CN101363870A - Bio-sensing chip and method for making same - Google Patents
Bio-sensing chip and method for making same Download PDFInfo
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- CN101363870A CN101363870A CNA2008102225254A CN200810222525A CN101363870A CN 101363870 A CN101363870 A CN 101363870A CN A2008102225254 A CNA2008102225254 A CN A2008102225254A CN 200810222525 A CN200810222525 A CN 200810222525A CN 101363870 A CN101363870 A CN 101363870A
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Abstract
The invention discloses an optical biosensing chip, and a preparation method thereof. the preparation method comprises the following steps of (1) optical-filming a glass substrate with oxide; (2) chemically modifying the filmed glass substrate with silane agent having active functional group; (3) further modifying the glass substrate with functional high molecular polymer; and (4) immobilizing protein molecule to the high molecular polymer modified glass substrate, and immobilizing organic small molecule ligand to the high molecular polymer modified glass substrate. The silane agent in combination with the functional high molecular polymer are adopted by the method to modify the surface of the glass substrate so that the a dense high molecular polymer layer is formed on the surface of the glass substrate, and a large amount of active groups for immobilizing biomolecule or organic small molecule ligand are contained to effectively inhibit non-specific adsorption. The prepared biosensing chip high capacity, high stability, low non-specific adsorption, repeated use, and high application value.
Description
Technical field
The present invention relates to bio-sensing chip and preparation method thereof.
Background technology
Biology sensor is a kind of ability of utilizing bioactivator identification molecule, optionally detects the sensor of specific biochemical; It mainly comprises immobilized biological sensitive materials (sensing chip), physics and chemistry transducer and signal amplifying apparatus.At aspects such as biomedical research, medical diagnosis on disease, environmental monitoring and food hygiene detection, biology sensor has wide application and application prospect.Most crucial parts are bio-sensing chips in the biology sensor, be fixed with biomolecule (as protein, nucleic acid or test substance molecule) on the surface of bio-sensing chip, it is the interface of out-phase biological respinse, and its character is very big to the overall performance influence of sensor.
Sensing chip in the optical biosensor adopts glass as base material, fixing biological molecules need be through two steps on substrate of glass: the activation of substrate and biomolecule fixing, the purpose of substrate activation is to form one deck molecular film at substrate surface, be used for fixing biomolecule, and suppress non-specific adsorption.Activation method to substrate of glass has a variety of at present, wherein film modified technology is the most effective, comprise self-assembled monolayer (Self-assembled monolayer, SAM) and L B film (with the naming of its inventor Langmuir and Blodgett), the reactive group of the glass basic surface after these method activation is limited, the fixed amount of biomolecule also is restricted, and on the other hand, the bio-sensing chip of the method preparation is relatively poor to the non-specific adsorption inhibition ability of biomolecule.
Summary of the invention
The preparation method of the bio-sensing chip of the preparation method who the purpose of this invention is to provide bio-sensing chip, particularly protein bio-sensing chip and organic molecule aglucon.
The method for preparing the protein bio-sensing chip provided by the invention in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that step 1) obtains is carried out chemical modification;
3) with functional high molecular polymer to step 2) rete that obtains carries out chemical modification;
4) with glutaraldehyde protein molecule is fixed on the rete that step 3) obtains, obtains protein bio-sensing chip provided by the invention.
In above-mentioned preparation method's the step 1), available silicon dioxide (SiO
2) or tantalum pentoxide (Ta
2O
5) wait transparent oxide to carry out plated film, the thickness of this rete is 50-150nm.
Step 2) in, functional groups in the used silylating reagent is epoxy radicals or amino, can be 3-diglycidyl hydroxypropyl trimethoxy silane (GOPTS), 3-aminopropyl dimethyl monosubstituted ethoxy silane (APDMS) or 3-aminopropyl triethoxysilane (APTES).When adopting APDMS or these two kinds of silylating reagents of APTES to carry out chemical modification, can earlier silylating reagent be dissolved in the toluene, the mass percent concentration of the APDMS of this silylating reagent or APTES solution is 1%; When adopting the GOPTS silylating reagent, adopt pure reagent to carry out chemical modification.
The reaction time of this step is 1-24h, and temperature of reaction is a room temperature.This step serves as the control purpose to form unimolecular layer as far as possible, so when the concentration of silylating reagent was higher, the reaction time can suitably shorten; When the concentration of silylating reagent was hanged down, the reaction time can suitably extend.
In the step 3), used functional high-polymer polymkeric substance is the hydrophilic high molecular polymer that has active amino, carboxyl and aldehyde radical, specifically can be glycosaminoglycan (AMDEX), carboxyl glucosan (CMDEX), two amino polyglycol (DAPEG), two carboxy polyethylene glycol (DCPEG), shitosan (CHI) or poly-piperazine (PEI).The molecular weight of above-mentioned high molecular polymer can freely be selected, preferred 1000-150000g/mol, more preferably 10000-100000g/mol.
After carrying out chemical modification with silylating reagent, before carrying out chemical modification with functional high molecular polymer, earlier above-mentioned water miscible functional high-polymer polymkeric substance is dissolved in the ultrapure water, fat-soluble functional high-polymer polymkeric substance is dissolved among the DMF, and the mass percent concentration of the water of this functional high-polymer polymkeric substance or DMF solution is 20-60%.
The reaction time of this step is 12-24h, and temperature of reaction is 25-37 ℃.After this step was finished, the above-mentioned functions high molecular polymer formed fine and close molecular film at substrate surface.
In the step 3), when the employing of functional high-polymer polymkeric substance is the polymkeric substance that contains active amino, as AMDEX, DAPEG, CHI or PEI, and step 2) when the middle silylating reagent that adopts is APDMS or APTES, need with " succinic anhydride method " amino on silanization meron surface to be changed into carboxyl, being about to substrate, to put into the pH value be 7.4 phosphate buffer, adds excessive succinic anhydride, control pH value of solution value is reacted 1h at least at 6-7 in the vibration dissolving.
In the step 4), available " glutaraldehyde method " is fixed to protein molecule on the rete that step 3) obtains.This method is that at first to adopt mass ratio be that the substrate that 1.25% glutaraldehyde water solution and step 3) obtain reacts, the amino of substrate surface is converted into aldehyde radical, the protein aqueous solution that with concentration is 1 μ g/mL-1mg/mL is again selected on the substrate, and cultivating at least under 37 ℃, 24h gets final product.
Used protein molecule is IgG, enzyme or inert protein in this step.
The protein bio-sensing chip that utilizes above-mentioned preparation method to obtain also belongs to protection scope of the present invention.
The method of the bio-sensing chip of preparation organic molecule aglucon provided by the invention comprises two kinds of methods; Wherein, method one in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that step 1) obtains is carried out chemical modification;
3) with functional high molecular polymer to step 2) rete that obtains carries out chemical modification;
4) organic molecule is fixed on the rete that step 3) obtains, obtains the bio-sensing chip of organic molecule aglucon provided by the invention;
In above-mentioned preparation method's the step 1), available SiO
2Or Ta
2O
5Carry out plated film Deng transparent oxide, the thickness of this rete is between the 50-150nm.
Step 2) in, functional groups in the used silylating reagent is epoxy radicals or amino, can be 3-diglycidyl hydroxypropyl trimethoxy silane (GOPTS), 3-aminopropyl dimethyl monosubstituted ethoxy silane (APDMS) or 3-aminopropyl triethoxysilane (APTES).When adopting APDMS or these two kinds of silylating reagents of APTES to carry out chemical modification, can earlier silylating reagent be dissolved in the toluene, the mass percent concentration of the APDMS of this silylating reagent or APTES solution is 1%; When adopting the GOPTS silylating reagent, adopt pure reagent to carry out chemical modification.
The reaction time of this step is 1-24h, and temperature of reaction is a room temperature.This step serves as the control purpose to form unimolecular layer as far as possible, so when the concentration of silylating reagent was higher, the reaction time can suitably shorten; When the concentration of silylating reagent was hanged down, the reaction time can suitably extend.
In the step 3), used functional high-polymer polymkeric substance is the hydrophilic high molecular polymer that has active amino, carboxyl and aldehyde radical, specifically can be glycosaminoglycan (AMDEX), carboxyl glucosan (CMDEX), two amino polyglycol (DAPEG), two carboxy polyethylene glycol (DCPEG), shitosan (CHI) or poly-piperazine (PEI).The molecular weight of above-mentioned high molecular polymer can freely be selected, preferred 1000-150000g/mol, more preferably 10000-100000g/mol.
After carrying out chemical modification with silylating reagent, before carrying out chemical modification with functional high molecular polymer, earlier above-mentioned water miscible functional high-polymer polymkeric substance is dissolved in the ultrapure water, fat-soluble functional high-polymer polymkeric substance is dissolved among the DMF, and the mass percent concentration of the water of this functional high-polymer polymkeric substance or DMF solution is 20-60%.
The reaction time of this step is 12-24h, and temperature of reaction is 25-37 ℃.After this step was finished, the above-mentioned functions high molecular polymer formed fine and close molecular film at substrate surface.
In the step 3), when the employing of functional high-polymer polymkeric substance is the polymkeric substance that contains active amino, as AMDEX, DAPEG, CHI or PEI, and step 2) in the silylating reagent that adopts when being APDMS or APTES, need the amino on silanization meron surface to be changed into carboxyl with " succinic anhydride method ", being about to substrate, to put into the pH value be 7.4 phosphate buffer, add excessive succinic anhydride, control pH value of solution value is at 6-7, and 1h is at least reacted in the vibration dissolving.
In the step 4), organic molecule is fixed to method on the rete that step 3) obtains, can adopts the method for directly fixing or indirect securement.
Wherein, direct Gu Ding method is with functionalized reagent organic molecule to be fixed on the rete that step 3) obtains.Used functionalized reagent is carbodiimide (EDC), glutaraldehyde, succinimide (NHS) or DIC (DIC).
The method of indirect securement is earlier with organic molecule and carrier protein coupling, obtains conjugate, this conjugate is fixed on the rete that step 3) obtains again.
Used carrier protein is bovine serum albumin(BSA) (BSA) or ovalbumin (OVA).Use water-soluble functionalized reagent afterwards again,, this conjugate is fixed on the rete as EDC, NHS or glutaraldehyde.
In the method for above-mentioned indirect securement, various coupling methods commonly used all are suitable for, as " EDC method " or " glutaraldehyde method ".Wherein, " EDC method " at first is (to be organic molecule with carbodiimide and hapten molecule, as 2, carboxyl reaction 4-D), generate the addition intermediate product, again with the carrier protein molecule on the amino acid residue reaction, form amido link, realize the covalent bond of organic molecule and inert protein (being carrier protein), obtain conjugate.The coupling ratio of conjugate (protein and micromolecular molecule mol ratio) is controlled at about 2-8.
" glutaraldehyde method " is that at first to adopt mass ratio be that the substrate that 1.25% glutaraldehyde water solution and step 3) obtain reacts, the amino of substrate surface is converted into aldehyde radical, the protein aqueous solution that with concentration is 1 μ g/mL-1mg/mL is again selected on the substrate, and cultivating at least under 37 ℃, 24h gets final product.
The method (method two) of the bio-sensing chip of another kind of preparation organic molecule aglucon provided by the invention in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that step 1) obtains is carried out chemical modification;
3) functional high-polymer polymkeric substance and organic molecule are carried out coupling, obtain conjugate;
4) with functionalized reagent the conjugate that step 3) obtains is fixed to step 2) on the rete that obtains, obtain the bio-sensing chip of organic molecule aglucon provided by the invention;
The step 1) and the step 2 of this method) identical with method one.
In the step 3), the coupling method of functional high-polymer polymkeric substance and organic molecule is identical with the coupling method of organic molecule in the preceding method and carrier protein.
In the step 4), used functionalized reagent is carbodiimide (EDC), glutaraldehyde, succinimide (NHS) or DIC (DIC).
In addition, adopt the bio-sensing chip of the organic molecule aglucon that above-mentioned preparation method obtains, also belong to protection scope of the present invention.
The method for preparing bio-sensing chip provided by the invention, adopt silylating reagent and functional high-polymer combination with polymers to modify glass substrate surface, modify the high polymer layer that the meron surface has one deck densification, contain and can be used for biomolecule or the fixing reactive group of organic molecule aglucon in a large number, and can effectively suppress non-specific adsorption, because the process of whole modification all adopts the mode of covalent bonds, the bio-sensing chip of preparation has high carrying capacity, high stability, low non-specific adsorption, reusable advantage, the fixed amount that has overcome biomolecule in the current substrate of glass method of modifying is limited, chip suppresses the relatively poor shortcoming of ability to the non-specific adsorption of biomolecule, has very high using value.
Description of drawings
The process synoptic diagram that when Fig. 1 prepares bio-sensing chip for embodiment 1 substrate of glass is carried out chemical modification.
The process synoptic diagram that when Fig. 2 prepares bio-sensing chips for embodiment 2 substrate of glass is carried out chemical modification.
Embodiment
The present invention is described further below in conjunction with the specific embodiment contrast, but the present invention is not limited to following examples.
Embodiment 1, making 2, the bio-sensing chip of 4-D
As shown in Figure 1, describe 2 in detail, the method for making of 4-D optics immuno-chip, this method in turn includes the following steps:
1) at first substrate of glass is carried out optical coating: wipe obvious stain on the glass basic surface with lens wiping paper, and (V (concentrated sulphuric acid): V (hydrogen peroxide)=3:2) and acetone thoroughly clean substrate to adopt highly basic (4mol/L NaOH), strong acid successively, after nitrogen dried up under the room temperature, adopting chemical gaseous phase deposition coating method (CVD) to plate a layer thickness at substrate surface was SiO about 100nm
2This substrate plating SiO
2Surface state behind the film is shown among Fig. 1 a 1.
2) water/toluene (0.25%) solution of the substrate of glass behind the plated film being put into APDMS reacts 24h, and the concentration of volume percent of APDMS is 1%, and successively with toluene, hydrochloric acid, ultrapure water flushing, nitrogen dries up after the reaction.The surface state of this substrate is shown among Fig. 1 a 21.Then substrate is faced up and put into staining jar, and add excessive succinic anhydride, and it is covered on the substrate, in staining jar, add phosphate buffer (pH=7.4), make the succinic anhydride dissolving in the oscillatory process, continue to add strong base solution control pH between 6～7.Reaction was carried out 1 hour at least.Reacted substrate is rinsed well with ultrapure water.The surface state of this substrate is shown among Fig. 1 b 22.
3) ultrapure water preparation mass percent concentration is 40% AMDEX solution, with the salt acid for adjusting pH value is 3-4, in solution, add 100mg/mL EDC again, it is dissolved in the reaction system, reaction system is applied on the substrate of glass behind the silanization, keeps cultivating 12-24h under the condition of the certain humidity of glass surface.Reacted substrate is rinsed well with ultrapure water, dries under the room temperature.The surface state of this substrate is shown among Fig. 1 b 3
4) 2 of preparation 100mg/mL, the DMF solution of 4-D, adding percent by volume in the solution is the DIC of 1:10, mixed solution is added drop-wise on the active position of glass substrate (surface state of this substrate as among Fig. 1 c 3 ' shown in), cultivates 24h at least then in the wet box of DMF.Reacted substrate is rinsed well under the room temperature of back and is dried, and obtains 2, the 4-D bio-sensing chip.The surface state of this substrate is shown among Fig. 1 c 4.
After the preparation 2,4-D sensing chip can not produce non-specific adsorption to inert protein (as bovine serum albumin(BSA) BSA, ovalbumin OVA) substantially, and to 2, the antibody of 4-D has good specific reaction.The result who adopts planar waveguide-type fluorescence immunoassay sensor to measure, less than 50 units, and to 2 of 5 μ g/mL, 4-D antibody then has the response of 330 units to this chip to the response of the BSA of 2mg/mL.
The bio-sensing chip of embodiment 2, making microcysin LR
As shown in Figure 2, describe the method for making of the biological LR sensing chip of Microcystin in detail, this method in turn includes the following steps:
1) wipes obvious stain on the glass basic surface with lens wiping paper, and (V (concentrated sulphuric acid): V (hydrogen peroxide)=3:2) and acetone thoroughly clean substrate to adopt highly basic (4mol/L NaOH), strong acid successively, after nitrogen dried up under the room temperature, adopting chemical gaseous phase deposition coating method (CVD) to plate a layer thickness at substrate surface was SiO about 100nm
2The surface state of this substrate is shown among Fig. 21.
2) drip pure GOPTS on the glass substrate behind the plated film, be advisable with all areas in covering substrate surface, react for preventing that GOPTS from contact with air, the glass substrate that another is measure-alike covers in the above, the formation sandwich structure, under the room temperature at N
2Reaction 1h in the environment fully washes with acetone afterwards.The surface state of this substrate is shown among Fig. 22.
3) ultrapure water preparation mass percent concentration is 50% glycosaminoglycan 0.1mL, evenly spreads upon on the active position of glass substrate, keeps cultivating 12h at least under the condition of glass surface supersaturation humidity, and reacted substrate is rinsed well with ultrapure water.The surface state of this substrate is shown among Fig. 23.
4) substrate that will be fixed with glycosaminoglycan is dipped in the aqueous solution that mass percent concentration is 1.25% glutaraldehyde, and overnight incubation under the room temperature finishes backlash and washes unreacted glutaraldehyde off.The microcysin LR of Dropwise 5 μ g/mL and the conjugate of OVA on the active position of substrate of glass, and in wet box, cultivate 24h at least.Substrate after the cultivation is dipped in the lysine solution of 2mg/mL, blocks unreacted aldehyde radical, and then substrate is put into the sodium borohydride (NaBH of 4mg/mL
4) in the solution, further block unreacted aldehyde radical.Ultrapure water flushing N
2Dry up, obtain the bio-sensing chip of microcysin LR.
Wherein, the conjugate of microcysin LR and OVA is prepared as follows:
1) molecular modification of microcapsule phycotoxin MC-LR: with mol ratio is that the 2-mercaptoethylmaine of 3000:1 and MC-LR add in the sodium carbonate buffer solution of 0.1M pH=9, and potpourri is reduced to room temperature 50 ℃ of reactions 1 hour after reaction finishes, and adds acetate and stops reaction.Product adopts the method for Solid-Phase Extraction to purify.
2) activation of carrier protein OVA: concentration is that 1.25% excessive glutaraldehyde solution mixes with the OVA solution of 20mg/mL, reacts 12-24h under the room temperature, perhaps at 37 ℃ of reaction 20min down.The reaction afterproduct adopts chromatographic column to carry out purifying.
3) coupling of microcysin LR and carrier protein OVA: the microcysin LR after the OVA after the 3mL activation and the 0.3mg modification is fully mixed, react 24h under the room temperature at least, reaction product is carried out purifying with chromatographic column.
The molecule ratio (coupling ratio) of microcysin LR and OVA is between 2-8 in the conjugate of this scheme preparation.
The microcysin LR sensing chip of the method preparation can not produce non-specific adsorption to inert protein (as bovine serum albumin(BSA) BSA, ovalbumin OVA etc.) substantially, and the antibody of microcysin LR is had good specific reaction.
1) wipes obvious stain on the glass basic surface with lens wiping paper, and (V (concentrated sulphuric acid): V (hydrogen peroxide)=3:2) and acetone thoroughly clean substrate to adopt highly basic (4mol/L NaOH), strong acid successively, after nitrogen dried up under the room temperature, adopting chemical gaseous phase deposition coating method (CVD) to plate a layer thickness at substrate surface was SiO about 100nm
2
2) drip pure GOPTS on the glass substrate behind the plated film, be advisable with all areas in covering substrate surface, react for preventing that GOPTS from contact with air, the glass substrate that another is measure-alike covers in the above, the formation sandwich structure, under the room temperature at N
2Reaction 1h in the environment fully washes with acetone afterwards.
3) substrate surface behind the process GOPTS silanization is with 5mg/cm
2Concentration add DAPEG, put into 60 ℃ of baking ovens, wait to melt the back and the PEG that melts be evenly distributed on substrate surface with glass bar, put into 60 ℃ of baking ovens then and react 36h at least, last ultrapure water rinses out DAPEG unnecessary on the substrate, N
2Dry up.
4) substrate that will be fixed with DAPEG is dipped in the aqueous solution that mass percent concentration is 1.25% glutaraldehyde, and overnight incubation under the room temperature finishes backlash and washes unreacted glutaraldehyde off.On the active position of substrate of glass 2 of Dropwise 5 μ g/mL, the 4-D monoclonal antibody, and in wet box, cultivate 24h at least.Substrate after the cultivation is dipped in the lysine solution of 2mg/mL, blocks unreacted aldehyde radical, and then substrate is put into the sodium borohydride (NaBH of 4mg/mL
4) in the solution, further block unreacted aldehyde radical.Ultrapure water flushing N
2Dry up, obtain 2, the bio-sensing chip of 4-D monoclonal antibody.
Embodiment 4, making 2, the bio-sensing chip of 4-D
1) wipes obvious stain on the glass basic surface with lens wiping paper, and (V (concentrated sulphuric acid): V (hydrogen peroxide)=3:2) and acetone thoroughly clean substrate to adopt highly basic (4mol/L NaOH), strong acid successively, after nitrogen dried up under the room temperature, adopting chemical gaseous phase deposition coating method (CVD) to plate a layer thickness at substrate surface was SiO about 100nm
2
2) drip pure GOPTS on the glass substrate behind the plated film, be advisable with all areas in covering substrate surface, react for preventing that GOPTS from contact with air, the glass substrate that another is measure-alike covers in the above, the formation sandwich structure, under the room temperature at N
2Reaction 1h in the environment fully washes with acetone afterwards.
3) ultrapure water preparation mass percent concentration be 50% 2, the conjugate 2 of 4-D and AMDEX, 4-D-AMDEX 0.1mL, evenly spread upon on the active position of glass substrate, keep cultivating 12h at least under the condition of glass surface supersaturation humidity, reacted substrate is rinsed well with ultrapure water.
Wherein, 2, the conjugate of 4-D and AMDEX is prepared as follows:
1) preparation of active ester: get 10mg 2,4-D is dissolved among the dry DMF of 2mL, adds 50.82mg N-hydroxy-succinamide (NHS) and 91.11mg dicyclohexylcarbodiimide (DCC) in solution, and every kind is 1.1 times of molar excess that add derivant.After stirring 30 minutes on ice, solution is placed under the room temperature and spends the night.Solution filters and preserves in refrigerator.
2) AMDEX and 2,4-D connects: 500mg AMDEX is dissolved in the mixed liquor of 5mL deionized water and 5mLDMF.Add active ester solution, fully mix, be placed under the room temperature and spend the night.Reaction back solution is used the organic phase filtering with microporous membrane then with the methanol extraction of 10 times of volumes, obtains conjugate after the drying.
Utilize in the conjugate of this method preparation 2, the molecule ratio (coupling ratio) of 4-D and AMDEX is between 2-10.In addition, among two kinds of preparation methods provided by the invention, various silylating reagents are identical with the principle that the functional high-polymer polymkeric substance reacts, and its concrete preparation method is same as the previously described embodiments, only corresponding compounds need be replaced to get final product.
Claims (18)
1, a kind of method for preparing the protein bio-sensing chip in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that described step 1) obtains is carried out chemical modification;
Described silylating reagent is 3-diglycidyl hydroxypropyl trimethoxy silane, 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane;
3) with functional high molecular polymer to described step 2) rete that obtains carries out chemical modification;
Described functional high-polymer polymkeric substance is glycosaminoglycan, carboxyl glucosan, two amino polyglycol, two carboxy polyethylene glycol, shitosan or poly-piperazine;
4) with glutaraldehyde protein molecule is fixed on the rete that described step 3) obtains, obtains described protein bio-sensing chip.
2, method according to claim 1, it is characterized in that: described step 2), carry out before the chemical modification with 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane, earlier described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane are dissolved in the toluene; The mass percent concentration of the toluene solution of described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane is 0.25-5%;
In the described step 3), after carrying out chemical modification, before carrying out chemical modification, earlier with the functional high-polymer polymkeric substance among the water-soluble or DMF with functional high molecular polymer with silylating reagent; The mass percent concentration of the water of described functional high-polymer polymkeric substance or DMF solution is 20-60%.
3, method according to claim 1 and 2 is characterized in that: described step 2), the reaction time of carrying out chemical modification with silylating reagent is 1-24h, and temperature of reaction is a room temperature;
In the described step 3), the reaction time of carrying out chemical modification with functional high molecular polymer is 12-24h, and temperature of reaction is 25-37 ℃.
4, method according to claim 1 is characterized in that: in the described step 1), use SiO
2Or Ta
2O
5After the substrate of glass plated film, SiO on the described substrate of glass
2Or Ta
2O
5The thickness of rete is 50-150nm;
In the described step 4), protein molecule is IgG, enzyme or inert protein.
5, the protein bio-sensing chip that obtains of the arbitrary described preparation method of claim 1-4.
6, a kind of method for preparing the bio-sensing chip of organic molecule aglucon in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that described step 1) obtains is carried out chemical modification;
Described silylating reagent is 3-diglycidyl hydroxypropyl trimethoxy silane, 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane;
3) with functional high molecular polymer to described step 2) rete that obtains carries out chemical modification;
Described functional high-polymer polymkeric substance is glycosaminoglycan, carboxyl glucosan, two amino polyglycol, two carboxy polyethylene glycol, shitosan or poly-piperazine;
4) organic molecule is fixed on the rete that described step 3) obtains, obtains the bio-sensing chip of described organic molecule aglucon.
7, method according to claim 6, it is characterized in that: described step 2), carry out before the chemical modification with 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane, earlier described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane are dissolved in the toluene; The mass percent concentration of the toluene solution of described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane is 0.25-5%;
In the described step 3), after carrying out chemical modification, before carrying out chemical modification, earlier with the functional high-polymer polymkeric substance among the water-soluble or DMF with functional high molecular polymer with silylating reagent; The mass percent concentration of the water of described functional high-polymer polymkeric substance or DMF solution is 20-60%.
8, according to claim 6 or 7 described methods, it is characterized in that: described step 2), the reaction time of carrying out chemical modification with silylating reagent is 1-24h, and temperature of reaction is a room temperature;
In the described step 3), the reaction time of carrying out chemical modification with functional high molecular polymer is 12-24h, and temperature of reaction is 25-37 ℃.
9, according to claim 6 or 7 described methods, it is characterized in that: in the described step 1), use SiO
2Or Ta
2O
5After the substrate of glass plated film, SiO on the described substrate of glass
2Or Ta
2O
5The thickness of rete is 50-150nm.
10, according to claim 6 or 7 described methods, it is characterized in that: in the described step 4), described organic molecule being fixed to method on the rete that described step 3) obtains, is with functionalized reagent organic molecule to be fixed on the rete that described step 3) obtains; Described functionalized reagent is carbodiimide, glutaraldehyde, succinimide or DIC.
11, according to claim 6 or 7 described methods, it is characterized in that: in the described step 4), described organic molecule is fixed to method on the rete that described step 3) obtains, be earlier with organic molecule and carrier protein coupling, obtain conjugate, more described conjugate is fixed on the rete that described step 3) obtains; Described carrier protein is bovine serum albumin(BSA) or ovalbumin.
12, according to claim 6 or 7 described methods, it is characterized in that: in the described step 4), functionalized reagent is carbodiimide, glutaraldehyde, succinimide or DIC; Described organic molecule is 2, the product of 4-D, Atrazine, simajine, algae endotoxin, nitrobenzene amine or chlorobenzene class.
13, the bio-sensing chip of the organic molecule aglucon that obtains of the arbitrary described preparation method of claim 6-12.
14, a kind of method for preparing the bio-sensing chip of organic molecule aglucon in turn includes the following steps:
1) on substrate of glass, uses SiO
2Or Ta
2O
5Plated film;
2) with silylating reagent the rete that described step 1) obtains is carried out chemical modification;
Described silylating reagent is amino third dimethyl monosubstituted ethoxy silicon of 3-diglycidyl hydroxypropyl trimethoxy silane, 3-or 3-aminopropyl triethoxysilane;
3) functional high-polymer polymkeric substance and organic molecule are carried out coupling, obtain conjugate;
Described functional high-polymer polymkeric substance is glycosaminoglycan, carboxyl glucosan, two amino polyglycol, two carboxy polyethylene glycol, shitosan or poly-piperazine;
4) conjugate that described step 3) is obtained with functionalized reagent is fixed to described step 2) on the rete that obtains, obtain the bio-sensing chip of described organic molecule aglucon;
Described functionalized reagent is carbodiimide, glutaraldehyde, succinimide or DIC.
15, method according to claim 14, it is characterized in that: described step 2), carry out before the chemical modification with 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane, earlier described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane are dissolved in the toluene; The mass percent concentration of the toluene solution of described 3-aminopropyl dimethyl monosubstituted ethoxy silane or 3-aminopropyl triethoxysilane is 0.25-5%.
16, according to claim 14 or 15 described methods, it is characterized in that: described step 2), the reaction time of carrying out chemical modification with silylating reagent is 1-24h, and temperature of reaction is a room temperature;
17, according to claim 14 or 15 described methods, it is characterized in that: in the described step 1), use SiO
2Or Ta
2O
5After the substrate of glass plated film, SiO on the described substrate of glass
2Or Ta
2O
5The thickness of rete is 50-150nm;
In the described step 3), the molecular weight of described functional high-polymer polymkeric substance is 1000 to 150 000g/mol, preferred 10 000 to 100 000g/mol;
Described organic molecule is 2, the product of 4-D, Atrazine, simajine, algae endotoxin, nitrobenzene amine or chlorobenzene class.
18, the bio-sensing chip of the organic molecule aglucon that obtains of the arbitrary described preparation method of claim 14-17.
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