide sequence that all comprises in conjunction with functional group in the array.Particularly, this array can comprise oligonucleotide.For example, can select oligonucleotide, make it cover the sequence of interested specific gene.Scheme as an alternative, this array can comprise cDNA or the est sequence that is applicable to express spectra.

In another embodiment, this is selected from nucleic acid substances in conjunction with functional group, as discerns the adaptive son (aptamer) and the adaptive nuclease (aptazyme) of special target compound.

In another exemplary, this is a drug moiety or derived from the pharmacophore of drug moiety in conjunction with functional group.This drug moiety can be the reagent that clinical use has been accepted, and perhaps they can be that its application is experimental or their activity or the mechanism of action are in medicine in the research.This drug moiety can have the checking effect in given symptom, perhaps can only suppose it shows expectation in given symptom effect.In preferred embodiments, the compound of this drug moiety for having sieved for the interaction ability of they and selected target compound.Thereby, be applicable to that enforcement drug moiety of the present invention comprises the medicine in the wide region drug categories with various pharmacologically actives.

Exemplary hydrophobicity adsorption function monomer comprises CH ₃(CH ₂) ₁₇OH, 1-Stearyl alcohol, 1-V-1326, perfluor polyoxyethylene glycol (Sovay, USA).

Exemplary wetting ability adsorption function monomer comprises polyvinyl alcohol and polyvinylpyrrolidone.

Exemplary anionresin adsorption function monomer comprises 3-chloro-2-hydroxypropyl-trimethyl ammonium chloride and 2-hydroxy-n-methyl chloropyridine (pyridium).

Exemplary cation exch ange adsorption function monomer comprises 1,4-butyleneglycol-2-sulfonic acid, 3,5-dihydroxymethyl Phenylsulfonic acid, resorcylic acid and dihydroxymethyl acetate.

Exemplary metal-chelating adsorption function monomer comprises N-Oxyethylethylenediaminetriacetic acid (NTA), N, two (the carboxymethyl)-L-Methionins of N-, iminodiethanoic acid, amino-iso hydroximic acid, salicylic aldehyde, oxine, N, N, N '-three (carboxyl trimethylammonium) thanomin and N-(2-pyridylmethyl) aminoacetate.The solution that adds metal ion such as copper, nickel, zinc, iron and gallium makes the gel functionalization.

2.EAM functional group

In some embodiments, EAM (energy absorption molecule) functional group can help lend some impetus to desorb and the ionization of analyte to gas phase during laser desorption/ionization process.The EAM monomer comprises the photoreactivity part as functional group.This photoreactivity partly preferably includes nuclear or the prothetic group that specificity absorbs the optical radiation of self-excitation light source.This photoreactive group absorbs from the energy in high-throughput source producing heat energy, and heat energy is transformed to promote and the desorb and the ionization of the analyte that gel effectively contacts.Under the attached situation of UV laser desorption, the EAM monomer preferably includes the aryl nucleus of electronics absorption UV optical radiation.Under the attached situation of IR laser desorption, the EAM monomer preferably includes by preferred directly vibration resonance or with the aryl nucleus or the prothetic group of slight off-resonance pattern absorbing IR radiation.UV photoreactivity part can be selected from phenylformic acid (for example 2,5-resorcylic acid), styracin (for example alpha-cyano-4-hydroxycinnamic acid), methyl phenyl ketone, benzoquinones, vanillic acid, coffic acid, nicotinic acid, sinapinic acid pyridine, forulic acid (ferrulic acid), 3-amino-quinoline and its derivative.IR photoreactivity part can be selected from phenylformic acid (for example 2, the 5-resorcylic acid), styracin (for example alpha-cyano-4-hydroxycinnamic acid), methyl phenyl ketone (for example 2,4,6-trihydroxy-acetophenone and 2,6-resacetophenone), coffic acid, forulic acid, nicotinic acid, sinapinic acid 3-amino-quinoline and its derivative.

IV. blending condition

Blending condition is not particularly limited, and can carry out functional control to it as those skilled in the art are known, to obtain desired result.For example, at Concise Encyclopediaof Polymer Science andEngineering, Ed.J.I.Kroschwitz (Wiley), 1990, " Polymer Blends ", the reference of being quoted in 830-835 page or leaf and the bibliography for example comprise the Polymer Blends of D.R.Paul etc., Vol.I and II, Academic Press, New York has described the blend of polymkeric substance in 1978.Usually, preferred solution blend and casting film method obtain the film and the layer of desired thickness.For example, can filtering solution to improve the quality.Can control drying rate.Those skilled in the art can adjust order, use blocking group and other method well known in the art of reagent interpolation and protect functional group, make to obtain desired functional group in final hydrogel blend.

The amount of the functional group of the amount of first and second polymkeric substance and photo-crosslinking functional group and selective binding biomolecule analysis thing give respectively the hydrogel precursor polymer blend compositions with by photo-crosslinking in the hydrogel performance and the performance of hydrogel selective binding biomolecule analysis thing.For example, first polymkeric substance can comprise the monomer subunits that contains Photocrosslinkable functional group of about 0.5 mole of %～about 15 moles of %, perhaps more specifically is the monomer subunits of about 1 mole of %～about 7 moles of %.The weight fraction of first polymkeric substance can for example be about 5wt%～about 60wt%, more specifically is about 15wt%～about 45wt%.The weight fraction of second polymkeric substance can for example be about 40wt%～about 95wt%, more specifically is about 55wt%～about 85wt%.But if for example first and second polymkeric substance are with diluted polymer (for example unmodified dextran) when using, the weight fraction of second polymkeric substance can for example be about 25wt%～about 80wt%, more specifically is about 40wt%～about 60wt%.Certain embodiments is described below, and, can further controls the amount of first and second polymkeric substance, the amount of photo-crosslinking functional group and the amount of selective binding functional group for application-specific.

Can control with another parameter that realizes desired blended characteristic is molecular weight, and described blended characteristic comprises soltion viscosity and forms the ability of high-quality coating.For example.The weight-average molecular weight of first polymkeric substance and second polymkeric substance respectively can for about 1,000～about 10,000,000, be specially about 10,000～about 1,000,000, and more specifically be about 20,000～about 100,000.

Except first and second polymkeric substance, operable alloying agent comprises, for example the polymkeric substance of fluorescence modification or the polymkeric substance by the fluorophor modification (as above about Fig. 1 record).Usually, preferred polymer modification alloying agent be have with first and second polymer phases with the reagent of main polymer chain.For example, for first and second polymkeric substance based on dextran, the dextran of operable fluorescence modification for example is the fluorescein dextran.Dextran can use fluorescent indicator such as fluorophore, dyestuff to wait modification or conjugation, and such product is commercially available or can customize synthetic.Can select the fluorophor compatible with the photo-crosslinking group.For example, can use absorption and emmission spectrum to select the fluorescent polymer that suits.

Another type alloying agent is that use has the main polymer chain identical with first and second polymerizations, still it does not carried out the polymkeric substance that modification becomes to have the functional group of selective binding biomolecule analysis thing or Photocrosslinkable.These alloying agents can be the thinners of functional group, and realize crosslinked and in conjunction with the better control of density.

V. functionalized surface and prepare the method for matrix

In order to use hydrogel blend according to the present invention to come functionalized surface, can use four step rule.The first step can prepare stayed surface, chooses wantonly to have undercoat.In another step, can prepare polyalcohol hydrogel blend precursor by body.In another step, mass polymer hydrogel blend precursor can be attached on the stromal surface as homogeneous polymer coating physics.At last, can implement the photo-crosslinking condition, for example be exposed under the UV light, to form the hydrogel surface coating, impel surface bonding and/or fixing, and generate polymer network.

In order to use hydrogel to come functionalized surface, can be at first with the dissolving of polyalcohol hydrogel precursor be suspended in single or the blended solvent system in.Solvent system can be water-based or organic.Before using the photo-crosslinking condition, can partly or entirely remove solvent system.Subsequently, the polyalcohol hydrogel precursor can be contacted with the surface, with form layers from the teeth outwards.

Forming the layer of polyalcohol hydrogel precursor and the method for film on stromal surface is not particularly limited.For example, in order to obtain uniform layer, operable method comprises spin coating, dip-coating, roller coat, spraying, silk screen printing, ink jet printing, chemical vapour deposition and other known coating process.Can use coating procedure to one chip matrix.Scheme as an alternative, one chip matrix can be formed the high surface area fixture of implementing coating procedure thereon.In another embodiment, can use general semiconductor technology: can on big flat surfaces, be coated with coating solution with the formation uniform coating, and the wafer that applies can be cut into the small pieces of a plurality of appropriate sizes subsequently.The sheet that is cut can directly be used as chip, perhaps shifts and is installed on the chip carrier matrix.Wafer material can for example be plastics, glass, silicon, metal or metal oxide.When all are adsorbed on the energy source of analyte on the detecting probe surface and gas phase ion spectrometer when equidistant, the hydrogel surface coating can obtain more accurate sample time-of-flight analysis uniformly.The hydrogel precursor of copolymerization be used to deposit, in-situ polymerization compares with crosslinked monomer solution, the former has the more compatible enough viscosity of coating process that makes connate water gel coat and foundation, this helps to form even and consistent hydrogel surface.

Can the polyalcohol hydrogel precursor be applied from the teeth outwards with the form of discontinuous discrete point or successive layers.Known patterning process be can use, photolithography and mask means comprised.

As known in the art, can use the local control of photomask photopolymerization to form pattern.Can wash unexposed zone and stay crosslinked hydrogel.This can be the form of point.For example, can form lateral dimensions and be about 100nm～about 3mm and more specifically be the point of about 500nm～about 500 μ m.

The matrix generality is described in 15～17 pages of WO 00/66265.Matrix can be made by the appropriate materials that can support hydrogel material arbitrarily.This matrix can have various performances, comprises porous or non-porous, inflexible or flexible.Stromal surface can be a different shape, comprises the plane.But the matrix with flat surfaces will provide polymer coated uniformly better.

Can use matrix material or polycomponent matrix, for example two-pack matrix.In two-pack matrix, for example, the solid fragment of silicon or glass can be embedded in the aluminum frame.Can on aluminum matrix, process long striped, to adapt to the embedding of slide glass or silicon wafer.Can use tackiness agent to adhere to.

Applying on aluminum matrix is particularly preferred embodiment.

Can carry out physics or chemical modification to matrix, to improve the adhesive power of hydrogel blend and matrix.In chemical modification, for example, stromal surface can be the surface by the undercoat of support layer supports.This undercoat is not particularly limited, but can for example be the hydrophobicity undercoat.Preferred hydrophobicity undercoat because it also can play the effect of passivation layer, is avoided the influence of the aqueous solution with the protection stromal surface.For example, it can be fluoridized/hydrocarbon silane undercoat or copolymer bottom coating for silane undercoat, hydrocarbon silane undercoat, fluorinated silane undercoat, blended.When using matrix of oxide, can use organoalkoxysilane and chlorinated silane chemistry to form undercoat.When using precious metal for example during the matrix of gold and silver, can use alkanethiol or disulphide to form undercoat.

The example of physically modified stromal surface comprises regulates the surface so that it is coarse, micropore or porous.

The not specific qualification of the thickness of initial bed, but can for example be about 4 ～about 10 μ m and more specifically for about 5nm～about 10 μ m and more specifically be about 10nm～about 10 μ m.

The type of supporting layer is not particularly limited, but can be for inorganic or organic.For example, it can be aluminium, silicon, glass, metal oxide, metal, polymkeric substance or matrix material.When hydrogel is used as the SELDI probe, can use conductive carrier.Can use plastic material as carrier.Can be by dissimilar polymkeric substance being mixed together or blend and further change the characteristic of plastic material by adding other material.For example, can introduce granular filler for example carbon dust, silicon-dioxide, pottery and powder metal to regulate the modulus and the specific conductivity of matrix material.Can use other additive, to improve chemical resistance performance and thermostability.

No matter whether stromal surface scribble bottom, can adjust by the functional group of Photocrosslinkable, but make the hydrogel coating photochemical fixation.For example, the Photocrosslinkable functional group of benzophenone type can combine with the C-H group.Stromal surface can comprise photoreactivity functional group, to promote during the photo-crosslinking and the combining of hydrogel.

Photo-crosslinking can be optionally, makes some polyalcohol hydrogel precursors by photo-crosslinking, and some polyalcohol hydrogel precursors are not by photo-crosslinking.Can form the discrete point of cross-linked hydrogel.For example remove residue by in water, cleaning.The result is the surface of pattern form.Can use traditional photolithography, comprise the photomask method.If stromal surface is hydrophobic, then the zone between the hydrophilic hydrogel can be hydrophobic.The drop that therefore, on specified point, can keep the aqueous solution.

In preferred embodiments, crosslinked polyalcohol hydrogel precursor is a basic homogeneous layer on the stromal surface, and average bed thickness be about 5nm～about 50 μ m and more specifically for about 5nm～about 10 μ m and more specifically be about 10nm～about 10 μ m and more specifically be about 100nm～about 10 μ m and more specifically be about 100nm～about 2 μ m.

The thickness of these hydrogel coatings can be importance of the present invention.Usually estimate the thickness of hydrogel coating by means commonly known in the art, for example by the multiple measurement of reflection measurement with reflection FTIR.Thickness can for example be about 1 μ m～about 10 μ m and more specifically be about 2 μ m～about 5 μ m.

The present invention is bound by theory not, but thick relatively hydrogel coating can give higher quantity that the bigger surface-area of detecting probe surface and can be used for captures sample in conjunction with functional group.Therefore, higher binding capacity will be expected.But, in the SELDI process, for conjugated protein importantly, by laser excitation by desorption and ionization before, from hydrogel layer, extract and with the EAM cocrystallization.For thick hydrogel, may be difficult in the sample extraction step, discharge fully the analyte of capturing, and thin hydrogel can more effectively extract and use the analyte of being captured.

Therefore, the thickness of hydrogel coating not only influences binding capacity, and influences extraction efficiency.Obtain best thickness by balance binding capacity and extraction efficiency.

Matrix can be the matrix that is used for biochip.In this, hydrogel can be covalently bond to stromal surface.

VI. use the hydrogel of blend:

Can use hydrogel blend according to the present invention to prepare one or more external members.For example, in one embodiment, a kind of hydrogel coating external member is provided, it comprises: (a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second composition, it comprises second polymkeric substance, this second polymkeric substance comprises the functional group of (i) selective binding biomolecule analysis thing, wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, or (ii) one or more energy absorbing portion.Two kinds of components can be packaged in the independent container, and provide about the specification sheets of the guidance (consumption that comprises component) of the composition and the blending ingredients of for example external member and sell.This specification sheets can further describe the type of the biomolecule analysis thing that said composition can selective binding.If desired, can provide matrix, comprise the matrix that is used for mass spectroscopy with this external member.Utilize this external member, can prepare biochip.

In another external member, the hydrogel coating external member comprises: (a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, (b) second composition, it comprises second polymkeric substance, and this second polymkeric substance can be synthesized modification to comprise the functional group of selective binding biomolecule analysis thing.Can provide other reagent to come synthesis modification second polymkeric substance, make it comprise the functional group of selective binding biomolecule analysis thing.In addition, the book that can furnish an explanation.

The biochip that can use blend hydrogel of the present invention to apply detects the analyte that is applied on this gel by the whole bag of tricks well known in the art.When using functionalized this hydrogel of conjugated group, this chip will be captured the surface analysis thing that is bonded to special groups.Can wash unconjugated material off.Can come the check and analysis thing by the method for any appropriate, comprise gas phase ion spectrometry method, optical means, electrochemical method, atomic power microscopy and frequency of radio method.The gas phase ion spectrometry method comprises that for example mass spectrum, ion mobility spectrometry and full ion(ic)current are measured.Optical means comprises microscopy (confocal with non-confocal), imaging method and non-imaging method.Optical detection can comprise and detects fluorescence, luminous, chemoluminescence, absorption, reflection, transmission and double refraction or specific refractory power (for example surface plasmon resonance (" SPR "), ellipsometry, quartz crystal trace Libra, resonant mirror method, grating coupler waveguide method (for example wavelength inquiry optics system (" WIOS ")) and interference measure).Electrochemical process comprises, for example voltammetry and amperometry law technology.The frequency of radio method comprises, for example multipole resonance spectrum method.Some of these methods are the keying action between check and analysis thing and the capture molecule in real time.Other method as the attached mass spectroscopy of laser desorption, comprises the analysis tool (SBAT) based on the surface, and this instrument need be communicated with the stromal surface direct physical of capturing analyte thereon.

Sample does not limit especially, is selected from following fluidic biofluid but can contain: as saliva, sputum, blood, serum, urine, lymph fluid, prostate gland fluid, seminal fluid, breast, cell extract, cell culture medium and its derivative.In some embodiments, can be with before adsorption surface contacts, by granularity exclusion chromatography and/or ion exchange chromatography pre-classification sample.

Sample can contact with the hydrogel sorbent material on the probe matrix.Treatment process in this depends on detection method.For example, in the situation of SELDI, can make sample dry on the hydrogel sorbent material.Can cause analyte in the sample by hydrogel sorbent material specificity and non-specific adsorption like this, and need not wash the analyte that is not incorporated into the absorbed agent off.Usually, contain several 10 ^-18Mole～100 * 10 ^-12The sample volume of about 1 μ L～about 500 μ L of mole analyte is enough to be used for the combination water gel adsorber.

In some embodiments, remove after the liquid sample, energy absorbing material can be applied on the probe.But the example of energy absorbing material comprises and being not limited to: cinnamic acid derivative, sinapinic acid and resorcylic acid.

Analyte is applied on the probe and drying after, adopt the gas phase ion spectrometry method that it is detected.Can use gas phase ion spectrometer analyze with probe on sorbent material bonded analyte or other material.Can adopt the gas phase ion spectrometry method to come the quantity and the characteristic of Measurement and analysis thing.Also can adopt gas phase ion spectrometry rule such as laser desorption ionisation mass spectroscopy to detect other material outside the interested analyte.

The gas phase ion spectrometry method detects:

The generation of data starts from by the ion detector detect ion in the mass spectrum.Typical laser desorption mass spectrograph can use the nitrogen laser of 337.1nm.The pulse width that is suitable for is about 4 nanoseconds.Usually, use the output rating of about 1～25 μ J.The ion of collision detector produces current potential, by digital high speed time-array (time-array) recording unit of simulating signal of capturing with this current potential digitizing.Ciphergen ' s ProteinChip  system has adopted analog-digital converter (ADC) to realize this process.The detector output that ADC locates the timed interval that rule is separated is integrated into the piece (bin) of the time of depending on.It is long that the timed interval was generally for 1～4 nanosecond.In addition, the flight time spectrum of ultimate analysis is not represented the individual pulse signal at the ionization energy of sample usually, but a plurality of pulse signals and.Reduce noise like this, and increased dynamicrange.Subsequently the flight time data are carried out data processing.In Ciphergen ' s ProteinChip  software, data processing generally includes TOF and subdues (baseline subtraction), high frequency noise filtration to conversion, the baseline of M/Z.

TOF comprises that to the conversion of M/Z use will transfer the algorithm of the ratio (M/Z) of quality and electric charge (mass-to-charge) flight time to.In this step, signal is converted into mass domain from time domain.In other words, be converted into the ratio or the M/Z of quality and electric charge each flight time.Can be interior or calibrate outside.In internal calibration, the sample of being analyzed contains the analyte of one or more known M/Z.To be appointed as known M/Z at the fignal center that the flight time of representing these set analysis things is located.Based on these specified M/Z ratios, calculate the parameter that is used for the flight time is converted into the mathematical function of M/Z.When external calibration, the function (as being set up by previous internal calibration) that will be converted into M/Z the flight time is used to flight time spectrum, and does not use internal calibration.

Baseline is subdued the quantitative evaluation that improves data by artificial, the reproducible instrumental bias of eliminating interference spectrum.It comprises that the use algorithm calculates the spectrum baseline, and this algorithm combines parameter such as peak width; And from mass spectrum, reduce baseline subsequently.

Eliminate HF noise signal by using smooth function.Typical smooth function is applied to average move function in each piece that depends on the time.In improved plan, average moving filter device is the digital filter of variable-width, and the bandwidth of its middle filtrator changes along with the variation of for example peak bandwidth, becomes wideer along with the increase of flight time usually.For example referring to WO 00/70648,2000.11.23 (Gavin etc. " Variable Width DigitalFilter for Time-of-flight Mass Spectrometry ").

Computer can be converted to the spectrum that is obtained the various forms that are used to show.In a kind of form, it is called as " spectrogram or keep figure ", can the display standard spectrogram, and wherein view illustrates the quantity of the analyte of each specified molecular weight that arrives detector.In another kind of form, it is called as " peak value figure ", has just kept peak heights and quality information from spectrogram, forms clearer image, and can more easily find to have the analyte of molecular weight much at one.In another kind of form, it is called as " gel figure ", can be the gray level image based on each peak height with each quality conversion among the peak figure, obtains to be similar to the profile of the wavestrip on the running gel.In another kind of form, it is called as " the stacked figure of 3D ", can several spectrum are stacked, and to study the trickle change on the relative peak height.In another kind of form, it is called as " diference mapping figure ", can more two or more spectrum, show the analyte of being regulated up or down between unique analyte and the sample expediently.

Analysis generally includes the identification representative from the peak in the spectrum of the signal of analyte.Certainly, can carry out the peak by eyes selects.But, can obtain the software of detected peaks automatically, it is the part of Ciphergen ' sProteinChip  software.Generally speaking, this software is by ratio the signal selected threshold value on of discernible signal with noise, and is marked at the peak quality at peak-to-peak signal center.In a kind of useful application, a plurality of spectrum are compared, to distinguish the identical peak that in the mass spectrum of some selected per-cent, exists.A kind of this software convergence of version all peaks that appear in the mass range in the various spectrum that define, and quality (M/Z) is assigned to all peaks near the mid point of quality (M/Z) bunch.

Can further analyze the peak data in one or more spectrum, for example, set up spreadsheet, wherein every row has been represented specific mass spectrum, and every row have been represented the peak in the spectrum that quality limits, and every lattice are included in the intensity at peak in the special spectrum.Various statistics or mode identification method can be applied to these data.

The spectrum that generates in embodiment of the present invention can use and adopt the mode identification method of disaggregated model to come it is classified.Usually, spectrum will be represented the sample from least two kinds of different sets, and it is sought sorting algorithm.For example, this set can be pathology to nonpathologic (for example tumour or non-tumour), drug responses device to the non-transponder of medicine, toxic reaction to non-toxic reaction, symptom source to non-symptom source, exist the phenotype condition to there not being the phenotype condition.

In some embodiments, subsequently can be with next " training " disaggregated model of the data from spectrum (for example mass spectrum or flight time spectrum) that utilizes such as the sample generation of " known sample ".The sample that " known sample " classified in advance.That from spectrum, obtain and be used to form the data of disaggregated model, can be called as " training dataset ".In case trained, this disaggregated model just can be discerned the pattern in the data that obtain from the spectrum that uses unknown sample to generate.This disaggregated model can be used for unknown sample is classified subsequently.This point can be used for for example forecasting that specific biological material is whether relevant with some biotic condition (for example ill or non-ill).

The training dataset that is used to form disaggregated model can comprise raw data or preprocessed data.In some embodiments, can from flight time spectrum or mass spectrum, directly obtain raw data, and can randomly carry out " pre-treatment " to it as mentioned above subsequently.

Can use statistical classification (or " study ") method of any suitable to form disaggregated model, these methods attempt data volume is isolated into classification based on the objective parameter that exists in the data.Can monitor or not monitor disaggregated model.The sorting technique that monitors and do not monitor be described in Jain, " Statistical Pattern Recognition:AReview ", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol.22, No.1, January 2000.

In the classification that monitors, the training data that will contain the known type sample offers study mechanism, and it learns one group of dependency that defines each known class.New data can be offered study mechanism subsequently, it uses the dependency of study this new data of classifying subsequently.The example of the sorting technique that monitors comprises linear regression method (for example polyteny returns (MLR), partial least square side (PLS) returns and principal component regression (PCR)), binary decision tree (for example recurrence partitioning, as the CART-classification and regression tree), artificial neural network such as back-propagating network, discriminant analysis (for example Bayesian sorter or Fischer analyze), logical division device and support vector sorter (support vector machine device).

Preferred supervision sorting technique is the recurrence partitioning.The spectrum that the recurrence partitioning uses the recurrence partition tree to classify and obtain from unknown sample.Other detailed content about the recurrence partitioning is described in US 6,675,104.

In other embodiments, the disaggregated model of foundation can use the learning method that does not monitor to form.The classification that the classification trial learning that does not monitor is concentrated similarity based on training data does not therefrom obtain the spectrum of training dataset and do not classify in advance.The learning method that does not monitor comprises a bunch analysis.Bunch analyze to attempt data are divided into " bunch " or group, this bunch or group should have very similar each other component in theory, and completely different with the component of other bunch.Use some to measure similarity subsequently apart from the metric system, the distance between its take off data item, and with collection of data items closer proximity to each other together.Clustering technique comprises MacQueen ' s K-means algorithm and Kohonen ' sSelf-Organizing Map algorithm.

Can on the digital machine of any suitable, form and use this sorting technique.Suitable digital machine comprises and uses arbitrary standards or specific operation system (as based on Unix, Windows ^TMOr Linux ^TMOperating system) miniature, small-sized or giant-powered computer.Employed digital machine can with set up interested spectrographic mass spectrograph physical isolation, perhaps it can be connected to mass spectrograph.

Training dataset and disaggregated model can embody by the computer code of being carried out by digital machine or use according to embodiments of the present invention.Computer code can be stored on the computer-readable medium of any suitable, comprises dish, rod, band of optics or magnetic etc.; And this computer code can be write with the computer programming language of any suitable, comprises C, C++, visual basic etc.

At last, the present invention also comprises particle and the bead that contains above-mentioned polyalcohol hydrogel precursor and hydrogel.Particulate mean diameter or size can for example be about 0.01 μ m～about 1,000 μ m, more specifically be about 0.1 μ m～about 100 μ m and more specifically for about 1 μ m～about 10 μ m.For consistent mass resolution and intensity is provided, preferably this particulate size or diameter are homogeneous.For example, particulate diameter deviation coefficient can less than about 5%, preferably less than about 3%, be more preferably less than about 1%.In one embodiment, this particle can be made by hydrogel, and this particle is substantially free of non-gelatinous material.In another embodiment, this can be coated with granules preparation on the non-aqueous gel particle of hydrogel.

Embodiment

Use following non-restrictive example to further describe the present invention.

1. the Sax copolymer blend of Photocrosslinkable

Embodiment 1

General introduction

In first group of experiment; by poly-(3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride) multipolymer and poly-(3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride) homopolymer blend, prepare the SAX copolymer blend of one group of Photocrosslinkable thus with 10 moles of % benzophenone (BP) modification.The BP molar fraction of the multipolymer of this blend changes between 1～10 mole of %.In addition, also prepared the SAX multipolymer of 3 moles of % benzophenone modification, and used it for the preparation hydrogel coating of making comparative research by copolymerization process.

Experiment 1:

Preparation [2-(methacryloyl oxygen base) ethyl] (4-benzoyl phenmethyl) dimethyl brometo de amonio monomer (monomer of Photocrosslinkable)

According to Fig. 2, with 132mL toluene/acetone (5: 1, V/V) add in the dry round-bottomed flask of the 250mL that magnetic stirring apparatus is housed with 15.45 gram 4-(brooethyl) benzophenone (Aldrich).At room temperature in this solution, dropwise add the gram of 10.59 in 20mL toluene methacrylic acid 2-(dimethylamino) ethyl ester.After 2 hours, filtering precipitate, and with washing with acetone four times.Desciccate powder under vacuum subsequently.Productive rate is about 90%. ¹H NMR confirms to have formed desired product.

Experiment 2:

The preparation of 3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride (SAX) monomer and [2-(methacryloyl oxygen base) ethyl] (4-benzoyl phenmethyl) monomeric multipolymer of dimethyl brometo de amonio

Preparation has 10 moles of %[2-(methacryloyl oxygen base) ethyl] the SAX Photocrosslinkable multipolymer of (4-benzoyl phenmethyl) dimethyl brometo de amonio.As shown in Figure 3; with 44.0 gram 3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride solution (Aldrich; the 50wt% aqueous solution) mix with 60 gram distilled water; mix with 4.79 gram [2-(methacryloyl oxygen base) ethyl] (4-benzoyl phenmethyl) dimethyl brometo de amonios and 0.36 gram V-50 (WakoChemical, a kind of water-soluble, cationic azo initiator) subsequently.Purify this solution 5 minutes with argon gas stream.Sealed vessel, and heated 40 hours down at 58 ℃ subsequently.Solution becomes gets very sticking after the polymerization.Use deionized water to dilute this polymers soln, and lyophilize under vacuum, white solid product obtained.

Experiment 3:

The preparation of 3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride (SAX) homopolymer

180.0 gram 3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride solutions (Aldrich, the 50wt% aqueous solution) are mixed with 400 gram distilled water, mix with 0.17 gram V-50 (Wako Chemical, a kind of water-soluble, cationic azo initiator) subsequently.Purify this solution 5 minutes with argon gas stream.Sealed vessel, and heated 40 hours down at 58 ℃ subsequently.Solution becomes gets very sticking after the polymerization.Use deionized water to dilute this polymers soln, and lyophilize under vacuum, white solid product obtained.

Experiment 4:

The SAX multipolymer for preparing 3 moles of %BP-modifications by blend

The SAX multipolymer and 3.886 of 10 moles of % benzophenone of 1.665 grams modification is restrained the SAX homopolymer at 37mL deionization H ₂The O/2-Virahol (7/3, W/W) the middle mixing, obtain transparent solution.This solution is filtered, and be used for the coating experiment.Use identical step to prepare the blended copolymer of SAX multipolymer with 1 mole of %BP-modification.

Experiment 5:

The SAX multipolymer for preparing 3 moles of %BP-modifications by copolyreaction

Also the SAX multipolymer for preparing 3 moles of %BP-modifications by copolyreaction is studied as a comparison.

With 70 gram 3-(methacryloyl amino) oxypropyl trimethyl ammonium chloride solution (Aldrich; the 50wt% aqueous solution) mix with 93 gram distilled water; mix with 2.04 gram [2-(methacryloyl oxygen base) ethyl] (4-benzoyl phenmethyl) dimethyl brometo de amonios and 0.30 gram V-50 (Wako Chemical, a kind of water-soluble, cationic azo initiator) subsequently.Purify this solution 5 minutes with argon gas stream.Sealed vessel, and heated 40 hours down at 58 ℃ subsequently.Solution becomes gets very sticking after the polymerization.Use deionized water to dilute this polymers soln, and lyophilize under vacuum, white solid product obtained.

Experiment 6:

Apply SiO ₂Aluminum matrix on the preparation of SAX hydrogel coating

The blended copolymer solution that will have 3 moles of % Photocrosslinkable groups is dispersed on the surface of the aluminum matrix that applies methacrylic ester.Subsequently this matrix is carried out the spin coating proceeding 1 minute of 3000RPM.Chip with coated polymer is UV light (Hg short-arc lamp, the 20mW/cm under the 365nm of about 360nm at wavelength subsequently ²) exposed 20 minutes down.FTIR result confirms to have formed the SAX hydrogel coating on the aluminum matrix surface.

In order to compare research, also use 3 moles of %BP-modification SAX multipolymers to form hydrogel by the copolymerization process preparation.

In order to check the stability of SAX hydrogel coating on the aluminum matrix surface, the chip that the SAX polyalcohol hydrogel is applied is immersed in the deionized water, and has used surface reflection FTIR to carry out this experiment.FTIR spectrum shows in all scenario, and the IR peak intensity of hydrogel coating does not reduce after the dipping several hrs in water.This result confirms, the same stable by the hydrogel coating of blended copolymer preparation and copolymerization prepared in reaction.Thus, blend method provides and has regulated the path of BP average content, and does not need to carry out independent aggregation test so that each specific copolymer is regulated best BP content.

And when SEDLI analyzes, has substantially the same feature by blend method and the SAX chip by copolymerization process preparation.All combine the milk protein in the Tris-HCl of 50mM pH9.0 buffered soln securely.For the regulation of using protein chip, for example referring to WO 00/66265 (November 9,2000 for Rich etc. " Probes for a GasPhase Ion Spectrometer, ").Fig. 4 has shown the compound mass spectrum of the SAX chip for preparing by blend method when low and high molecular weight protein recognition test.This illustrates the protein that remains on the SAX probe.

2. dextran sill

Experiment 7:

Use 4-benzoyl phenylformic acid to come the derivatize dextran

As shown in Figure 5, by using 1,3-dicyclohexyl carbodiimide (DCC) makes the reaction of 4-benzoyl phenylformic acid and dextran as coupling agent, the dextran of preparation BP-modification.This synthesis step is as follows:

(MW is about 70,000, Sigma with 300mL DMSO and 17.54 gram dextran; Spend the night 100 ℃ of following vacuum-dryings before using), 8.57 gram 4-benzoyl phenylformic acid, 11.72 grams 1,3-dicyclohexyl carbodiimide (DCC) and 6.94 gram dimethyl aminopyridines join in the dry round-bottomed flask of the 500mL that magnetic stirring apparatus is housed together.Solution cools off by ice bath, and stirs 1 hour.Remove ice bath, and at room temperature stir this solution and spend the night.Then, filter out sedimentary by product, filtrate is poured in the acetone with precipitation polymers.The polymer precipitation thing is dissolved in the deionized water once more, and this solution mixture is carried out dialysis with respect to deionized water by seamless cellulose tube (intercepting molecular weight 12,000).With polymers soln lyophilize under vacuum of dialysis, obtain white solid product. ¹H NMR confirms to have formed desired product.The modification degree is 5 moles of % (being equivalent to 10 sugar units of a BP unit modification).

Use 4-(glycidyl ether oxygen base) benzophenone to come the derivatize dextran

A kind of replacement method of BP modified glucan is to make the reaction of 4-(glycidyl ether oxygen base) benzophenone and dextran.

Experiment 8:

Synthesizing of 4-(glycidyl ether oxygen base) benzophenone

As shown in Figure 6,100mL exsiccant acetone is added in the dry round-bottomed flask of the 250mL that magnetic stirring apparatus is housed with 15.05 gram 4-dihydroxy benaophenonels, 32.1 gram Epicholorohydrins and 6.2 gram salt of wormwood.With this vlil 6 hours, and cool to room temperature subsequently.Filter out sedimentary Repone K, and evaporate this solution to remove excessive Epicholorohydrin.Recrystallization crude product from ethanol. ¹H NMR confirms to have formed desired product.

Experiment 9:

The reaction of 4-(glycidyl ether oxygen base) benzophenone and dextran

According to Fig. 7, (MW is about 70,000, Sigma with 100mLDMSO and 23.85 gram dextran; Spend the night 100 ℃ of following vacuum-dryings before using), 6.04 gram 4-(glycidyl ether oxygen base) benzophenone and 3.96 grams 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene (DBU) joins in the dry round-bottomed flask of the 250mL that magnetic stirring apparatus is housed together.At room temperature stirring this solution spends the night.Then, filtrate is poured in the acetone with precipitation polymers.The polymer precipitation thing is dissolved in the deionized water once more, and this solution mixture is carried out dialysis with respect to deionized water by seamless cellulose tube (intercepting molecular weight 12,000).With polymers soln lyophilize under vacuum of dialysis, obtain white solid product. ¹H NMR confirms to have formed desired product.The modification degree is about 3 moles of %.

Experiment 10:

1, the preparation of 1 '-carbonyl dimidazoles (CDI)-preactivated dextran chip

The dextran solution for preparing the BP-modification by blend

(MW is about 70,000, Sigma) mixes in deionized water/isopropanol solvent mixture, to obtain the blend dextran solution of 2.5 moles of %BP modifications with the dextran of a 5 moles of %BP modifications and a natural dextran.

Also by (MW is about 70,000, and Sigma) blend in deionized water/isopropanol solvent mixture prepares the blend dextran solution of 1 mole of %BP modification with the dextran of a 5 moles of %BP modifications and four parts of natural dextran.

The concentration of BP is relevant with the swelling behavior of hydrogel coating, and it influences the perviousness of important parameters such as structure, mechanical property and surface attachment network.

Experiment 11:

The preparation of dextran hydrogel coating on the Al surface

With BP content is that the blend dextran solution of 2.5 moles of % is dispersed on the surface of the aluminum matrix that applies methacrylic ester, subsequently this matrix is carried out the spin coating proceeding 1 minute of 3000RPM.Chip with coated polymer is UV light (Hg short-arc lamp, the 20mW/cm under the 365nm of about 360nm at wavelength subsequently ²) exposed 20 minutes down.FTIR result (referring to Fig. 8 (a)) confirms to have formed the dextran hydrogel coating on the aluminum matrix surface.The coating that obtains in water the dipping 24 hours be stable.

Make the chip and 1 of dextran coating, (CDI, Aldrich) reaction is to prepare preactivated surface for 1 '-carbonyl dimidazoles.This synthesis step is as follows:

The chip of dextran coating is immersed in 1 among the DMSO, in 1 '-carbonyl dimidazoles (CDI) 5wt% solution 1 hour.From solution, take out chip subsequently, and, use washing with acetone subsequently with the DMSO washing, dry in nitrogen gas stream.Reflection FTIR spectrum (referring to Fig. 8 (b)) confirms that hydroxyl almost Quantitative yield is the imidazolyl carboxylic acid ester, and it shows as hydroxyl peak (3500-3300cm ^-1) almost completely disappear and at 1771cm ^-1The place forms strong carbonyl absorption peak.

These CDI activatory chips are used for and free primary amine group covalent attachment.Typical application comprises: immunoassay, receptor-ligand are in conjunction with research and transcription factor analysis.For the regulation of using protein chip, for example referring to WO 00/66265 (November 9,2000 for Rich etc. " Probes for a Gas Phase Ion Spectrometer, ").Fig. 9 has shown the SELDI spectrum of typical antibody-antigen recognition test.This SELDI spectral results shows, CDI-dextran chip has good specificity combination and than the non-specific binding of low degree.

Experiment 12:

The preparation of diethylamino ethyl-dextran (DEAE-dextran) chip

The deae dextran solution for preparing the BP-modification by blend

(Aldrich) mix with 5 moles of %BP-modified glucans of 9.6mg, to obtain the deae dextran blend solution of 1 mole of %BP-modification MW＞500,000 with the deae dextran of 38.4mg.

In order to prepare the DEAE hydrogel coating, the above-mentioned solution of 3wt% is dispersed on the surface of the aluminum matrix that applies methacrylic ester, after the drying, the chip with coated polymer is UV light (Hg short-arc lamp, the 20mW/cm under the 365nm of about 360nm at wavelength subsequently ²) exposed 20 minutes down.FTIR result confirms to have formed the deae dextran hydrogel coating on the aluminum matrix surface.

In order to estimate the chemical stability of this hydrogel coating, the DEAE chip is immersed in all kinds of SOLVENTS system, use reflection FTIR to check that the surperficial chemistry of going up keeps subsequently.Four kinds of solvent conditions have been used: deionized water, 0.2M

NaCl buffered soln, acetonitrile and DMSO.This chip at room temperature is immersed in the solvent 2～20 hours.Use reflection FTIR to check before the immersion solvent and chemical transformation afterwards.IR result shows that the DEAE hydrogel floods up to 20 hours for the stability of 0.2M NaCl buffered soln, acetonitrile and DMSO.This hydrogel also flooded up to 40 hours the stability of deionized water.

And when SEDLI analyzed, the DEAE chip was securely in conjunction with removing albuminous human serum protein in the Tris-HCl buffered soln of 50mM pH7.5.For the regulation of using protein chip, for example referring to WO00/66265 (November 9,2000 for Rich etc. " Probes for a Gas Phase Ion Spectrometer, ").Figure 10 has shown the compound mass spectrum when low and high-molecular weight are removed albuminous human serum protein's recognition test.This test figure has shown the serum protein that are retained on the deae dextran probe.

Experiment 13:

The preparation of asuro chip

Dextran sulfate sodium salt is the polyanionic derivative of dextran.

The asuro solution for preparing the BP-modification by blend

With the 12.8mg asuro (MW＞500,000, Aldrich), the natural dextran of 25.6mg (MW500,000, Aldrich) mix, to obtain the asuro aqueous solution of 1 mole of %BP of blend with 5 moles of %BP modified glucans of 9.6mg.Use natural dextran to regulate density in conjunction with functional group as thinner.

In order to prepare the asuro hydrogel coating, the above-mentioned solution of 3wt% is dispersed on the surface of the aluminum matrix that applies methacrylic ester, after the drying, the chip with coated polymer is UV light (Hg short-arc lamp, the 20mW/cm under the 365nm of about 360nm at wavelength subsequently ²) exposed 20 minutes down.FTIR result confirms to have formed the asuro hydrogel coating on the aluminum matrix surface.

Experiment 14:

The regulation of preparation MEP/MBI chip array

The preparation of-matrix:

* use the smooth aluminum matrix of silica-coating, adopt CVD (chemical vapour deposition) to apply methacryloyl oxygen base propyl group-Trimethoxy silane subsequently.

The dextran of-benzophenone-modification: referring to experiment 7

-dextran modification: MEP

* allylation, MEP modification subsequently

* allylation

20g dextran (Sigma, MW70,000) is dissolved in the 100mL deionized water.

Add the 200mg sodium borohydride in case oxidation.

Add 12.5mL sodium hydroxide subsequently.

After 5 minutes, add the 2mL allyl bromide 98.

After stirring 16 hours, use 200mL acetone that product (allyl group-dextran) is precipitated out.

For purifying, with this solid once more solvent in 100mL water, pour into subsequently in the 200mL acetone.Repeat this process once.

At last, this solid is dissolved in the 200mL deionized water, with postlyophilization once more.

* MEP modification

The dextran (2g) of this allylation is dissolved in the 20mL deionized water.

1.0g mercaptoethyl pyridine-HCl (Biosepra) and 20mg AZAP (2,2 '-azo two (2-methyl propanamide), two hydrochloric acid) are added in the solution successively.

Subsequently with this solution of nitrogen purge, and cover lid.

This solution is remained on 85 ℃ to be stirred 2 hours down.

Use the 50mL acetone precipitation to go out product, subsequently product is dissolved in the 20mL deionized water once more.

By using MWCO 5000 cellulose membrane dialysis to be further purified this product, with postlyophilization.

-dextran modification: MBI

* allylation, bromination, MBI modification subsequently

* allylation

10g dextran (Sigma, MW70,000) is dissolved in the 500mL deionized water.

Add the 100mg sodium borohydride in case oxidation.

Add 12.5mL sodium hydroxide subsequently.

After 5 minutes, add the 3mL allyl bromide 98.

After stirring 16 hours, use 100mL acetone that product (allyl group-dextran) is precipitated out.

For purifying, with this solid once more solvent in 500mL water, pour into subsequently in the 100mL acetone.Repeat this process once.

At last, this solid is dissolved in the 200mL deionized water, with postlyophilization once more.

* bromination

The dextran (5g) of this allylation is dissolved in the 100mL deionized water.

1.5g N-bromination succinimide (Aldrich) and 2.5g Potassium Bromide are added in this solution successively.

Use subsequently phosphoric acid (is 1/3 with deionized water dilution) with pH regulator between 3.7～3.9.

Under shield cap (because generating bromine), this solution of stirring at room 1 hour.

* MBI modification

2.7g mercaptobenzimidazole sulfonic acid (Aldrich) is added in this solution.

Use 10M NaOH with pH regulator between 11～11.5.

Check pH one hour, and regulate as required.

After 16 hours, 200mL acetone is added in this solution so that product is precipitated out.

Use 100mL water dissolution product, test MWCO 5000 Mierocrystalline cellulose dialyzers subsequently and carry out dialysis.

Obtain the product (MBI-dextran) of white powder after the lyophilize.

* benzophenone modified glucan: referring to experiment 7

-with the MEP/MBI polymer-coated on matrix:

* in deionized water, prepare 2% modified glucan solution

* prepare the solution that uses

* for MEP

900 μ L 2%MEP-dextran, 100 μ L, 2% benzophenone-dextran, 400 μ L10% glycerine and 100 μ L deionized waters are added in the 4mL amber bottle.

* for the MBI array

900 μ L 2%MBI-dextran and 100 μ L, 2% benzophenone-dextran are added in the 4mL amber bottle.

* above-mentioned dextran solution of 1 μ L and 1 μ L ethanol are deposited on the matrix of silanization successively.

* should the surface dry in baking oven, and solidified in 20 minutes by nearly UV illumination radiation.

* after the radiation, use the deionized water clean surface, and dry.

The following example has been described in US patent application serial number NO.10/660, in 738 (the 2003.09.12 submissions), and in the description of embodiment A record.

Experiment 15: preparation MBISA biochip array

By the sputtering method smooth aluminum matrix (" chip ") of silica-coating.Coating number waterborne polymeric (Cytonix, Beltsville, MD), on chip, to form addressable site.The chip that this mode is prepared cleans in plasma cleaner, and is introduced in the chemical vapor deposition (CVD) stove.By CVD methacryloyl oxygen base propyl group-Trimethoxy silane is deposited on the chip, applies vacuum, and subsequently at the chip of 60 ℃ of following cured coated.

Dextran modification

Allylation

10g dextran (Sigma, MW-75,000) is dissolved in the 50mL deionized water with 100mg sodium borohydride (to restrain oxidation).Sodium hydroxide solution (12.5mL) is added in this dextran mixture, add allyl bromide 98 (3mL) after 5 minutes.After stirring 16 hours, come precipitated product (allyl group-dextran) by adding 100mL acetone.By adding acetone (100mL) with twice precipitation crude product, the dextran of purifying allylation thus from the aqueous solution (50mL).At last, the product with purifying is dissolved in the 200mL deionized water, with postlyophilization once more.

Bromination and mercaptobenzimidazole sulfonic acid modified

Use 1.5g N-bromination succinimide (Aldrich) and 2.5g Potassium Bromide to handle the aqueous solution (100mL) of allylation dextran (5g) successively.Use phosphoric acid (is 1/3 with the deionized water dilution) that pH is added between 3.7～3.9 subsequently.At room temperature stir the solution 1 hour (, therefore under stink cupboard, carrying out) that is obtained owing to form bromine.

2.7g mercaptobenzimidazole sulfonic acid (Aldrich) is added in this solution.Use 10M NaOH (aqueous solution) between 11～11.5, checked pH one hour with pH regulator, and carried out necessary adjusting.After 16 hours, 200mL acetone is added in this solution so that crude product is precipitated out, subsequently product is dissolved in 100mL once more, use MWCO 5000 Mierocrystalline cellulose dialysis bags to carry out dialysis subsequently.Obtain the product (MBI-dextran) of white powder after the lyophilize.

The benzophenone dextran

(Aldrich is DCC) with the dry DMSO of 50mL for interpolation 2.26g 4-benzoyl phenylformic acid (Aldrich, benzophenone 4-carboxylic acid), 2.26g dicyclohexyl carbodiimide in the 250mL round-bottomed flask.

By another funnel 8.1g dextran (Sigma, MW～75,000) and the solution of 0.12g dimethyl aminopyridine (DMAP) in 100mL DMSO are dropwise added in the benzophenone solution of stirring.After stirring 16 hours, filter the throw out that is obtained, in rotatory evaporator, remove the solvent in the filtrate subsequently.Sometimes, can be further purified crude product by recrystallization.

The coating of MBI polymkeric substance on matrix

The 1.35g MBI dextran and the 0.15g benzophenone dextran of as above preparation are dissolved in the 100mL deionized water.1 this dextran solution of μ L and 1 μ L ethanol are deposited on the chip of silanization successively.The dry chip that applies in baking oven, and radiation (near UV) 20 minutes are to solidify.After the radiation, use the deionized water wash chip, and dry.

Describe and set forth the present invention though preferably used with reference to embodiment preferred and its, it is not limited to this, because within the four corner of the present invention of stating in as claims, can improve and changes at this.

Claims (94)

1, a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, and this precursor composition comprises:

A) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

B) second polymkeric substance, it comprises the functional group of selective binding biomolecule analysis thing, and wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, and

Wherein the amount of the functional group of the amount of first and second polymkeric substance and Photocrosslinkable functional group and selective binding biomolecule analysis thing is given this hydrogel precursor polymer blend compositions respectively and is become the performance of hydrogel and the performance of hydrogel selective binding biomolecule analysis thing by photo-crosslinking.

2, according to the composition of claim 1, wherein first polymkeric substance prepares by homopolymerization or one or more monomers of copolymerization.

3, according to the composition of claim 1, wherein first polymkeric substance prepares with the multipolymer that formation comprises the functional group of Photocrosslinkable functional group and selective binding biomolecule analysis thing by making monomer copolymerization.

4, according to the composition of claim 1, wherein first polymkeric substance prepares to introduce Photocrosslinkable functional group by the synthesis modification first pre-functionalized polymeric.

5, according to the composition of claim 1, wherein first polymkeric substance, second polymkeric substance or the two are water-soluble polymers.

6, according to the composition of claim 1, wherein first polymkeric substance comprises the linear polymer main chain with side group, this side group comprises Photocrosslinkable functional group, and second polymkeric substance comprises the linear polymer main chain with side group, and this side group comprises the functional group of selective binding biomolecule analysis thing.

7, according to the composition of claim 1, wherein first polymkeric substance comprises the monomer subunits that comprises Photocrosslinkable functional group of about 0.5 mole of %～about 15 moles of %.

8, according to the composition of claim 1, wherein first polymkeric substance and second polymkeric substance weight-average molecular weight separately are about 1,000～about 10,000,000.

9, according to the composition of claim 1, wherein Photocrosslinkable functional group is the UV-curable functional group.

10, according to the composition of claim 1, wherein Photocrosslinkable functional group is at least a or derivatives thereof in benzophenone, diazo ester, aromatic yl azide and the diazomethane.

11, according to the composition of claim 1, wherein Photocrosslinkable functional group comprises benzophenone group or derivatives thereof.

12, according to the composition of claim 1, wherein selective binding functional group and biomolecule analysis thing covalent attachment.

13, according to the composition of claim 1, wherein selective binding functional group combines with the biomolecule analysis thing is non-covalent.

14, according to the composition of claim 1, wherein hydrogel composition also comprises energy absorbing portion.

15, according to the composition of claim 1, wherein selective binding functional group be stratographic or biologic specificity in conjunction with functional group.

16, according to the composition of claim 1, wherein said hydrogel composition does not contain light trigger substantially, and comprises fluorophor in addition.

17, according to the composition of claim 16, wherein first polymkeric substance comprises the linear polymer main chain with side group, this side group comprises Photocrosslinkable functional group, and second polymkeric substance comprises the linear polymer main chain with side group, this side group comprises selective binding functional group, and wherein first and second polymkeric substance are water-soluble polymers.

18, according to the composition of claim 16, wherein first and second polymkeric substance are the water-soluble polysaccharide polymkeric substance, and first polymkeric substance prepares to introduce Photocrosslinkable functional group by the synthesis modification first pre-functionalized copolymers.

19, according to the composition of claim 1, wherein said crosslinked be photo-crosslinking.

20, according to the composition of claim 1, wherein said crosslinked be heat cross-linking.

21, a kind of aquogel polymer blend composition, it is prepared as follows:

A) make aquogel polymer blend precursor composition crosslinked, this precursor composition comprises:

I) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

Ii) second polymkeric substance, it can carry out synthesis modification to comprise the functional group of selective binding biomolecule analysis thing, forms crosslinked hydrogel thus;

B) by the described crosslinked hydrogel of synthesis modification, make its functional group that comprises selective binding biomolecule analysis thing,

Wherein the amount of the amount of first and second polymkeric substance and Photocrosslinkable functional group and selective binding functional group is given described hydrogel precursor blend polymer and is become the performance of hydrogel and the performance of hydrogel selective binding biomolecule analysis thing by photo-crosslinking.

22, according to the aquogel polymer blend composition of claim 21, wherein said crosslinked be photo-crosslinking.

23, according to the aquogel polymer blend composition of claim 21, wherein said crosslinked be heat cross-linking.

24, according to the composition of claim 21, wherein said composition does not contain light trigger substantially.

25, according to the composition of claim 21, wherein first polymkeric substance, second polymkeric substance or the two are water-soluble polymers or water-swellable polymer.

26, according to the composition of claim 21, wherein first polymkeric substance prepares by homopolymerization or one or more monomers of copolymerization.

27, according to the composition of claim 21, wherein first polymkeric substance prepares with the multipolymer that formation comprises the functional group of Photocrosslinkable functional group and selective binding biomolecule analysis thing by making monomer copolymerization.

28, according to the blend composition of claim 21, wherein first polymkeric substance prepares to introduce Photocrosslinkable functional group by the synthesis modification first pre-functionalized polymeric.

29, according to the composition of claim 21, wherein first polymkeric substance comprises the linear polymer main chain with side group, this side group comprises Photocrosslinkable functional group, and second polymkeric substance comprises the linear polymer main chain with side group, and this side group comprises selective binding functional group.

30, according to the composition of claim 21, wherein first polymkeric substance comprises the monomer subunits that contains Photocrosslinkable functional group of about 0.5 mole of %～about 15 moles of %.

31, according to the composition of claim 21, wherein first polymkeric substance and second polymkeric substance weight-average molecular weight separately are about 1,000～about 10,000,000.

32, according to the composition of claim 21, wherein first polymkeric substance and second polymkeric substance are water miscible.

33, according to the composition of claim 21, wherein Photocrosslinkable functional group is the UV-curable functional group.

34, according to the composition of claim 21, wherein Photocrosslinkable functional group is at least a or derivatives thereof in benzophenone, diazo ester, aromatic yl azide and the diazomethane.

35, according to the composition of claim 21, wherein said composition also comprises fluorophor.

36, according to the composition of claim 21, wherein said composition also comprises energy absorbing portion.

37, according to the composition of claim 21, wherein selective binding functional group and biomolecule analysis thing covalent attachment.

38, according to the composition of claim 21, wherein selective binding functional group combines with the biomolecule analysis thing is non-covalent.

39, according to the composition of claim 21, wherein selective binding functional group is that biologic specificity is in conjunction with functional group or chromatogram conjugated group.

40, according to the composition of claim 21, wherein selective binding functional group is for absorbing group.

41, according to the composition of claim 21, wherein first polymkeric substance comprises the linear polymer main chain with side group, this side group comprises Photocrosslinkable functional group, and second polymkeric substance comprises the linear polymer main chain with side group, this side group comprises selective binding functional group, and wherein first and second polymkeric substance are water-soluble polymers.

42, according to the composition of claim 21, wherein first and second polymkeric substance are water-soluble polymers, and first polymkeric substance prepares with the Photocrosslinkable functional group that introduces first monomer subunits by the synthesis modification first pre-functionalized copolymers.

43, according to the composition of claim 21, wherein first and second polymkeric substance comprise hydroxyl.

44, according to the composition of claim 21, wherein first and second polymkeric substance are polysaccharide.

45, according to the composition of claim 21, wherein first and second polymkeric substance comprise hydroxyl.

46, according to the composition of claim 21, wherein first and second polymkeric substance are polysaccharide.

47, according to the composition of claim 21, wherein first and second polymkeric substance are the dextran of modification.

48, according to the composition of claim 45, wherein said composition does not contain light trigger substantially.

49, according to the composition of claim 46, wherein said composition does not contain light trigger substantially.

50, according to the composition of claim 47, wherein said composition does not contain light trigger substantially.

51, a kind of matrix, it comprises stromal surface and the aquogel polymer blend composition on stromal surface, wherein said composition comprises that (i) comprises first polymkeric substance of crosslinked functional group, wherein first polymkeric substance is chosen the functional group that also comprises selective binding biomolecule analysis thing wantonly, second polymkeric substance that (ii) comprises the functional group of selective binding biomolecules, the functional group of the selective binding biomolecules of this functional group and first polymkeric substance can be identical or different.

52, according to the matrix of claim 51, wherein stromal surface is the undercoat surface.

53, according to the matrix of claim 51, wherein stromal surface is the plane.

54, according to the matrix of claim 53, wherein stromal surface is the undercoat surface, and wherein hydrogel is a homogeneous layer.

55, according to the matrix of claim 53, wherein stromal surface is the undercoat surface, and wherein hydrogel is the discrete point form.

56, according to the matrix of claim 53, wherein hydrogel and stromal surface covalent attachment.

57, according to the matrix of claim 51, wherein first and second polymkeric substance comprise polysaccharide, and first polymkeric substance comprises crosslinked benzophenone functional group.

58, according to the matrix of claim 51, wherein hydrogel does not contain light trigger substantially.

59, according to the matrix of claim 52, wherein hydrogel does not contain light trigger substantially.

60, according to the matrix of claim 56, wherein hydrogel does not contain light trigger substantially.

61, according to the matrix of claim 51, wherein the functional group of selective binding biomolecules is used for the covalently bonding to biomolecules analyte.

62, according to the matrix of claim 51, wherein the functional group of selective binding biomolecules is used for non-covalent binding biomolecules analyte.

63, according to the matrix of claim 51, wherein the aquogel polymer blend composition is that thickness is about 1 micron～about 10 microns film.

64, according to the matrix of claim 51, wherein the aquogel polymer blend composition also comprises fluorescent polymer.

65, according to the matrix of claim 51, wherein the aquogel polymer blend composition also comprises fluorophor.

66, according to the matrix of claim 51, wherein the aquogel polymer blend composition also comprises energy absorbing portion.

67, according to the matrix of claim 51, wherein said matrix is biochip.

68, according to the matrix of claim 51, wherein the biomolecule analysis thing is a protein.

69, according to the matrix of claim 51, wherein said matrix is biochip, and the biomolecule analysis thing is a protein, and wherein the aquogel polymer blend composition is that thickness is about 1 micron～about 10 microns film.

70, according to the matrix of claim 69, wherein stromal surface is the undercoat surface.

71, a kind of method of using hydrogel composition to make functionalisation of surfaces, this method comprises:

(A) provide (i) to have the matrix on surface and (ii) according to the composition of claim 75 or 76,

(B) contact 5 or 86 composition according to Claim 8, forming the layer of described composition from the teeth outwards,

(C) make lip-deep at least some compositions crosslinked, to form the hydrogel that contacts with the surface.

72, according to the method for claim 71, wherein stromal surface is the undercoat surface, and this undercoat is by support layer supports.

73, according to the method for claim 71, the wherein said crosslinked photo-crosslinking and be optionally of being makes some compositions be crosslinked and some compositions is not crosslinked.

74, according to the method for claim 73, the crosslinked discrete point that photocrosslinkable hydrogel is provided of selective light wherein.

75, according to the method for claim 71, wherein crosslinked composition is a basic layer uniformly on the stromal surface, and average bed thickness is about 5 nanometers～about 10 microns.

76, according to the method for claim 71, wherein said matrix is the matrix that is used for biochip.

77, according to the method for claim 71, wherein said composition does not contain light trigger substantially.

78, according to the method for claim 72, wherein said composition does not contain light trigger substantially.

79, according to the method for claim 73, wherein said composition does not contain light trigger substantially.

80, according to the method for claim 75, wherein said composition does not contain light trigger substantially, and described matrix is the matrix that is used for biochip.

81, prepare 5 or 86 method for compositions according to Claim 8, it comprises that mixing first and second polymkeric substance are to form the step of hydrogel precursor blend polymer.

82, a kind of particle, it comprises the aquogel polymer blend composition according to claim 1 or 21.

83, a kind of method that is used for detection of biological analysis of molecules thing, it comprises: the matrix according to claim 51 is contacted with the sample that contains the biomolecule analysis thing, and (ii) detect this biomolecule analysis thing according to itself and combining of selective binding functional group subsequently.

84, the crosslinked blends of first polysaccharide and second polysaccharide, wherein the one or more crosslinked group of second glycocalix replaces.

85, a kind of hydrogel precursor polymer blend compositions, it comprises:

A) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

B) second polymkeric substance, it comprises the functional group of (i) selective binding biomolecule analysis thing, wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, or (ii) one or more energy absorbing portion

Wherein the consumption of the functional group of the amount of first and second polymkeric substance and Photocrosslinkable functional group and selective binding biomolecule analysis thing is given described hydrogel precursor polymer blend compositions respectively and is become the performance of hydrogel and the performance of hydrogel selective binding biomolecule analysis thing by photo-crosslinking.

86, a kind of hydrogel precursor polymer blend compositions, it comprises:

A) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

B) second polymkeric substance, it can carry out synthesis modification comprising the functional group of selective binding biomolecule analysis thing,

Wherein the amount of the amount of first and second polymkeric substance and Photocrosslinkable functional group is given described hydrogel precursor blend polymer is become hydrogel by photo-crosslinking performance.

87, a kind of aquogel polymer blend composition, it comprises:

A) first polymkeric substance, its comprise Photocrosslinkable functional group and

B) second polymkeric substance, it comprises (i) one or more functional groups by non-covalent combination selective binding biomolecule analysis thing, (ii) one or more functional groups by covalent attachment mode selective binding biomolecule analysis thing, or (iii) one or more energy absorbing portion, or its combination.

88,7 aquogel polymer blend composition according to Claim 8, wherein second polymkeric substance comprises (i) one or more functional groups by non-covalent combination selective binding biomolecule analysis thing.

89,7 aquogel polymer blend composition according to Claim 8, wherein second polymkeric substance comprises (ii) one or more functional groups by covalent attachment mode selective binding biomolecule analysis thing.

90,7 aquogel polymer blend composition according to Claim 8, wherein second polymkeric substance comprises (iii) one or more energy absorbing portion.

91, a kind of hydrogel coating external member, it comprises:

(a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

(b) second composition, it comprises second polymkeric substance, described second polymkeric substance comprises the functional group of (i) selective binding biomolecule analysis thing, wherein the functional group of the selective binding biomolecule analysis thing in first polymkeric substance and second polymkeric substance can be identical or different, or (ii) one or more energy absorbing portion.

92, a kind of hydrogel coating external member, it comprises:

(a) first composition, it comprises first polymkeric substance that contains Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

(b) second composition, it comprises second polymkeric substance, described second polymkeric substance can carry out synthesis modification to comprise the functional group of selective binding biomolecule analysis thing.

93, a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, and this precursor composition comprises:

A) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

B) second polymkeric substance, it comprises energy absorption functional group,

Wherein the amount of the amount of first and second polymkeric substance and Photocrosslinkable functional group and energy absorption functional group is given described hydrogel precursor polymer blend compositions respectively and is become the performance of hydrogel and hydrogel to promote analyte of interest performance to the gas phase desorption when being subjected to high energy gamma source such as laser bombardment by photo-crosslinking.

94, a kind of aquogel polymer blend composition, it prepares by cross-linked hydrogel polymer blend precursor composition, and this precursor composition comprises:

A) first polymkeric substance, it comprises Photocrosslinkable functional group, wherein the optional functional group that also comprises selective binding biomolecule analysis thing of first polymkeric substance and

B) second polymkeric substance, and

Wherein said hydrogel also comprise can with biomolecules form covalent linkage reactive group and

Wherein the amount of the amount of first and second polymkeric substance and Photocrosslinkable functional group and energy absorption functional group is given described hydrogel precursor polymer blend compositions is become hydrogel by photo-crosslinking performance.

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