CN108152485B - A kind of immunoassay method reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic - Google Patents
A kind of immunoassay method reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic Download PDFInfo
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- CN108152485B CN108152485B CN201711316296.8A CN201711316296A CN108152485B CN 108152485 B CN108152485 B CN 108152485B CN 201711316296 A CN201711316296 A CN 201711316296A CN 108152485 B CN108152485 B CN 108152485B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
Abstract
Present invention discover that the catalytic reaction products of alkaline phosphatase cause the metal oxide for being deposited on CdS electrode surface or hydrated metal oxide to decompose, to excite the anode photoelectric current of CdS material modified electrode significantly.Phenomenon accordingly establishes a kind of high-throughput optical electro-chemistry immunoassay method that immune response is separated with optical electro-chemistry detection using mouse IgG as model.By the progress sandwich immunoassay reaction of 96 microwell plates, the photoelectric current of signaling molecule excitation CdS electrode is generated using the reaction that is catalyzed of the immobilized alkaline phosphatase of Au nanoparticle, to realize high throughput, high sensitivity, selectively measure.Also, this method successfully avoids adverse effect of the large biological molecule to signal of electrode surface fixation in the detection of conventional photo chemo-immunity.
Description
Technical field:
The present invention relates to nanoanalysis detection fields, more particularly to the catalysis reaction triggering CdS for passing through alkaline phosphatase
The photoelectric current of nano material optoelectronic pole, to realize high-throughput optical electro-chemistry immunoassay.
Background technique:
Immunoassay has very in numerous areas such as clinical disease diagnosis, biological medicine, food safety and environmental protections
Consequence.But traditional immunoassay method is not able to satisfy the demand of low content target analyte detection, therefore establishes letter
Just, sensitively detection method is still very necessary.Optical electro-chemistry (PEC) analysis is a kind of new detection means, with many
Advantage, such as simple, cheap, the easy realization micromation of equipment and sensitivity height, so that the technology is since appearance in biology
Detection field has obtained fervent concern and fast development.
Current PEC immune detection is concentrated mainly on two major classes type: first, using electrode surface formed capture antibody/
Antigen/enzyme labelling assay for determining antibody sandwich structure is detected, after immune response occurs, the detection antibody on sandwich structure
The native enzyme meeting catalysis substrate connected generates electron donor, enhances the photoelectricity of the optical electro-chemistry active material of electrode face finish
Flow [Zhao W W, Ma Z Y, Yan D Y, Xu J J, Chen H Y.Anal.Chem., 2012,84:10518-10521；
Zhuang J Y,Tang D Y,Lai W Q,Xu M D,Tang D P.Anal.Chem.,2015,87:9473-9480；
Zhang N,Ma Z Y,Ruan Y F,Xu J J,Chen H Y.Anal.Chem.,2016,88:1990-1994.].Other one
Class optical electro-chemistry immunoassay is increased using steric hindrance caused by the fixed antibody antigen of electrode surface, it is suppressed that electronics
The reaction of donor and optical electro-chemistry active material, measurement [Wang G L, the Xu J that photo-signal is suppressed to realize object
J,Chen H Y,Fu S Z.Biosens.Bioelectron.,2009,25:791-796；Li W,Sheng P,Cai J,
Feng H,Cai Q.Biosens.Bioelectron.,2014,61:209-214.].It is above-mentioned based on electrode surface sessile antibody/
The optical electro-chemistry immune detection of antigen haves the defects that certain: 1. in the detection method of signal enhancing type, being fixed on electrode table
The antibody in face, antigen, enzymic-labelled antibody have biggish space steric effect as large biological molecule, can inhibit electron donor and
Identification reaction between optical electro-chemistry active material, to reduce the sensitivity of detection；2. immune response and optical electro-chemistry detection
Signal output be to be carried out on same plate electrode, which results within the regular hour, optical electro-chemistry inspection can only be carried out
Survey or biological respinse, so as to cause the small throughput of method；3. large biological molecule be fixed on electrode surface will lead to the absorption of light/
Scattering etc. influences the accuracy of detection.Therefore, develop has the high-throughput detection that immune response is separated with optical electro-chemistry detection very much
It is necessary.
The present invention is established a kind of immune using the photoelectric current of the catalysis reaction triggering CdS optoelectronic pole of alkaline phosphatase
Immunoassay method that reaction is separated with optoelectronic pole, high-throughput.Surface deposited metal oxide or hydrated metal oxide
CdS optoelectronic pole substantially without photo-signal, and the reducing agent that the catalytic hydrolysis reaction of alkaline phosphatase generates makes metal
Oxide or hydrated metal oxide decompose, so as to cause greatly improving for CdS optoelectronic pole photoelectric current.Using mouse IgG as model,
Detection antibody is marked by alkaline phosphatase, detection is realized to the interlayer structure immune response carried out in 96 microwell plates.
The maximum advantage of this method, which is to be immunoreacted from electrode surface, to be separated, and the fixed biomolecule pair of electrode surface is avoided
The sensitivity of optical electro-chemistry measurement and the influence of accuracy.More importantly the introducing and immune response of microwell plate reaction
Separation with Photoelectric Detection substantially increases detection efficiency.
Summary of the invention:
The object of the present invention is to provide a kind of optical electro-chemistry method of immunity efficiently, easy, high-throughput；Especially mention
For a kind of strategy of the photoelectric current of catalysis reaction triggering CdS electrode using alkaline phosphatase, it is immune to open optical electro-chemistry
The new way of measurement.It can produce biggish anode photoelectric current after CdS material modified electrode illumination prepared by the present invention；And pass through
After further depositing metal oxide or hydrated metal oxide, apparent inhibition is had occurred in the photoelectric current of electrode.By micro-
After the immune response of the alkaline phosphatase label carried out in orifice plate, enzymic catalytic reaction produces being capable of electrode decomposing surface deposition
Metal oxide or hydrated metal oxide, significantly restored so as to cause photoelectric current.The degree that photoelectric current restores
It is related with the content of antigen in immune response, signal enhancing type immunoassay method is established accordingly.
The purpose of the present invention can be achieved by the following technical measures:
A, the optoelectronic pole of CdS modification, specific steps are as follows: by the Triton- of 2% mass concentration are prepared using chemical deposition
100 solution of X and certain density CdSO4It is a certain range that ammonia spirit is added after solution mixing and adjusts the pH of solution；Then past
The thiourea solution of 0.5M is added in above-mentioned reaction solution；The ITO electrode cleaned is immersed in above-mentioned solution at 90 DEG C later
Reaction 5-15 minutes takes out and rinses electrode, and the optoelectronic pole of CdS modification is obtained after drying overnight；
B, CdS modified electrode obtained is immersed in six methylene four of the metal ion solution containing 10mM, 20mM
In the mixed solution of the oxidant formation of amine, 0.2M, reacts at 90 DEG C and take out after ten minutes, it is careful to wash；
C, using mouse IgG as model object, optical electro-chemistry immunoassays have been carried out: it is anti-to draw the capture of 0.5mg/mL mouse IgG
Body is added drop-wise in 96 microwell plates, is washed 96 microwell plates with Tris-HCl buffer solution after 4 DEG C of overnight incubations；Then, add
Enter 4 DEG C of incubation 2h of bovine serum albumin confining liquid, is washed with Tris-HCl buffer solution；Then, the mouse IgG of various concentration is added
Enter to secure capture antibody microwell plate in, 37 DEG C be incubated for 1 hour after wash；Mouse IgG detection antibody and alkali will have been loaded
Property phosphate Au nano-particle complex be added in microwell plate, 37 DEG C be incubated for 1 hour after wash；Finally, by Vitamin C
Acid phosphoric acid ester is added in the microwell plate of above-mentioned completion immune response, and 37 DEG C are reacted 30 minutes；
D, the electrode for obtaining b step is immersed in a period of time in the solution of step c generation, later carefully washs electrode
Completely；Finally, it is molten to put the electrodes into the Tris buffering that the pH containing 0.05mol/L KCl and 0.1mol/L triethanolamine is 7.0
In liquid, using silver/silver chloride electrode as reference electrode, platinum filament is used as to electrode, is surveyed under+0.1V current potential in homemade optical electro-chemistry
Determine to carry out photoelectric current measurement on instrument.
Certain density CdSO described in step a of the present invention4Solution is 0.5-1.2mol/L；It is adjusted after addition ammonium hydroxide
The range of the pH of reaction solution is 10-13；Metal ion solution described in inventive step b is Fe2+Or Co2+Or Mn2+；Described
Oxidizing agent solution is H2O2Or NaClO.
Detailed description of the invention:
Fig. 1 curve a is the photoelectric current of CdS material modified electrode, and curve b is to be immersed in MnSO4, hexamethylene tetramine,
H2O2Mixed solution in react after be immersed in 1.0 × 10 again-4Mol/L ascorbic acid phosphoric acid esters, the mixing of 5U alkaline phosphatase
In solution after 3min electrode photoelectric current, curve c is to be immersed in MnSO4, hexamethylene tetramine, H2O2Mixed solution in it is anti-
Photoelectric current after answering.
Fig. 2 is the photoelectric current of system with the variation (a-j) of mouse IgG concentration: 0,5.0 × 10-4, 7.0 × 10-4, 1.0 ×
10-3, 1.0 × 10-2, 0.1,1.0,10,50,100ng/mL relational graph；Illustration is linearity curve corresponding with photoelectricity flow graph.
Fig. 3 is the selectivity that this method measures mouse IgG.
Specific embodiment:
Example 1
A, the optoelectronic pole of CdS modification, specific steps are as follows: by the Triton- of 2% mass concentration are prepared using chemical deposition
The CdSO of X 100 solution and 1.0M4It is 13 that ammonia spirit is added after solution mixing and adjusts the pH of solution；Then molten toward above-mentioned reaction
The thiourea solution of 0.5M is added in liquid；The ITO electrode cleaned is immersed in above-mentioned solution at 90 DEG C later and is reacted 5 minutes,
It takes out and rinses electrode, the optoelectronic pole of CdS modification is obtained after drying overnight；
B, CdS modified electrode obtained is immersed in the FeSO containing 10mM4Solution, 20mM hexamethylene tetramine,
The H of 0.2M2O2In the mixed solution of formation, reacts at 90 DEG C and take out after ten minutes, it is careful to wash；
C, using mouse IgG as model object, optical electro-chemistry immunoassays have been carried out: it is anti-to draw the capture of 0.5mg/mL mouse IgG
Body is added drop-wise in 96 microwell plates, is washed 96 microwell plates with Tris-HCl buffer solution after 4 DEG C of overnight incubations；Then, add
Enter 4 DEG C of incubation 2h of bovine serum albumin confining liquid, is washed with Tris-HCl buffer solution；Then, the mouse IgG of various concentration is added
Enter to secure capture antibody microwell plate in, 37 DEG C be incubated for 1 hour after wash；Mouse IgG detection antibody and alkali will have been loaded
Property phosphate Au nano-particle complex be added in microwell plate, 37 DEG C be incubated for 1 hour after wash；Finally, by Vitamin C
Acid phosphoric acid ester is added in the microwell plate of above-mentioned completion immune response, and 37 DEG C are reacted 30 minutes；
D, the electrode for obtaining b step is immersed in a period of time in the solution of step c generation, later carefully washs electrode
Completely；Finally, it is molten to put the electrodes into the Tris buffering that the pH containing 0.05mol/L KCl and 0.1mol/L triethanolamine is 7.0
In liquid, using silver/silver chloride electrode as reference electrode, platinum filament is used as to electrode, is surveyed under+0.1V current potential in homemade optical electro-chemistry
Determine to carry out photoelectric current measurement on instrument.
Example 2
A, the optoelectronic pole of CdS modification, specific steps are as follows: by the Triton- of 2% mass concentration are prepared using chemical deposition
The CdSO of X 100 solution and 0.5M4It is 11 that ammonia spirit is added after solution mixing and adjusts the pH of solution；Then molten toward above-mentioned reaction
The thiourea solution of 0.5M is added in liquid；The ITO electrode cleaned is immersed in above-mentioned solution at 90 DEG C later and is reacted 10 minutes,
It takes out and rinses electrode, the optoelectronic pole of CdS modification is obtained after drying overnight；
B, CdS modified electrode obtained is immersed in the MnSO containing 10mM4Solution, 20mM hexamethylene tetramine,
In the mixed solution that the NaClO of 0.2M is formed, taken out after being reacted 15 minutes at 90 DEG C, it is careful to wash；
C, using mouse IgG as model object, optical electro-chemistry immunoassays have been carried out: it is anti-to draw the capture of 0.5mg/mL mouse IgG
Body is added drop-wise in 96 microwell plates, is washed 96 microwell plates with Tris-HCl buffer solution after 4 DEG C of overnight incubations；Then, add
Enter 4 DEG C of incubation 2h of bovine serum albumin confining liquid, is washed with Tris-HCl buffer solution；Then, the mouse IgG of various concentration is added
Enter to secure capture antibody microwell plate in, 37 DEG C be incubated for 1 hour after wash；Mouse IgG detection antibody and alkali will have been loaded
Property phosphate Au nano-particle complex be added in microwell plate, 37 DEG C be incubated for 1 hour after wash；Finally, by Vitamin C
Acid phosphoric acid ester is added in the microwell plate of above-mentioned completion immune response, and 37 DEG C are reacted 30 minutes；
D, the electrode for obtaining b step is immersed in a period of time in the solution of step c generation, later carefully washs electrode
Completely；Finally, it is molten to put the electrodes into the Tris buffering that the pH containing 0.05mol/L KCl and 0.1mol/L triethanolamine is 7.0
In liquid, using silver/silver chloride electrode as reference electrode, platinum filament is used as to electrode, is surveyed under+0.1V current potential in homemade optical electro-chemistry
Determine to carry out photoelectric current measurement on instrument.
Claims (3)
1. a kind of immunoassay method for reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic, it is characterised in that:
A, the optoelectronic pole of CdS modification, specific steps are as follows: by the Triton-X of 2% mass concentration are prepared using chemical deposition
The CdSO of 100 solution and 0.5-1.2mol/L4It is 10-13 that ammonia spirit is added after solution mixing and adjusts the pH of solution；Then past
The thiourea solution of 0.5M is added in above-mentioned reaction solution；The ITO electrode cleaned is immersed in above-mentioned solution at 90 DEG C later
Reaction 5-15 minutes takes out and rinses electrode, and the optoelectronic pole of CdS modification is obtained after drying overnight；
B, by CdS modified electrode obtained be immersed in the metal ion solution containing 10mM, 20mM hexamethylene tetramine,
In the mixed solution that the oxidant of 0.2M is formed, reacts at 90 DEG C and take out after ten minutes, it is careful to wash；
C, using mouse IgG as model object, optical electro-chemistry immunoassays have been carried out: having drawn 0.5mg/mL mouse IgG capture antibody drop
It is added in 96 microwell plates, is washed 96 microwell plates with Tris-HCl buffer solution after 4 DEG C of overnight incubations；Then, ox is added
4 DEG C of incubation 2h of haemocyanin confining liquid, are washed with Tris-HCl buffer solution；Then, the mouse IgG of various concentration is added to
Secure capture antibody microwell plate in, 37 DEG C be incubated for 1 hour after wash；Mouse IgG detection antibody and alkaline phosphorus will have been loaded
The Au nano particle of acid esters enzyme is added in microwell plate, 37 DEG C be incubated for 1 hour after wash；Finally, ascorbic acid phosphoric acid esters are added
Enter in the microwell plate being immunoreacted to above-mentioned completion, 37 DEG C are reacted 30 minutes；
D, the electrode for obtaining b step is immersed in a period of time in the solution of step c generation, later carefully washs electrode dry
Only；Finally, putting the electrodes into the Tris buffer solution that the pH containing 0.05mol/L KCl and 0.1mol/L triethanolamine is 7.0
In, using silver/silver chloride electrode as reference electrode, platinum filament is used as to electrode, is measured under+0.1V current potential in homemade optical electro-chemistry
Photoelectric current measurement is carried out on instrument.
2. a kind of immunoassay for reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic according to claim 1
Method, it is characterised in that metal ion described in step b is Fe2+Or Co2+Or Mn2+。
3. a kind of immunoassay for reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic according to claim 1
Method, it is characterised in that oxidant described in step b is H2O2Or NaClO.
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