CN1763527A - Method for making sample cell of solid phase white amino reflective layer - Google Patents
Method for making sample cell of solid phase white amino reflective layer Download PDFInfo
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- CN1763527A CN1763527A CN 200510107412 CN200510107412A CN1763527A CN 1763527 A CN1763527 A CN 1763527A CN 200510107412 CN200510107412 CN 200510107412 CN 200510107412 A CN200510107412 A CN 200510107412A CN 1763527 A CN1763527 A CN 1763527A
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Abstract
The present invention relates to a kind of method for making sample cell of solid phase white amino reflective layer.Its method is: adopt pyridine/acetone activation processing PS surface in advance, with acetate/metacresol/pyridine dissolving, activation nylon, make hydrophobic interaction takes place between the two, can make nylon-6 be incorporated into the inside surface of PS sample cell firmly, uniformly; Directly handle the oxygen atom alkylation that makes on the nylon membrane with TOTFB; Alkyl on the nylon-6 film is through the effect of glutaraldehyde double-functional group, a covalently bound end is in conjunction with the free amine group on " arm ", the free amine group of the aldehyde radical conjugated protein that the other end is free forms hands over firm covalent bond, obtains sample cell of solid phase white amino reflective layer.This sample cell can be repeatedly used.Method of the present invention can be used for the immobilized enzyme method, improves the effect of enzyme engineering, reduces its cost.
Description
Technical field
The invention belongs to method for making sample cell of solid phase white amino reflective layer.
Background technology
Technology such as solid-phase immunoassay, affine layer and immobilized enzyme all need bioactivator (immune aglucon, nucleic acid, enzyme etc.) is incorporated into the inertia macromolecular material, as agarose, cellulose, nylon and various frosting; Wherein polystyrene (PS) is easy because of raw material sources, and physics, chemical property are stablized, and are easy to machine-shaping etc., make its application the most general.Be incorporated into the quantity of solid phase surface molecule and biologically active or affinity and be one of factor that influence improves the immunoassay detection sensitivity (haptens, poly-covalently bound with the styrene solid phase of modifying of protein and nucleic acid, the immunological method magazine, Covalent Linking of haptens, protein andnucleic acid to a modified polystyrene support.J Immunol Methods1993.159:177-187).Physisorphtion is because absorption back molecular activity reduces, and the repeatedly flushing generation molecule in long preservation and operation comes off, and causes the obviously reduction of its sensitivity, repeatability and stability.
From the seventies in last century, people adopt various chemistry and physical method that biomolecule is covalently bonded in solid phase, comprise plastics are become reactive group (amino through chemical modification, carboxyl and sulfydryl), reactive group transplanting and methods such as bioactive molecule and polystyrene copolymerization, but because the hydrogen bond of polystyrene surface is highly stable, many results are not satisfactory.Though what have forms commodity, but because cost exceeds the conventional material several times, be not suitable for widespread usage, so the conventional now ELSIA solid phase of using still adopts physisorphtion (protein and the covalently bound technology of frosting that are used for immunoassay, immunization method is learned magazine, 1987,98:129-135 Covalent binding of protein to grafted plasticsurfaces suitable suitable suitable suitable for.Immunol.Methods.1987,98:129-135).Therefore, development is suitable for the conventional solid phase material that contains reactive group of using becomes one of gordian technique that improves the sensitivity of ultramicron immunoassay.
Nylon (polycaprolactam) comes from the many ammonia by amino as dissociating, cause people's attention very early.But most researchs all concentrate in advance with biomolecule in conjunction with nylon membrane on after be covered in polystyrene (PS) microwell plate again (thermolysin be fixed on the polyamide, biotechnology and bioengineering 2003,82 (2) 190-199.Immobilization?of?Thermolysin?to?polyamideNonwoven?Materials.Biotechnol.and?Bioeng.2003，82(2)190-199)。Be not suitable for doing ELSIA solid phase sample pond.(nylon of haptens combination is coated polystyrene board as the enzyme-linked immuno assay solid phase about nylon being incorporated into the report that polystyrene is a sample cell, immunization method is learned magazine 1990,127:43-49.Haptenated nylon-coated polystyrene plates as asolid phase for ELSIA.J.Immunol Methods.1990,127:43-49), owing to adopt the natural combination under the stationary state, be proved to be in conjunction with inhomogeneous, the nylon hydrolysis be can not tolerate and amino strong acid treatment and the organic solvent in the chemical coupling discharged, cause polystyrene (PS) surface to be etched, destroy, be not suitable for doing conventional solid phase material.
Summary of the invention
In order to improve the sensitivity of ultramicron immunoassay, development is suitable for the solid phase material that solid-phase time-resolved fluorescence immunoassay is used, solution is with reflection replacement transmission, with technical matterss such as chemical covalent bond alternative physical absorption, the invention provides the fixedly nylon layer of one deck White-opalescent of a kind of surface at polystyrene micropore plate, after chemical modification as the manufacture method of the sample cell of solid phase white amino reflective layer that uses in the DSLFIA technology.
The structure of the sample cell of solid phase white amino reflective layer that method of the present invention provides as shown in Figure 1.It is one the circular platform type at the end or the goblet of column type; Be to constitute by matrix 1, middle level 2, interior surface layers 3; Described matrix 1 is polystyrene (PS) microwell plate, and middle level 2 is the nylon-6 retes that are linked in the inside surface of matrix 1 polystyrene (PS) microwell plate, interior surface layers 3 be with the nylon-6 rete in middle level 2 with covalently bound polyamines basic unit;
The connection of the inside surface of described matrix 1 polystyrene (PS) microwell plate and the nylon-6 rete in middle level 2 is to use pyridine/acetone inside surface of activation processing matrix 1 in advance, with acetate/metacresol/pyridine dissolving, activation nylon-6, make hydrophobic interaction takes place between the two, simultaneously pyridine has activation PS concurrently and gentle " erosions " acts on, and can make nylon-6 be linked in the inside surface of PS microwell plate securely fast and forms interior surface layers 2; Nylon layer to interior surface layers 2 carries out the alkylation processing, alkyl-the OR that can make the generation of nylon-6 surface is at an easy rate with 2, an amino covalence combination of 6-diaminocaproic acid, form the arm of other end free amine group, it is polyamines basic units that the free amine group of this " arm " can form more firm covalently bound interior surface layers 3 with the free amine group of protein, so obtain the opaque reflective sample cell of solid phase white.
Introduce the step and the condition of manufacturing sample cell of solid phase white amino reflective layer method of the present invention below more in detail, so that the present invention is further described.
Agents useful for same of the present invention:
A. matrix 1 activator: the acetone soln of 6.0-8.0% pyridine (V/V).
B. the activator of the nylon-6 in middle level 2: the acetate/m-cresol solution (V/V) of (0.5-0.9% pyridine).
C. the diethyl ether solution of epichlorokydrin: epichlorokydrin and diethyl ether solution volume ratio are 1: 9.
D. triethyl oxygen drone tetraboric acid fat (TOTFB) reagent preparation: 40ml 5% (v/v) boron trifluoride diethyl etherate (v/v) solution is continuing under the stirring, the diethyl ether solution that adds the epichlorokydrin of 12.5ml, in 30 minutes, add, reflux is one hour afterwards, room temperature stirred 3 hours again, leach TOTFB, again with absolute ether flushing 3 times, the TOTFB that obtains.Its productive rate is more than the 90%--95%.
E.TOTFB dichloromethane solution: contain 0.01-0.05g/ml TOTFB triethyl oxygen father-in-law's tetrafluoro boric acid ester (TOTFB) dichloromethane solution.
F. pentanedial liquid: the 0.5M pentanedial liquid is dissolved in 0.2M borate buffer solution (pH=7.6) mutually with 5% (v/v).
G.1,6 hexane diamine solution: concentration is the carbonate buffer solution of (pH=9.5) of 0.5M 1,6 hexane diamine.
Preparation method's of the present invention step and condition:
(1) .nylon-6 and PS matrix 1 is connected
In by PS matrix 1 hole, add matrix 1 activator, room temperature was placed 1-3 minute, discard the activator that adds the middle level 2nylon-6 that contains 12-18% polyamide-6 powder behind the solution immediately, the rotary substrate 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface in matrix 1 hole equably, forming nylon-6 thickness is the rete in the sample cell of solid phase white amino reflective layer middle level 2 of 0.1-0.2mm;
(2). nylon membrane surface alkylation
Add in the sample cell of TOTFB dichloromethane solution above-mentioned (1), room temperature was placed 4-10 minute, discarded, and used dichloromethane rinse 2 times, with dioxane flushing 1 time;
(3). modify the nylon membrane surface
Glutaraldehyde solution is joined in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes, discard, add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times, add 1,6 hexane diamine (pH=9.5) solution again, room temperature was placed 3 hours, outwell, with PBS solution flushing 3 times, promptly get sample cell of solid phase white amino reflective layer then.
Preparation method's of the present invention relevant course of reaction is as follows:
Above-mentioned procedure declaration: be incorporated into the PS surface n ylon-6 polycaprolactam ammonia structure reactive group that dissociates for making, adopt the nylon layer of handling the microwell plate inside surface from synthetic triethyl oxygen father-in-law tetrafluoro boric acid ester (TOTFB) alkanisation, do not influence the nylon-6 structure, and can fully expose reactive group
Connect 1 with glutaraldehyde, the 6-hexane diamine, promptly spreading " arm " has improved free amine group reaction probability.Utilize the effect of glutaraldehyde double-functional group, an end is in conjunction with the free amine group on " arm ", and the free amine group of the aldehyde radical conjugated protein that the other end is free forms and hands over firm covalent bond.
1. adopt pyridine/acetone activation processing PS surface in advance,, make hydrophobic interaction takes place between the two with acetate/metacresol/pyridine dissolving, activation nylon.Simultaneously pyridine has activation PS and gentle " erosions " effect concurrently, can make nylon-6 quick firm be incorporated into the PS surface.
2. for making the nylon-6 polycaprolactam ammonia structure reactive group that dissociates, used hydrochloric acid partial hydrolysis method in the past, because excessively the hydrolysis meeting makes the nylon structural break, it is insufficient that the control hydrolysis time causes free amine group to expose again, handle the nylon layer of microwell plate inside surface so adopt triethyl oxygen father-in-law tetrafluoro boric acid ester (TOTFB) alkanisation that oneself synthesizes, do not influence the nylon-6 structure, and can fully expose reactive group
The latter is easy to connect 1 with glutaraldehyde, 6-hexane diamine, i.e. spreading " arm ".
3. utilize the effect of glutaraldehyde double-functional group, an end is in conjunction with the free amine group on " arm ", and the free amine group of the aldehyde radical conjugated protein that the other end is free forms firm covalent bond.
Technical characterstic of the present invention and effect:
1. utilize nylon-6 to be incorporated into the sample cell of solid phase white amino reflective layer of the inside surface of PS, adopt new many ammonia of dissolving technology and specific PS activator, many ammonia coagulating agent, make polyamines evenly, firmly transplant surface, form white catoptrical polyamines base internal layer in nylon-6.
This polyamines inside surface solid phase room temperature expose in the air can long preservation and can tolerate strong acid, alkali, organic solvent indeformable, do not come off.For making the nylon-6 polycaprolactam ammonia structure reactive group that dissociates, used hydrochloric acid partial hydrolysis method in the past, because excessively the hydrolysis meeting makes the nylon structural break, it is insufficient that the control hydrolysis time causes free amine group to expose again, so adopt TOTFB alkanisation processing nylon layer.This method can not influence the nylon-6 structure, and can fully expose reactive group
Utilize the effect of glutaraldehyde double-functional group, the free amine group in the end combination " arm ", the free amine group of the aldehyde radical conjugated protein that the other end is free, the activity of raising binding immunoassay aglucon.
3. adopt pyridine to replace methylene chloride.Because pyridine not only volatilizees slow and can promote the nylon of dissolved state to assemble and with the combining of PS, form on the PS surface even, tough and tensile, can not be through the film of organic solvent; And when it combines with PS, to active amino content, physical strength and desirable effect is received in aspects such as organic solvent tolerance.
4. handle the nylon surface with TOTFB, existing report in the document, but all be to use the nylon film reaction, wash-out need pass through a large amount of soaked with liquid, and the present invention adopts in the pond flushing three times, and non-specific bond is 3.41%, the experiment/non-special=12.9, be significantly higher than 2.1 times of diagnostic criteria of ELISA.
5. the sample cell of solid phase white amino reflective layer with method manufacturing of the present invention is used for the radioactive label immunoassay, and the aglucon combination rate reaches 43.98%, for the 2.5-13.1 of existing PS microwell plate doubly.The sample cell of solid phase white amino reflective layer that proves method manufacturing of the present invention has greater activity and sensitivity.
6. batch interior error statistics of the sample cell of solid phase white amino reflective layer of the inventive method manufacturing is tested and the average CV=5.0% of control group, and each organizes 8 parallel samples CV＜10%, is significantly higher than existing PS microwell plate.
7. the destructive test result of the sample cell of solid phase white amino reflective layer of the inventive method manufacturing proves: the temperature of the complete sex change of protein is 65 ℃, experiment adopts 50 ℃ of maintenances to heat 48 hours, the combination rate of sample cell coupling antibody still keeps 41%, than having reduced by 4.6% before the experiment.And the PS sample cell combination rate 5% that existent method is made, complete failure.
The performance of sample cell of solid phase white amino reflective layer of the present invention can with compare with the NuncCovalink-NH microwell plate that Denmark produces, practicality is then superior than it.For example the reactive group of Nunc plate is-NH, and the present invention is-NH
2, the amino of this group linking protein matter is easier, more stable; Expensive 2 times of Nunc plate prices than PS microwell plate.The present invention draws materials easily because of easy and simple to handle, and cost is only than PS microwell plate expensive about 20%.
Sample cell of solid phase white amino reflective layer with method manufacturing of the present invention both can be used for being applied to biochemical analysis fields such as solid-phase immunoassay (radiommunoassay, fluoroimmunoassay, enzyme-linked immuno assay, time resolved fluoro-immunoassay) DNA chip, can also be used as affinity chromatography solid phase bond, being about to diameter is that 5-8 μ m PS microballon is transplanted active amino by this method, can be used as high-effect affinity chromatographic material, and can use repeatedly, its function and cost are excellent than polyacrylamide gel commonly used all, are with a wide range of applications.
Description of drawings
Fig. 1 is the sample cell of solid phase white amino reflective layer structural representation.
Embodiment
Employed reagent of embodiments of the invention and preparation thereof:
A. matrix 1 activator: the acetone soln of 6.0-8.0% pyridine (V/V).
B. the activator of the nylon-6 in middle level 2: the acetate/m-cresol solution (V/V) of (0.5-0.9% pyridine).
C. epichlorokydrin diethyl ether solution: epichlorokydrin and diethyl ether solution volume ratio are 1: 9.
D. triethyl oxygen drone tetraboric acid fat (TOTFB) and preparation: 40ml 5% (v/v) boron trifluoride diethyl etherate (v/v) solution is continuing under the stirring, the diethyl ether solution that adds the epichlorokydrin of 12.5ml, in 30 minutes, add, reflux is one hour afterwards, room temperature stirred 3 hours again, leach TOTFB, again with absolute ether flushing 3 times, the TOTFB that obtains.Its productive rate is more than the 90%--95%.The TOTFB that obtains must use up in 48 hours, otherwise lost efficacy.
E.TOTFB dichloromethane solution: contain 0.01-0.05g/ml TOTFB triethyl oxygen father-in-law's tetrafluoro boric acid ester (TOTFB) dichloromethane solution.
F. glutaraldehyde borate buffer solution: the 0.5M pentanedial liquid is dissolved in 0.2M borate buffer solution (pH=7.6) mutually with 5% (v/v).
G.1,6 hexane diamine solution: concentration is the carbonate buffer solution of (pH=9.5) of 0.5M 1,6 hexane diamine.
Embodiment 1:
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator, room temperature was placed 1 minute, add the 12%nylon-6 solution of 200 μ l immediately after discarding solution by the dissolving of middle level 2nylon-6 activator, rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the sample cell of solid phase white amino reflective layer middle level 2nylon-6 rete of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l, 5% triethyl oxygen father-in-law tetrafluoro boric acid ester (TOTFB) is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discards, and uses dichloromethane rinse 2 times, dioxane flushing 1 time;
3. 200 μ l 0.5M glutaraldehydes are added in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes discard.Add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l0.058g/ml again, room temperature was placed 3 hours, outwelled.With PBS buffer solution flushing 3 times, promptly get sample cell of solid phase white amino reflective layer of the present invention then.
(1). add 400 μ l matrixes, 1 activator in the matrix 1 that is made of PS, room temperature was placed 1 minute, added the 13%nylon-6 solution of 200 μ l with the dissolving of middle level 2nylon-6 activator immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming nylon-6 thickness is the sample cell of solid phase white amino reflective layer middle level 2nylon-6 rete of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 4%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded. use dichloromethane rinse 2 times, dioxane flushing 1 time;
3. add 200 μ l 0.5M glutaraldehyde borate buffer solutions in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes discard.Add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l 0.5M again, room temperature was placed 3 hours, outwelled, and with PBS buffer solution flushing 3 times, promptly obtained sample cell of solid phase white amino reflective layer of the present invention then.
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator.Room temperature was placed 1 minute, added the 14%nylon-6 solution of 200 μ l by the dissolving of middle level 2nylon-6 activator immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the sample cell of solid phase white amino reflective layer middle level 2nylon-6 rete of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 3%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded.With dichloromethane rinse 2 times, dioxane flushing 1 time; Described TOTFB synthetic method is by document: (enzyme is fixed in the chemistry of nylon, biological chemistry .J.1975,147:593-603 A chemistry for the Immobilization of Enzymeson nylon.Biochem.J.1975,147:593-603) synthetic method is synthetic;
3. 200 μ l 0.5M glutaraldehyde borate solutions are joined in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; 1, the 6 hexane diamine solution that adds 200 μ l 0.5M again, room temperature was placed 3 hours, outwelled, and with PBS solution flushing 3 times, promptly got sample cell of solid phase white amino reflective layer of the present invention then.
Embodiment 4
(1). add 400 μ l matrixes, 1 activator in the matrix 1 that is made of PS, room temperature was placed 1 minute, added the 15%nylon-6 solution of 200 μ l with the dissolving of middle level 2nylon-6 activator immediately after discarding solution.Rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming nylon-6 thickness is the sample cell of solid phase white amino reflective layer middle level 2nylon-6 rete of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 2%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discards, and uses dichloromethane rinse 2 times, dioxane flushing 1 time;
3. in the sample cell that joins above-mentioned (2) with 200 μ l glutaraldehyde borate buffer solutions (pH=7.6), 37 ℃ of incubations add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times after 15 minutes; Add 200 μ again and be dissolved in 1, the 6 hexane diamine solution of 0.1M carbonate buffer solution (pH=9.5) 0.5M, room temperature was placed 3 hours, outwelled, and with PBS solution flushing 3 times, promptly got sample cell of solid phase white amino reflective layer of the present invention then.
Embodiment 5
(1). in the matrix 1 that constitutes by PS, add 400 μ l matrixes, 1 activator.Room temperature was placed 1 minute, add the 16%nylon-6 solution of 200 μ l immediately after discarding solution by the dissolving of middle level 2nylon-6 activator, rotate (40 times/minute) matrix 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface of matrix 1 equably, forming the nylon-6 thicknesses of layers is the sample cell of solid phase white amino reflective layer middle level 2nylon-6 rete of 0.1-0.2mm;
(2). the dichloromethane solution that adds 400 μ l 1%TOTFB is in the sample cell of above-mentioned (1), and room temperature was placed 4-10 minute, discarded.With dichloromethane rinse 2 times, dioxane flushing 1 time;
3. add 200 μ l 0.5M glutaraldehyde borate buffer solutions in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times; Add 200 μ l 0.5M1 again, 6 hexane diamine carbonate buffer solutions (pH=9.50), room temperature was placed 3 hours, outwelled.With PBS solution flushing 3 times, promptly get sample cell of solid phase white amino reflective layer of the present invention then.
Claims (2)
1, a kind of method for making sample cell of solid phase white amino reflective layer is characterized in that preparation method's step and condition are as follows:
(1) connection of .nylon-6 and PS matrix 1
In the PS of sample cell of solid phase white amino reflective layer matrix 1, add matrix 1 activator pyridine/acetone soln, room temperature was placed 1-3 minute, discard the activator that adds the middle level 2nylon-6 that contains 12-18% polyamide-6 powder behind the solution immediately, the rotary substrate 1 at a slow speed along the axis, make nylon-6 quick, firm, be incorporated into the inside surface in matrix 1 pond equably, forming nylon-6 thickness is the rete in the sample cell of solid phase white amino reflective layer middle level 2 of 0.1-0.2mm;
(2). nylon membrane surface alkylation
Add in above-mentioned (1) sample cell of TOTFB dichloromethane solution, room temperature was placed 4-10 minute, discarded, and used dichloromethane rinse 2 times, with dioxane flushing 1 time;
(3). modify the nylon membrane surface
Glutaraldehyde solution is joined in the sample cell of above-mentioned (2), 37 ℃ of incubations 15 minutes, discard, add 0.2M pH=7.2 phosphate (PBS) buffer solution flushing three times, add 1,6 hexane diamine (pH=9.5) solution again, room temperature was placed 3 hours, outwell, with PBS solution flushing 3 times, get sample cell of solid phase white amino reflective layer then.
2, a kind of method for making sample cell of solid phase white amino reflective layer as claimed in claim 1 is characterized in that described:
Matrix 1 activator is: the acetone soln of 6.0-8.0% pyridine (V/V).
The activator of the nylon-6 in middle level 2 is: the acetate/m-cresol solution (V/V) of (0.5-0.9% pyridine).
The TOTFB dichloromethane solution is: contain 0.01-0.05g/ml TOTFB triethyl oxygen father-in-law's tetrafluoro boric acid ester (TOTFB) dichloromethane solution.
Pentanedial liquid is: the 0.5M pentanedial liquid is dissolved in 0.2M borate buffer solution (pH=7.6) mutually with 5% (v/v).
1,6 hexane diamine solution is: 1, the 6 hexane diamine solution (v/v) of the 0.5M that incites somebody to action is dissolved in the carbonate buffer solution of 0.1M (pH=9.5).
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| CN1173182C (en) * | 2002-12-18 | 2004-10-27 | 陕西超英生物医学研究开发有限公司 | Albumen chip for detecting autoimmunity antibody of diabetes, as well as preparation and detection method |
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