CN101694494B - Method for manufacturing micro-cantilever modified with antibody - Google Patents
Method for manufacturing micro-cantilever modified with antibody Download PDFInfo
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- CN101694494B CN101694494B CN200910236140A CN200910236140A CN101694494B CN 101694494 B CN101694494 B CN 101694494B CN 200910236140 A CN200910236140 A CN 200910236140A CN 200910236140 A CN200910236140 A CN 200910236140A CN 101694494 B CN101694494 B CN 101694494B
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Abstract
The invention discloses a method for manufacturing a micro-cantilever modified with an antibody. The method comprises steps as follows: 1) an anti-antibody is connected to a micro-cantilever with a gold film through sulfhydrylation so as to obtain the micro-cantilever modified with the anti-antibody, and 2) the antibody is connected to the micro-cantilever modified with the anti-antibody by aid of association action of the antibody and the anti-antibody so as to obtain the micro-cantilever modified with the antibody. According to the result, compared with the existing method, the method for manufacturing a micro-cantilever modified with an antibody has the advantages of having few reaction steps and short operation time, preventing loss of antibody activity due to sulfhydrylation reaction, and exposing more antigenic action sites by leading the antibody to be combined directionally. Therefore, the method and products thereof have wide application prospects on the technical field of micro-cantilever immunol sensing.
Description
Technical field
The present invention relates to the method that a kind of preparation is modified with the micro-cantilever of antibody.
Background technology
The micro-cantilever sensing technology is a kind of novel sensing technology that on the basis of microelectromechanical systems (MEMS) technology and AFM (AFM) development, develops rapidly, is the new focus of nanosensor technical study.Discover that when having biochemical reaction to take place on the micro-cantilever single side surface, the change of its surface stress will cause micro-cantilever to produce flexural deformation or vibration performance changes, and reads in conjunction with optics or electrical method.Based on the micro-cantilever immune sensing technology that relies on immunologic opsonin identification to set up, desmoenzyme joins the advantage of immunoassay technology (ELISA), makes detection technique obtain bigger lifting.This technology can not need affinity tag, and is highly sensitive, and realizes the significant advantage that big scale, high-throughput, parallel array are measured easily.In recent years, this emerging technology is all obtaining using widely to aspects such as cancer detection, DNA hybridization and transcription factors, but actually rare at food and environmental safety field Study of Monitoring.
The prerequisite of micro-cantilever immune sensing broad application is how antibody highly sensitive, high specificity effectively to be attached on the golden film of micro-cantilever, reduces the loss of this antibody activity simultaneously as far as possible.At present; The domestic and foreign literature reported method is mainly carried out through the sulfhydrylization reagent with bifunctional group; Through on antibody, introducing sulfydryl realizing the combination of antibody and golden film, thereby reach the purpose (Chinese patent CN101407548) of detection, but the method for this direct sulfhydrylation antibody; Need all carry out to detect behind the sulfhydrylation to every kind of antibody; Use trouble, directly sulfhydrylation antibody causes the loss of antibody activity easily in addition, thereby has limited micro-cantilever immune sensing broad application.
Summary of the invention
The purpose of this invention is to provide the method that a kind of preparation is modified with the micro-cantilever of antibody.
Preparation provided by the present invention is modified with the method for the micro-cantilever of antibody, is made up of following steps:
1) through sulfhydrylation anti-antibody is connected on the micro-cantilever that has golden film, obtains being modified with the micro-cantilever of anti-antibody;
2) effect of mutually combining through antibody and anti-antibody is connected to antibody on the said micro-cantilever that is modified with anti-antibody, obtains being modified with the micro-cantilever of antibody.
In the aforesaid method, said anti-antibody can be mouse source anti-antibody; Said antibody can be mouse source antibody.
In the aforesaid method, said mouse source anti-antibody can be the sheep anti mouse anti-antibody; Said mouse source antibody can be mouse resource monoclonal antibody or mouse source polyclonal antibody.
In the aforesaid method, said mouse resource monoclonal antibody specifically can be anti-dormin monoclonal antibody or anti-ZR monoclonal antibody.
In the aforesaid method; The said effect of mutually combining through antibody and anti-antibody; Antibody is connected to method on the said micro-cantilever that is modified with anti-antibody for the said micro-cantilever that is modified with anti-antibody being placed the solution of said antibody, under 30 ℃-37.5 ℃ condition, reacts 0.5h-2.0h; Be preferably under 30 ℃-37 ℃ condition and react 1h-2.0h.
In the aforesaid method, the concentration of antibody described in the solution of said antibody is 1ug/ml-3ug/ml.
In the aforesaid method; Saidly anti-antibody is connected on the micro-cantilever that has golden film through sulfhydrylation; The method that obtains being modified with the micro-cantilever of anti-antibody is: utilize sulfhydrylization reagent to carry out sulfhydrylation said anti-antibody, obtain the sulfhydrylation anti-antibody, the said micro-cantilever that has golden film is put in the sulfhydrylation anti-antibody solution; React, obtain being modified with the micro-cantilever of anti-antibody.
Said sulfhydrylization reagent comprise hydrochloric acid mercaptan imine, 11-thiol carboxylic acid (11-MUA), 2-aminoothyl mercaptan (AET), 3-thiohydracrylic acid (MPA), sulfo group HOSu NHS base-6-(3 '; 2-pyridine two sulphur-propionic acid amide)-the two sulfosuccinimide propionic esters (DTSSP) of capronate (Sulfo-LC-SPDP) and 3,3 '-two sulphur etc.
In the aforesaid method, said the said micro-cantilever that has golden film is put in the sulfhydrylation anti-antibody solution in the reaction, the condition of said reaction is: temperature is 30-37.5 ℃, and the time is 0.5-2.0h; Said temperature is preferably 37 ℃, and the said time is preferably 1h.
The micro-cantilever that is modified with antibody that is prepared by above-mentioned arbitrary said method also belongs to protection scope of the present invention.
In view of micro-cantilever immune sensing technical antagonism body fixed importance and sulfhydrylization reagent participation antibody fixed complicacy; The present invention modifies anti-antibody on the micro-cantilever earlier; Combine with the specific recognition reacting phase of antibody through anti-antibody then, antibody is fixed on the golden film of micro-cantilever.The result shows, thereby the method for the prepared micro-cantilever that is modified with antibody of the present invention is compared with existing method and had that reactions step is few, the running time short, avoid because of the active loss of mercaptolation antagonist, make the directed advantage that combines to expose more antigenic action site of antibody.Therefore, method of the present invention and product have broad application prospects in micro-cantilever immune sensing technical field.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Embodiment 1, preparation are modified with the micro-cantilever of sheep anti mouse anti-antibody
1, the sulfhydrylation of sheep anti mouse anti-antibody
(1) accurately take by weighing sheep anti mouse anti-antibody (available from U.S. Sigma company) 10.0mg and be dissolved in the 1.0mL PBS damping fluid, obtaining concentration is the anti-antibody solution of 10mg/mL;
(2) take by weighing 2.0mg hydrochloric acid mercaptan imine (available from U.S. Sigma company) and be dissolved in the 1.0mL zero(ppm) water, obtain the sulfhydrylization reagent that concentration is 2.0mg/mL (at present joining existing usefulness);
(3) sulfhydrylization reagent with the anti-antibody solution of 1.0mL above-mentioned steps (1) and 15.3 μ L above-mentioned steps (2) mixes, and makes that the mol ratio of sheep anti mouse anti-antibody and hydrochloric acid mercaptan imine is 1: 1 in the mixing solutions, and gentle agitation is reacted 0.5h under the room temperature condition;
(4) with the PBS damping fluid (contain 0.15M NaCl and 1.0mM EDTA, pH 7.2) of 20mM reaction solution dialysis 48h to above-mentioned steps (3), during every 2h change a dialyzate, obtain sulfhydrylation sheep anti mouse anti-antibody solution.PBS damping fluid (contain 0.15M NaCl and 1.0mM EDTA, pH 7.2) with 20mM is subsequent use to 1.0mg/mL with sulfhydrylation sheep anti mouse anti-antibody solution dilution.
2, sulfhydrylation sheep anti mouse anti-antibody is modified on the golden film of micro-cantilever
The micro-cantilever (available from American I BM company) that clean and air dried is coated with golden film is put in the sulfhydrylation sheep anti mouse anti-antibody solution that step (4) obtains in the experiment 1 (concentration of sulfhydrylation sheep anti mouse anti-antibody in solution is 5 μ g/mL); 37 ℃ of incubation 1h obtain being modified with the micro-cantilever of sheep anti mouse anti-antibody.
Embodiment 2, preparation are modified with the micro-cantilever of anti-dormin monoclonal antibody
One, preparation
Anti-dormin monoclonal antibody is available from U.S. Sigma company, and catalog number is PGR1-1KT;
Put into anti-dormin monoclonal anti liquid solution (concentration of anti-dormin monoclonal antibody in solution is 1 μ g/mL) after the micro-cantilever that is modified with the sheep anti mouse anti-antibody of preparation among the embodiment 1 cleaned with PBS; 37 ℃ of incubation 1h; Anti-dormin monoclonal antibody and anti-antibody mutually combine; And then be connected on the golden film of micro-cantilever, obtain being modified with the micro-cantilever of anti-dormin monoclonal antibody.
Two, ELISA effect detection
(1) sealing: get the micro-cantilever that is modified with anti-dormin monoclonal antibody that two above-mentioned steps one obtain, PBS cleans the back and dries up with nitrogen, is immersed in 100 μ L quality percentage compositions then and is in 5% the BSA solution (dissolving with PBS), 37 ℃ of sealing 30min;
(2) clean: carefully take out micro-cantilever with tweezers, with the washings flushing for several times, nitrogen dries up; Washings is formed: contain 8.0g NaCl, 0.2g KH in every 1L washings
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20, all the other are water;
(3) compete: the micro-cantilever after nitrogen is dried up is put into two apertures (marked suppresses Kong Yufei and suppresses the hole) of enzyme plate respectively, suppresses the hole and adds 50 μ L dormin standard model solution, and non-inhibition hole adds 50 μ L sample diluting liquids as contrast; And then adding the dormin that concentration is 1.0 μ g/mL-horseradish peroxidase connection thing respectively, 37 ℃ of incubations are competed 30min;
Dormin standard model solution: obtain with sample diluting liquid dilution dormin standard substance, the concentration of dormin in dormin standard model solution is 10ng/mL.
The dormin standard substance are available from U.S. Sigma company.
Sample diluting liquid is formed: by final concentration is that the gelatin that the PBS of 0.1mol/L pH 7.5, tween 20 that final concentration is 0.1% volumn concentration and final concentration are 0.1% quality percentage composition is formed, and said final concentration is the concentration of each material in diluent;
(4) clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the washings flushing for several times, nitrogen dries up;
(5) develop the color: two micro-cantilevers after nitrogen is dried up are put into two apertures of enzyme plate respectively; Every hole adds 100 μ L chromophoric solutions, and (tmb substrate uses liquid and substrate buffer solution to press 1: 9 volume ratio mixing; Add the 4.0mg urea peroxide in the above-mentioned mixed solution of every 10mL), color development at room temperature;
(6) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, takes out micro-cantilever, and the 450nm wavelength reads the OD value respectively.Stop buffer is formed: the sulphuric acid soln of 2.0M.
Three repetitions are established in experiment, and the result takes the mean.The result shows, suppresses hole and the absorbance of non-inhibition hole under the 450nm wavelength and is respectively 0.172 and 0.879, explains that anti-dormin monoclonal anti physical efficiency effectively is attached on the golden film of the micro-cantilever that is modified with the sheep anti mouse anti-antibody.
Three, micro-cantilever beam sensor effect detection
(1) clean: absolute ethyl alcohol, acetone and PBS be the little beam sensor sample pool of wash-out respectively, flow velocity 1mL/min.
(2) standard model solution: obtain with PBS dilution dormin standard substance; The concentration of dormin standard substance in standard model solution is respectively 100ng/mL, 10ng/mL, 1ng/mL.
(3) sample determination: the micro-cantilever that will be modified with anti-dormin monoclonal antibody is inserted in the sample cell of little beam sensor; Moving phase is PBS; Flow velocity 0.2mL/min; Behind little beam sensor signal stabilization, add the above-mentioned standard model solution of preparing of 2mL respectively, utilize micro-cantilever beam sensor to monitor, obtain displacement.
(4) system balancing: after reaction experiment finishes, take out micro-cantilever, with PBS balanced reaction containment system.After concentration of every mensuration, take out little beam, carry out new operation by (1) then.
Three repetitions are established in experiment, and the result takes the mean.The result detects the standard model solution of three concentration, and the displacement that obtains is respectively 240,46 and 22nm.The result shows that the dormin standard model of different concns can produce micro-cantilever displacement in various degree, explains that anti-dormin monoclonal anti physical efficiency effectively is attached on the golden film of the micro-cantilever that is modified with the sheep anti mouse anti-antibody.
Embodiment 3, preparation are modified with the micro-cantilever of anti-ZR monoclonal antibody
One, preparation
Anti-ZR monoclonal antibody is available from U.S. Sigma company, and catalog number is PGR5-1KT;
Put into anti-ZR monoclonal anti liquid solution (concentration of anti-ZR monoclonal antibody in solution is 1 μ g/mL) after the micro-cantilever that is modified with the sheep anti mouse anti-antibody of preparation among the embodiment 1 cleaned with PBS; 30 ℃ of incubation 2h; Anti-ZR monoclonal antibody and anti-antibody mutually combine; And then be connected on the golden film of micro-cantilever, obtain being modified with the micro-cantilever of anti-ZR monoclonal antibody.
Two, ELISA effect detection
(1) sealing: get the micro-cantilever that is fixed with anti-ZR monoclonal antibody that two above-mentioned steps 1 obtain; PBS cleans the back and dries up with nitrogen; Be immersed in 100 μ L quality percentage compositions then and be in 5% the BSA solution (dissolving), 37 ℃ of sealing 30min with PBS;
(2) clean: carefully take out micro-cantilever with tweezers, with the washings flushing for several times, nitrogen dries up; Washings is formed: contain 8.0g NaCl, 0.2g KH in every 1L washings
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20, all the other are water;
(3) compete: the micro-cantilever after nitrogen is dried up is put into two apertures (marked suppresses Kong Yufei and suppresses the hole) of enzyme plate respectively, suppresses the hole and adds 50 μ L ZR standard model solution; Non-inhibition hole adds 50 μ L sample diluting liquids as contrast; And then adding the ZR that concentration is 2.0 μ g/mL-horseradish peroxidase connection thing respectively, 37 ℃ of incubations are competed 30min;
ZR standard model solution: obtain with sample diluting liquid dilution ZR standard substance, the concentration of ZR in ZR standard model solution is 20ng/mL.
The ZR standard substance are available from U.S. Sigma company.
Sample diluting liquid is formed: by final concentration is that the gelatin that the PBS of 0.1mol/L pH 7.5, tween 20 that final concentration is 0.1% volumn concentration and final concentration are 0.1% quality percentage composition is formed, and said final concentration is the concentration of each material in diluent;
(4) clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the washings flushing for several times, nitrogen dries up;
(5) develop the color: two micro-cantilevers after nitrogen is dried up are put into two apertures of enzyme plate respectively; Every hole adds 100 μ L chromophoric solutions, and (tmb substrate uses liquid and substrate buffer solution to press 1: 9 volume ratio mixing; Add the 4.0mg urea peroxide in the above-mentioned mixed solution of every 10mL), color development at room temperature;
(6) stop: behind the 15min, every hole adds 50 μ L stop buffer termination reactions, takes out micro-cantilever, and the 450nm wavelength reads the OD value respectively.Stop buffer is formed: the sulphuric acid soln of 2.0M.
Three repetitions are established in experiment, and the result takes the mean.The result shows, suppresses hole and the absorbance of non-inhibition hole under the 450nm wavelength and is respectively 0.167 and 0.802, explains that anti-ZR monoclonal anti physical efficiency effectively is attached on the golden film of the micro-cantilever that is modified with the sheep anti mouse anti-antibody.
Three, micro-cantilever beam sensor effect detection
(1) clean: absolute ethyl alcohol, acetone and PBS be the little beam sensor sample pool of wash-out respectively, flow velocity 1mL/min;
(2) sample solution: obtain with PBS dilution ZR standard substance, the concentration of ZR in sample solution is respectively 1000ng/mL, 100ng/mL, 10ng/mL;
(3) sample determination: the micro-cantilever that will be modified with anti-ZR monoclonal antibody is inserted in the sample cell of little beam sensor; Moving phase is PBS; Flow velocity 0.2mL/min; Behind little beam sensor signal stabilization, add the above-mentioned standard model solution of preparing of 2mL respectively, utilize micro-cantilever beam sensor to monitor, obtain displacement.
(4) system balancing: after reaction experiment finishes, take out micro-cantilever, with PBS balanced reaction containment system.After concentration of every mensuration, take out micro-cantilever, carry out new operation by (1) then.
Three repetitions are established in experiment, and the result takes the mean.The result shows, detects displacement that the sample of three concentration obtains and is respectively 102,71 and 46nm; The ZR standard model of different concns can produce micro-cantilever displacement in various degree, explains that anti-ZR monoclonal anti physical efficiency effectively is attached on the golden film of the micro-cantilever that is modified with the sheep anti mouse anti-antibody.
Claims (6)
1. method for preparing the micro-cantilever that is modified with antibody, form by following steps:
1) through sulfhydrylation anti-antibody is connected on the micro-cantilever that has golden film, obtains being modified with the micro-cantilever of anti-antibody;
Saidly anti-antibody is connected on the micro-cantilever that has golden film through sulfhydrylation; The method that obtains being modified with the micro-cantilever of anti-antibody is: utilize sulfhydrylization reagent to carry out sulfhydrylation said anti-antibody; Obtain the sulfhydrylation anti-antibody; The said micro-cantilever that has golden film is put in the sulfhydrylation anti-antibody solution, under 30-37.5 ℃ condition, reacted 0.5-2.0h;
2) effect of mutually combining through antibody and anti-antibody is connected to antibody on the said micro-cantilever that is modified with anti-antibody, obtains being modified with the micro-cantilever of antibody;
The said effect of mutually combining through antibody and anti-antibody; Antibody is connected to method on the said micro-cantilever that is modified with anti-antibody for the said micro-cantilever that is modified with anti-antibody being placed the solution of said antibody, under 30-37.5 ℃ condition, reacts 0.5-2.0h;
The concentration of antibody described in the solution of said antibody is 1ug/ml-3ug/ml;
Said anti-antibody is the sheep anti mouse anti-antibody.
2. method according to claim 1 is characterized in that: said antibody is mouse source antibody.
3. method according to claim 2 is characterized in that: said mouse source antibody is mouse resource monoclonal antibody or mouse source polyclonal antibody.
4. method according to claim 3 is characterized in that: said mouse resource monoclonal antibody is anti-dormin monoclonal antibody or anti-ZR monoclonal antibody.
5. method according to claim 1; It is characterized in that: said sulfhydrylization reagent be hydrochloric acid mercaptan imine, 11-thiol carboxylic acid, 2-aminoothyl mercaptan, 3-thiohydracrylic acid, sulfo group HOSu NHS base-6-(3 '; 2-pyridine two sulphur-propionic acid amide)-the two sulfosuccinimide propionic esters of capronate or 3,3 '-two sulphur.
6. the micro-cantilever that is modified with antibody that makes by arbitrary said method among the claim 1-5.
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| CN102778569B (en) * | 2012-05-11 | 2015-01-07 | 中国科学院理化技术研究所 | Method for detecting insecticidal crystal protein Cry1Ab based on micro-cantilever |
| CN107515297B (en) * | 2017-08-17 | 2019-08-27 | 扬州大学 | It is a kind of to drive autobiography sense microcantilever sensors, production method and its application certainly |
| CN113514632A (en) * | 2021-04-20 | 2021-10-19 | 中国科学技术大学 | Nano-antibody-based micro-cantilever immunosensing method |
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| CN101407548A (en) * | 2008-11-25 | 2009-04-15 | 中国农业大学 | Method for preparing micro-cantilever beam modified with antibody |
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