CN104407152B - A kind of preparation method of double antibody single channel encephalitis antigen immune sensor and application - Google Patents
A kind of preparation method of double antibody single channel encephalitis antigen immune sensor and application Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method and application of double antibody single channel encephalitis antigen immune sensor, belong to new function material, novel sensor constructing technology field.Based on specificity good between antigen-antibody, the sandwich structure that this sensor utilizes human immunoglobulin(HIg) immunity right is to increase encephalitis antigen sensing range, significantly improve the sensitivity of sensor, reduce the detection limit of sensor, the early diagnosis of encephalitis B tumour is had great importance.
Description
Technical field
The present invention designs a kind of preparation method and application of double antibody single channel electrochemical immunosensor.Specifically adopt Pd icosahedron as connection carrier, utilize human immunoglobulin(HIg) antibody for skeletal support antibody, connect JE neutralizing antibody, thus realize the specific detection to encephalitis antigen, belong to the development of new bio sensing technology and novel method for sensing constructing technology field.
Background technology
Cancer is the general designation of a large class malignant tumour.In normal human, the content of tumor markers is lower than certain concentration, but, tumour is in generation evolution, tumour cell unconventionality expression or host cell are to the Cucumber produced after tumor response, as tumour specific antigen, some differentiation antigen, embryonal antigen, hormone etc., the content of these tumor markerses can sharply rise, and can be used for diagnosis and the auxiliary diagnosis of some tumour clinical.Therefore, in clinical research, it is very important for developing a kind of quick, easy, sensitive detection tumor markers method.
Electrochemical immunosensor has highly sensitive, selectivity is good, structure is simple, easy and simple to handle, be easy to miniaturization, can be continuous, the advantages such as rapid automatized detection analysis, by the development that it is novel electrochemical immunosensor new technology, breach traditional sensor Constructed wetlands, obtain good experimental result, for clinical practice in the future, there is good guide effect, therefore the present invention constructs a kind of novel electrochemical immunosensor, Pd icosahedron is utilized to be cross-linked human immunoglobulin(HIg) antibody and JE neutralizing antibody, utilize AuGS for base material simultaneously, achieve the detection to encephalitis antigen.
The clinical testing procedure of current existing tumor markers is a lot, as radiommunoassay, chemiluminescence immune assay, enzyme-linked immuno assay etc.Immunosensor is a kind of biology sensor combined with analytical chemistry method by immunological method, by the functionality combination between antigen and antibody, and it the is had advantage such as high sensitivity, high selectivity, analysis be quick and easy and simple to handle.
Summary of the invention
An object of the present invention is base material based on AuGS, constructs a kind of without enzyme, the electrochemical immunosensor of unmarked fast super sensitivity.
Two of object of the present invention is exactly construct double antibody single channel electrochemical immunosensor first, achieves the highly sensitive detection to encephalitis antigen.
Three of object of the present invention is exactly utilize Pd icosahedron for carrier, fixes, achieve the structure of double antibody single channel sensor while achieving human immunoglobulin(HIg) antibody and JE neutralizing antibody.
technical scheme of the present invention is as follows:
1. the preparation method of a double antibody single channel encephalitis antigen immune sensor
(1) Al is used
2o
3burnishing powder polishing diameter is the glass-carbon electrode of 4mm, and ultrapure water cleans up; 6 μ L, 1.0 ~ 1.6mg/mL gold hybrid modification sulfhydrylation Graphene AuGS dispersant liquid drop is added to electrode surface, under room temperature, dries film forming;
(2) drip human immunoglobulin(HIg) antibody A b (H) standard solution of 6 μ L, 1 ~ 5 μ g/mL successively, 3 μ L, massfraction are the BSA solution of 0.5% ~ 2%, and ultrapure water is cleaned, and dries in 4 DEG C of refrigerators;
(3) drip human immunoglobulin(HIg) antigen A g (H) standard solution of 6 μ L, 1 μ g/mL, ultrapure water, dry in 4 DEG C of refrigerators;
(4) Ab (H) Ab (E) the Pd double antibody dripping 6 μ L incubates compound solution, ultrapure water, dries in 4 DEG C of refrigerators;
(5) drip 6 μ L, 0.01 ~ 5ng/mL encephalitis antigen A g (E) standard solution of a series of variable concentrations to electrode surface, ultrapure water, dries in 4 DEG C of refrigerators, the obtained single pass immunosensor of a kind of double antibody.
The preparation of 2.AuGS dispersion liquid
(1) preparation of modification sulfhydrylation Graphene
Under magnetic stirring, the graphene oxide GO of 0.1 ~ 0.2g is scattered in 10 ~ 20mL ultrapure water, obtained GO aqueous solution, joins in GO aqueous solution by the 3-mercaptopropyltriethoxysilane MPTES of 0.3mL, ultrasonic 2 ~ 5h, under magnetic stirring, GO solution be heated to 70 DEG C and be incubated 2 ~ 4h, adding 0.1 ~ 0.3mL, volume fraction is 80% hydrazine hydrate, at 95 DEG C, being incubated 2 ~ 4h, centrifugal, vacuum drying, obtained modification sulfhydrylation Graphene;
(2) preparation of gold nano solution A u
Under magnetic stirring, be that the gold chloride of 1% joins in the ultrapure water of 100mL by 1mL, massfraction, and be heated to boiling, by 2.5 ~ 5mL, massfraction be 1% sodium citrate join in chlorauric acid solution, continue heating 15 ~ 30min to redden to solution, cooling, obtained gold nano solution A u;
(3) preparation of AuGS dispersion liquid
Joined by the GS of 1mg in the gold nano solution A u of 60 ~ 90mL, strong stirring 20 ~ 60min, namely centrifuging obtains AuGS.
3. the preparation of double antibody hatching thing solution A b (H) Ab (E) Pd
(1) the icosahedral preparation of Pd
The NaCl solution of 0.666gPVP and 0.3 ~ 0.4mL, 1mol/L is joined in the ethylene glycol solution of 30mL, add the chlorine palladium acid sodium solution of 0.25 ~ 0.35mL, 0.5mol/L, strong magnetic agitation 30 ~ 60min, by gained chlorine palladium acid sodium mixed solution strong stirring 48 ~ 72h at 120 DEG C, with acetone centrifuge washing 5 ~ 8 times, forced air drying, obtained palladium icosahedron;
(2) preparation of double antibody hatching thing Ab (H) Ab (E) Pd solution
The Pd icosahedron of 0.1 ~ 0.2mg is scattered in 2mL ultrapure water, obtain Pd icosahedron solution, JE neutralizing antibody Ab (E) solution of human immunoglobulin(HIg) antibody A b (H) standard solution of 20 ~ 30 μ L, 100 μ g/mL and 80 ~ 100 μ L, 100 μ g/mL is joined in Pd icosahedron solution, in 4 DEG C of concussion incubators, hatches 24h; Add the BSA of 10 ~ 100 μ L, 1.0mg/mL, in 4 DEG C of concussion incubators, hatch 24h, obtained double antibody hatching thing Ab (H) Ab (E) Pd solution.
4. the detection of encephalitis antigen
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared immunosensor is working electrode, 10mL be 4 ~ 6mmol/L potassium ferricyanide and 0.1mmol/L potassium nitrate solution containing concentration in test;
(2) detect encephalitis antigen standard solution with square wave voltammetry, its voltage tester scope is-0.2V ~ 0.6V;
(3) after background current tends towards stability, the peak point current of the sensor before and after encephalitis antigen adds, then record current change, drawing curve.
useful achievement of the present invention
(1) the sulfhydrylation Graphene of modification and the compound AuGS of gold nano grain, can be good at the electron transmission promoting electrode surface, and the existence of Au can be good at the connection carrying out antibody, improves the stability of sensor.
(2) the icosahedral use of Pd, can be good at connecting human immunoglobulin(HIg) antibody and JE neutralizing antibody, fixes while realizing double antibody.
(3) based on Pd icosahedron, construct the electrochemical immunosensor of a single channel double antibody, the method is the novel sensor construction method built first, achieves the detection to encephalitis antigen.
(4) this sensor utilizes human immunoglobulin(HIg) for supporting antibody, instead of traditional mesoporous material, thus for the film forming of sensing surface, the immobilized intensity of biomolecule is greatly improved, and greatly improves the sensitivity of sensor.
(5) the present invention utilizes the immune response of antigen, antibody for twice, improves the specificity of detection method.
(6) electrochemical immunosensor prepared of the present invention is for the detection of encephalitis antigen, and the response time is short, and detectability is low, and the range of linearity is wide, can realize simple, quick, highly sensitive and specific detection, and its detectability can reach 2.4pg/mL.
Embodiment
embodiment 1a kind of preparation method of double antibody single channel encephalitis antigen immune sensor
(1) Al is used
2o
3burnishing powder polishing diameter is the glass-carbon electrode of 4mm, and ultrapure water cleans up; 6 μ L, 1.0mg/mL gold hybrid modification sulfhydrylation Graphene AuGS dispersant liquid drops are added to electrode surface, under room temperature, dry film forming;
(2) drip human immunoglobulin(HIg) antibody A b (H) standard solution of 6 μ L, 1 μ g/mL successively, 3 μ L, massfraction are the BSA solution of 0.5%, and ultrapure water is cleaned, and dries in 4 DEG C of refrigerators;
(3) drip human immunoglobulin(HIg) antigen A g (H) standard solution of 6 μ L, 1 μ g/mL, ultrapure water, dry in 4 DEG C of refrigerators;
(4) Ab (H) Ab (E) the Pd double antibody dripping 6 μ L incubates compound solution, ultrapure water, dries in 4 DEG C of refrigerators;
(5) drip 6 μ L, 0.01 ~ 5ng/mL encephalitis antigen A g (E) standard solution of a series of variable concentrations to electrode surface, ultrapure water, dries in 4 DEG C of refrigerators, the obtained single pass immunosensor of a kind of double antibody.
A kind of preparation method of double antibody single channel encephalitis antigen immune sensor
embodiment 2a kind of preparation method of double antibody single channel encephalitis antigen immune sensor
(1) Al is used
2o
3burnishing powder polishing diameter is the glass-carbon electrode of 4mm, and ultrapure water cleans up; 6 μ L, 1.2mg/mL gold hybrid modification sulfhydrylation Graphene AuGS dispersant liquid drops are added to electrode surface, under room temperature, dry film forming;
(2) drip human immunoglobulin(HIg) antibody A b (H) standard solution of 6 μ L, 3.5 μ g/mL successively, 3 μ L, massfraction are the BSA solution of 1%, and ultrapure water is cleaned, and dries in 4 DEG C of refrigerators;
(3) drip human immunoglobulin(HIg) antigen A g (H) standard solution of 6 μ L, 1 μ g/mL, ultrapure water, dry in 4 DEG C of refrigerators;
(4) Ab (H) Ab (E) the Pd double antibody dripping 6 μ L incubates compound solution, ultrapure water, dries in 4 DEG C of refrigerators;
(5) drip 6 μ L, 0.01 ~ 5ng/mL encephalitis antigen A g (E) standard solution of a series of variable concentrations to electrode surface, ultrapure water, dries in 4 DEG C of refrigerators, the obtained single pass immunosensor of a kind of double antibody.
embodiment 3a kind of preparation method of double antibody single channel encephalitis antigen immune sensor
(1) Al is used
2o
3burnishing powder polishing diameter is the glass-carbon electrode of 4mm, and ultrapure water cleans up; 6 μ L, 1.6mg/mL gold hybrid modification sulfhydrylation Graphene AuGS dispersant liquid drops are added to electrode surface, under room temperature, dry film forming;
(2) drip human immunoglobulin(HIg) antibody A b (H) standard solution of 6 μ L, 5 μ g/mL successively, 3 μ L, massfraction are the BSA solution of 2%, and ultrapure water is cleaned, and dries in 4 DEG C of refrigerators;
(3) drip human immunoglobulin(HIg) antigen A g (H) standard solution of 6 μ L, 1 μ g/mL, ultrapure water, dry in 4 DEG C of refrigerators;
(4) Ab (H) Ab (E) the Pd double antibody dripping 6 μ L incubates compound solution, ultrapure water, dries in 4 DEG C of refrigerators;
(5) drip 6 μ L, 0.01 ~ 5ng/mL encephalitis antigen A g (E) standard solution of a series of variable concentrations to electrode surface, ultrapure water, dries in 4 DEG C of refrigerators, the obtained single pass immunosensor of a kind of double antibody.
embodiment 4the preparation of AuGS dispersion liquid
(1) preparation of modification sulfhydrylation Graphene
Under magnetic stirring, the graphene oxide GO of 0.1g is scattered in 10mL ultrapure water, obtained GO aqueous solution, joins in GO aqueous solution by the 3-mercaptopropyltriethoxysilane MPTES of 0.3mL, ultrasonic 2h, under magnetic stirring, GO solution be heated to 70 DEG C and be incubated 2h, adding 0.1mL, volume fraction is 80% hydrazine hydrate, at 95 DEG C, be incubated 2h, centrifugal, vacuum drying, obtained modification sulfhydrylation Graphene;
(2) preparation of gold nano solution A u
Under magnetic stirring, be that the gold chloride of 1% joins in the ultrapure water of 100mL by 1mL, massfraction, and be heated to boiling, by 2.5mL, massfraction be 1% sodium citrate join in chlorauric acid solution, continue heating 15min to redden to solution, cooling, obtained gold nano solution A u;
(3) preparation of AuGS dispersion liquid
Joined by the GS of 1mg in the gold nano solution A u of 60mL, strong stirring 20min, namely centrifuging obtains AuGS.
embodiment 5the preparation of AuGS dispersion liquid
(1) preparation of modification sulfhydrylation Graphene
Under magnetic stirring, the graphene oxide GO of 0.14g is scattered in 15mL ultrapure water, obtained GO aqueous solution, joins in GO aqueous solution by the 3-mercaptopropyltriethoxysilane MPTES of 0.3mL, ultrasonic 4h, under magnetic stirring, GO solution be heated to 70 DEG C and be incubated 3h, adding 0.2mL, volume fraction is 80% hydrazine hydrate, at 95 DEG C, be incubated 3h, centrifugal, vacuum drying, obtained modification sulfhydrylation Graphene;
(2) preparation of gold nano solution A u
Under magnetic stirring, be that the gold chloride of 1% joins in the ultrapure water of 100mL by 1mL, massfraction, and be heated to boiling, by 4mL, massfraction be 1% sodium citrate join in chlorauric acid solution, continue heating 25min to redden to solution, cooling, obtained gold nano solution A u;
(3) preparation of AuGS dispersion liquid
Joined by the GS of 1mg in the gold nano solution A u of 80mL, strong stirring 40min, namely centrifuging obtains AuGS.
embodiment 6the preparation of AuGS dispersion liquid
(1) preparation of modification sulfhydrylation Graphene
Under magnetic stirring, the graphene oxide GO of 0.2g is scattered in 20mL ultrapure water, obtained GO aqueous solution, joins in GO aqueous solution by the 3-mercaptopropyltriethoxysilane MPTES of 0.3mL, ultrasonic 5h, under magnetic stirring, GO solution be heated to 70 DEG C and be incubated 4h, adding 0.3mL, volume fraction is 80% hydrazine hydrate, at 95 DEG C, be incubated 4h, centrifugal, vacuum drying, obtained modification sulfhydrylation Graphene;
(2) preparation of gold nano solution A u
Under magnetic stirring, be that the gold chloride of 1% joins in the ultrapure water of 100mL by 1mL, massfraction, and be heated to boiling, by 5mL, massfraction be 1% sodium citrate join in chlorauric acid solution, continue heating 30min to redden to solution, cooling, obtained gold nano solution A u;
(3) preparation of AuGS dispersion liquid
Joined by the GS of 1mg in the gold nano solution A u of 90mL, strong stirring 60min, namely centrifuging obtains AuGS.
embodiment 7the preparation of double antibody hatching thing solution A b (H) Ab (E) Pd
(1) the icosahedral preparation of Pd
The NaCl solution of 0.666gPVP and 0.3mL, 1mol/L is joined in the ethylene glycol solution of 30mL, add the chlorine palladium acid sodium solution of 0.25mL, 0.5mol/L, strong magnetic agitation 30min, by gained chlorine palladium acid sodium mixed solution strong stirring 48h at 120 DEG C, with acetone centrifuge washing 5 times, forced air drying, obtained palladium icosahedron;
(2) preparation of double antibody hatching thing Ab (H) Ab (E) Pd solution
The Pd icosahedron of 0.1mg is scattered in 2mL ultrapure water, obtain Pd icosahedron solution, JE neutralizing antibody Ab (E) solution of human immunoglobulin(HIg) antibody A b (H) standard solution of 20 μ L, 100 μ g/mL and 80 μ L, 100 μ g/mL is joined in Pd icosahedron solution, in 4 DEG C of concussion incubators, hatches 24h; Add the BSA of 10 ~ 100 μ L, 1.0mg/mL, in 4 DEG C of concussion incubators, hatch 24h, obtained double antibody hatching thing Ab (H) Ab (E) Pd solution.
embodiment 8the preparation of double antibody hatching thing solution A b (H) Ab (E) Pd
(1) the icosahedral preparation of Pd
The NaCl solution of 0.666gPVP and 0.33mL, 1mol/L is joined in the ethylene glycol solution of 30mL, add the chlorine palladium acid sodium solution of 0.3mL, 0.5mol/L, strong magnetic agitation 40min, by gained chlorine palladium acid sodium mixed solution strong stirring 60h at 120 DEG C, with acetone centrifuge washing 6 times, forced air drying, obtained palladium icosahedron;
(2) preparation of double antibody hatching thing Ab (H) Ab (E) Pd solution
The Pd icosahedron of 0.15mg is scattered in 2mL ultrapure water, obtain Pd icosahedron solution, JE neutralizing antibody Ab (E) solution of human immunoglobulin(HIg) antibody A b (H) standard solution of 25 μ L, 100 μ g/mL and 90 μ L, 100 μ g/mL is joined in Pd icosahedron solution, in 4 DEG C of concussion incubators, hatches 24h; Add the BSA of 50 μ L, 1.0mg/mL, in 4 DEG C of concussion incubators, hatch 24h, obtained double antibody hatching thing Ab (H) Ab (E) Pd solution.
embodiment 9the preparation of double antibody hatching thing solution A b (H) Ab (E) Pd
(1) the icosahedral preparation of Pd
The NaCl solution of 0.666gPVP and 0.4mL, 1mol/L is joined in the ethylene glycol solution of 30mL, add the chlorine palladium acid sodium solution of 0.35mL, 0.5mol/L, strong magnetic agitation 60min, by gained chlorine palladium acid sodium mixed solution strong stirring 72h at 120 DEG C, with acetone centrifuge washing 8 times, forced air drying, obtained palladium icosahedron;
(2) preparation of double antibody hatching thing Ab (H) Ab (E) Pd solution
The Pd icosahedron of 0.2mg is scattered in 2mL ultrapure water, obtain Pd icosahedron solution, JE neutralizing antibody Ab (E) solution of human immunoglobulin(HIg) antibody A b (H) standard solution of 30 μ L, 100 μ g/mL and 100 μ L, 100 μ g/mL is joined in Pd icosahedron solution, in 4 DEG C of concussion incubators, hatches 24h; Add the BSA of 100 μ L, 1.0mg/mL, in 4 DEG C of concussion incubators, hatch 24h, obtained double antibody hatching thing Ab (H) Ab (E) Pd solution.
embodiment 10the detection of encephalitis antigen
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, 10mL be the 4mmol/L potassium ferricyanide and 0.1mmol/L potassium nitrate solution containing concentration in test;
(2) detect encephalitis antigen standard solution with square wave voltammetry, its voltage tester scope is-0.2V ~ 0.6V;
(3) after background current tends towards stability, the peak point current of the sensor before and after encephalitis antigen adds, then record current change, drawing curve.
embodiment 11the detection of encephalitis antigen
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, 10mL be the 5mmol/L potassium ferricyanide and 0.1mmol/L potassium nitrate solution containing concentration in test;
(2) detect encephalitis antigen standard solution with square wave voltammetry, its voltage tester scope is-0.2V ~ 0.6V;
(3) after background current tends towards stability, the peak point current of the sensor before and after encephalitis antigen adds, then record current change, drawing curve.
embodiment 12the detection of encephalitis antigen
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, and prepared immunosensor is working electrode, 10mL be the 6mmol/L potassium ferricyanide and 0.1mmol/L potassium nitrate solution containing concentration in test;
(2) detect encephalitis antigen standard solution with square wave voltammetry, its voltage tester scope is-0.2V ~ 0.6V;
(3) after background current tends towards stability, the peak point current of the sensor before and after encephalitis antigen adds, then record current change, drawing curve.
Claims (2)
1. a preparation method for double antibody single channel encephalitis antigen immune sensor, is characterized in that, comprise the following steps:
(1) preparation of modification sulfhydrylation Graphene
Under magnetic stirring, the graphene oxide GO of 0.1 ~ 0.2g is scattered in 10 ~ 20mL ultrapure water, obtained GO aqueous solution, joins in GO aqueous solution by the 3-mercaptopropyltriethoxysilane MPTES of 0.3mL, ultrasonic 2 ~ 5h, under magnetic stirring, GO solution be heated to 70 DEG C and be incubated 2 ~ 4h, adding 0.1 ~ 0.3mL, volume fraction is 80% hydrazine hydrate, at 95 DEG C, being incubated 2 ~ 4h, centrifugal, vacuum drying, obtained modification sulfhydrylation Graphene;
(2) preparation of gold nano solution A u
Under magnetic stirring, be that the gold chloride of 1% joins in the ultrapure water of 100mL by 1mL, massfraction, and be heated to boiling, by 2.5 ~ 5mL, massfraction be 1% sodium citrate join in chlorauric acid solution, continue heating 15 ~ 30min to redden to solution, cooling, obtained gold nano solution A u;
(3) preparation of AuGS dispersion liquid
Joined by the GS of 1mg in the gold nano solution A u of 60 ~ 90mL, strong stirring 20 ~ 60min, namely centrifuging obtains AuGS;
(4) the icosahedral preparation of Pd
The NaCl solution of 0.666gPVP and 0.3 ~ 0.4mL, 1mol/L is joined in the ethylene glycol solution of 30mL, add the chlorine palladium acid sodium solution of 0.25 ~ 0.35mL, 0.5mol/L, strong magnetic agitation 30 ~ 60min, by gained chlorine palladium acid sodium mixed solution strong stirring 48 ~ 72h at 120 DEG C, with acetone centrifuge washing 5 ~ 8 times, forced air drying, obtained palladium icosahedron;
(5) preparation of double antibody hatching thing Ab (H) Ab (E) Pd solution
The Pd icosahedron of 0.1 ~ 0.2mg is scattered in 2mL ultrapure water, obtain Pd icosahedron solution, JE neutralizing antibody Ab (E) solution of human immunoglobulin(HIg) antibody A b (H) standard solution of 20 ~ 30 μ L, 100 μ g/mL and 80 ~ 100 μ L, 100 μ g/mL is joined in Pd icosahedron solution, in 4 DEG C of concussion incubators, hatches 24h; Add the BSA of 10 ~ 100 μ L, 1.0mg/mL, in 4 DEG C of concussion incubators, hatch 24h, obtained double antibody hatching thing Ab (H) Ab (E) Pd solution;
(6) preparation of double antibody single channel encephalitis antigen immune sensor
A Al
2o
3burnishing powder polishing diameter is the glass-carbon electrode of 4mm, and ultrapure water cleans up; 6 μ L, 1.0 ~ 1.6mg/mL gold hybrid modification sulfhydrylation Graphene AuGS dispersant liquid drop is added to electrode surface, under room temperature, dries film forming;
B drips human immunoglobulin(HIg) antibody A b (H) standard solution of 6 μ L, 1 ~ 5 μ g/mL successively, and 3 μ L, massfraction are the BSA solution of 0.5% ~ 2%, and ultrapure water is cleaned, and dries in 4 DEG C of refrigerators;
C drips human immunoglobulin(HIg) antigen A g (H) standard solution of 6 μ L, 1 μ g/mL, and ultrapure water dries in 4 DEG C of refrigerators;
Ab (H) Ab (E) the Pd double antibody that d drips 6 μ L incubates compound solution, ultrapure water, dries in 4 DEG C of refrigerators;
E drip 6 μ L, 0.01 ~ 5ng/mL encephalitis antigen A g (E) standard solution of a series of variable concentrations to electrode surface, ultrapure water, dries in 4 DEG C of refrigerators, the obtained single pass immunosensor of a kind of double antibody.
2. the preparation method of a kind of double antibody single channel encephalitis antigen immune sensor as claimed in claim 1, is characterized in that, as follows for encephalitis antigen-measuring step:
(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared immunosensor is working electrode, 10mL be 4 ~ 6mmol/L potassium ferricyanide and 0.1mmol/L potassium nitrate solution containing concentration in test;
(2) detect encephalitis antigen standard solution with square wave voltammetry, its voltage tester scope is-0.2V ~ 0.6V;
(3) after background current tends towards stability, the peak point current of the sensor before and after encephalitis antigen adds, then record current change, drawing curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410695482.7A CN104407152B (en) | 2014-11-27 | 2014-11-27 | A kind of preparation method of double antibody single channel encephalitis antigen immune sensor and application |
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| CN201410695482.7A CN104407152B (en) | 2014-11-27 | 2014-11-27 | A kind of preparation method of double antibody single channel encephalitis antigen immune sensor and application |
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| CN104407152A CN104407152A (en) | 2015-03-11 |
| CN104407152B true CN104407152B (en) | 2015-12-30 |
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| CN103543268A (en) * | 2013-10-16 | 2014-01-29 | 北京华卫骥生物医药有限公司 | Method for detecting Japanese encephalitis virus (JEV), quantum dot labelled immunochromatographic test strip and preparation method thereof |
| CN104133070A (en) * | 2014-07-17 | 2014-11-05 | 济南大学 | Preparation method and use of environmental estrogen label-free immunosensor |
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| CN104155357A (en) * | 2014-05-23 | 2014-11-19 | 济南大学 | Preparation method and application of three-dimensional cubic duct based mesoporous silica sensor |
| CN104133070A (en) * | 2014-07-17 | 2014-11-05 | 济南大学 | Preparation method and use of environmental estrogen label-free immunosensor |
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| 电化学免疫传感器研究进展;钟桐生 等;《化学传感器》;20020331;第22卷(第1期);全文 * |
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