CN101158686A - Electrochemical sensor used for detecting blood fluke, preparation method and applications - Google Patents
Electrochemical sensor used for detecting blood fluke, preparation method and applications Download PDFInfo
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- CN101158686A CN101158686A CNA2007100360651A CN200710036065A CN101158686A CN 101158686 A CN101158686 A CN 101158686A CN A2007100360651 A CNA2007100360651 A CN A2007100360651A CN 200710036065 A CN200710036065 A CN 200710036065A CN 101158686 A CN101158686 A CN 101158686A
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- gold electrode
- mercaptoethylmaine
- schistosome
- electrochemical sensor
- detect
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Abstract
The invention discloses an electrochemistry sensor used for detecting schistosome. The invention comprises an Au electrode, a mercaptoethylamine self-assembling layer; a glutaraldehyde cross-linked layer and a schistosome antigen fixed layer are covered on the Au electrode orderly. The invention has the advantages that the detecting sensitivity is high, the speed of LOD low detecting is fast and the specificity is good. The reagent and the instrument which are needed in the detecting are portable and have low cost.
Description
Technical field
The invention belongs to the bioanalysis field, be specifically related to a kind of electrochemical sensor that is used to detect.
Background technology
Snail fever is a kind of tropical disease that is caused by blood fluke.Main blood fluke kind of causing a disease has 5 kinds, is respectively Schistosoma haematobium, Schistosoma mansoni, and Schistosoma japonicum interleaves blood fluke, the public blood fluke of river bank.According to the WHO report, the infected snail fever that goes up of 2,000,000,000 people that have an appointment every year.Schistosoma japonicum is mainly popularly in the area, Asia a kind ofly to parasitize the great a kind of parasitic disease of the caused harmfulness of human body by blood fluke.
Diagnosis is a key link of treatment snail fever.So diagnostic techniques is an effectively condition precedent of control snail fever fast and accurately.Be by whether having schistosome ovum in the microscopic examination excreta at present, but the sensitivity of this method is low, is difficult to detect slight patient to the classical detection method of this disease.The immune response of schistosome antibody and egg antigen is having very big application prospect aspect clinical practice and the epidemic research.The detection method that is used for schistosome antibody at present mainly contains ring ovum precipitation experiment (COPT), red cell agglutination is tested (IHA), cercarian huellen reaction (CHR), counter immunoelectrophoresis (CIE), immunoenzymatic assay (EIA), indirect fluorescent antibody experiment (IFAT), PEG precipitation experiment (PEGPT), radiommunoassay (RIA) etc. indirectly.But still there is problem in various degree in these detection methods, as consuming time, detection sensitivity is high inadequately.
With respect to above method, has immunoreactive specificity based on the bilharzial electrochemical immunoanalytical technology of enzymatic metal deposition amplification detection, have the convenient and swift of Electrochemical Detection again, add that the amplifying technique of enzymatic metal deposition makes that the detection of snail fever is sensitive more, accurate.Even the patients serum is diluted up to ten thousand times, also can detect.It is simple, cheap to test required instrument, easy and simple to handlely need not the professional and technical personnel.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of amplifying technique simple, sensitive, that utilize the enzymatic metal to deposit to detect bilharzial electrochemical sensor and preparation method and application.
For solving the problems of the technologies described above, the present invention by the following technical solutions:
A kind ofly be used to detect bilharzial electrochemical sensor, comprise: gold electrode, and mercaptoethylmaine self assembly layer, glutaraldehyde cross-linking layer and schistosome antigen fixed bed on covering successively on the gold electrode.
A kind ofly prepare the above-mentioned method that is used to detect bilharzial electrochemical sensor, may further comprise the steps:
1, the processing of gold electrode
Gold electrode is used the ultrasonic cleaning of second alcohol and water respectively after polishing with alumina powder, and is dry in nitrogen then.
2, the self assembly of mercaptoethylmaine
The gold electrode that above-mentioned pre-service is good is immersed in the ethanolic solution that contains mercaptoethylmaine, makes it form the self assembled monolayer (SAM) of mercaptoethylmaine in gold electrode surfaces.
3, the connection of glutaraldehyde and schistosome antigen is fixing
With above-mentioned surface coverage the gold electrode of mercaptoethylmaine self assembled monolayer place glutaraldehyde water solution, after reaction is finished, washing, drip phosphate buffer solution (the PB solution that contains schistosome antigen in gold electrode surfaces, pH7.3), after reaction is finished, drip BSA solution and seal unreacted glutaraldehyde, clean gold electrode, the interface of sensor has just prepared like this.
Described being used to detected the method that bilharzial electrochemical sensor detects schistosome antibody, may further comprise the steps:
1, immune response: the serum that has schistosome antibody of the infected body after will diluting drips in gold electrode surfaces, after reaction is finished, takes out cleaning electrode;
2, enzymatic deposition of silver: drip alkali phosphorus enzyme labeling goat-anti rabbit or people's antibody-solutions (AH-ALP) at electrode surface, after reaction is finished, electrode is fully cleaned up, (pH9.08 includes 1mMAgNO then electrode to be immersed in glycine buffer
3, 1mM ascorbic acid 2-phosphate (AA-P)) in, after reaction is finished, electrode is cleaned up;
3, Electrochemical Detection: (LSV) detects with linear sweep voltammetry, and potential range is 0～0.8V (vs.SCE), and sweep velocity is 100mV/s, and the supporting electrolyte of electrochemistry experiment is 0.6M KNO
3/ 0.1M HNO
3, experimental temperature is controlled at room temperature and gets final product, and whole testing process only needs 1 minute.
4, data analysis: schistosome antibody concentration becomes the good linear relation in the dynamics dilution range in 1: 12500～1: 100 in LSV peak current signal and the serum, detects lower limit and can reach 1: 10186.The data and the typical curve of gained LSV peak current signal are contrasted, according to typical curve diagnosable go out sick body whether be subjected to infection by Schistosoma and infected degree.
Advantage of the present invention:
1, consumptive material is few.In the experimentation, each sample and required reagent all are several microlitres.Cooperate chip technology, this method will realize high throughput testing, be fit to very much the examination of the extensive sample in epidemic-stricken area.2, detection sensitivity height, it is low to detect lower limit.Traditional ELISA method just can't have been measured signal after serum is diluted more than 300 times, and dilution ratio can be brought up to 1.25 * 10 based on the detection method that enzymatic metal deposition is amplified
5Detect lower limit and reached 1: 10186 dilution ratio.3, detection speed is fast.To be deposited on the silver-colored stripping on the electrode and read peak current 60 seconds the time that only needs with the LSV method, all more a lot of rapidly than general detection means.4, specificity is good.This sensor does not all have obviously response to contained other protein in the human body as fibrin ferment, protoheme, immunoglobulin G etc.5, it is portable and cheap to detect required reagent and instrument.
Embodiment
Embodiment 1: be used to detect the sensor preparation of schistosome antibody
1, the processing of gold electrode
After polishing with 0.3 μ m alumina powder, gold electrode, uses ethanol and redistilled water ultrasonic cleaning 5 minutes then respectively again with the polishing of 0.05 μ m alumina powder.It is dry in nitrogen that water cleans the back.
2, the self assembly of mercaptoethylmaine
The gold electrode that above-mentioned pre-service is good is immersed in (10mM mercaptoethylmaine) 10h in the ethanol solution that contains mercaptoethylmaine, makes it form the self assembled monolayer (SAM) of mercaptoethylmaine in gold electrode surfaces.
3, the connection of glutaraldehyde and schistosome antigen is fixing
The glutaraldehyde water solution that the gold electrode of the self assembled monolayer of mercaptoethylmaine on the above-mentioned surface coverage is placed 2.5% (v/v) is behind 37 ℃ of reaction 1h, washing, electrode surface drip PB (pH7.3) solution that 4 μ L contain 0.1 μ M schistosome antigen at 37 ℃ in calibration cell reaction 1h, drip 1mg/mL BSA solution in 37 ℃ of reaction 1h, seal unreacted glutaraldehyde.With 1/15M PB and redistilled water cleaning electrode several.The interface of sensor has just prepared like this.
Embodiment 2: the detection of infection by Schistosoma rabbit anteserum sample
Drip infection by Schistosoma rabbit anteserum after the 4 μ L dilution in electrode surface.Place 37 ℃ of calibration cells to react 1h, take out, with 1/15M PB and redistilled water cleaning electrode.Drip 4 μ L alkali phosphorus enzyme labeling goat anti-rabbit antibody solution (AR-ALP) (with the 1/15M that contains 1mg/mL BSA, the PB of pH7.3 does 50 times of dilutions with 1mg/mL alkali phosphorus enzyme labeling goat anti-rabbit antibody solution) at 37 ℃ of reaction 1h at electrode surface.Electrode is fully cleaned up with PB, deionized water, and (pH9.08 includes 1mM AgNO then electrode to be immersed in the 50mM glycine buffer of now joining
3, 1mM ascorbic acid 2-phosphate (AA-P)) at 37 ℃ in dark place reaction 30min.After the reaction electrode is cleaned up with ultrapure water.(LSV) detects with linear sweep voltammetry, and potential range is 0～0.8V (vs.SCE), and sweep velocity is 100mV/s.The supporting electrolyte of electrochemistry experiment is 0.6M KNO
3/ 0.1M HNO
3Experimental temperature all is controlled at room temperature.The result shows, schistosome antibody concentration becomes the good linear relation in the dynamics dilution range in 1: 12500～1: 100 in LSV peak current signal and the serum, detects lower limit and can reach 1: 10186.The data and the typical curve of gained LSV peak current signal are contrasted, according to typical curve diagnosable go out sick body be subjected to infection by Schistosoma and infected degree.In the 10 routine rabbit anteserum samples, 5 examples are positive, and explanation is the infection by Schistosoma rabbit anteserum, and 5 examples are negative, and explanation is not infect bilharzial rabbit anteserum.Can carry out accurate, sensitive detection to infection by Schistosoma rabbit anteserum sample based on the schistosome antibody detection technique that enzymatic metal deposition is amplified.
Embodiment 3: infection by Schistosoma human serum sample's detection
Utilize this enzymatic metal deposition amplification detection technology that the 20 routine suspicious patients' in epidemic-stricken area blood serum sample is detected.Suspicious patient's blood serum sample is added drop-wise on the gold electrode sensing device that is fixed with schistosome antigen 37 ℃ hatches 60min, after 60min is sealed with 1mg/mL BSA in unreacted site, with PB (pH7.3) buffer solution for cleaning sensor surface, again the goat anti-human igg of itself and ALP mark is hatched 60min in 37 ℃.Electrode is fully cleaned up with PB, deionized water, and (pH9.08 includes 1mM AgNO then electrode to be immersed in the 50mM glycine buffer of now joining
3, 1mM ascorbic acid 2-phosphate (AA-P)) at 37 ℃ in dark place reaction 30min.After the reaction electrode is cleaned up with ultrapure water.At last, on electrochemical workstation, measure peak current with linear sweep voltammetry and compare with working curve.In 20 routine suspicious patients' the blood serum sample, 5 examples are negative, and explanation is that blood fluke does not infect serum, and 15 examples are positive, and explanation is an infection by Schistosoma serum.The result shows, can the human serum sample of infection by Schistosoma be measured fast and accurately based on the schistosome antibody detection technique that enzymatic metal deposition is amplified.
Claims (3)
1. one kind is used to detect bilharzial electrochemical sensor, it is characterized in that comprising gold electrode, and mercaptoethylmaine self assembly layer, glutaraldehyde cross-linking layer and schistosome antigen fixed bed on covering successively on the gold electrode.
2. one kind prepares the described method that is used to detect bilharzial electrochemical sensor of claim 1, may further comprise the steps:
A, gold electrode clean up after polishing with alumina powder, and be dry in nitrogen then;
B, the gold electrode that above-mentioned pre-service is good are immersed in the ethanolic solution that contains mercaptoethylmaine, make it form the self assembled monolayer of mercaptoethylmaine in gold electrode surfaces;
C, with above-mentioned surface coverage the gold electrode of mercaptoethylmaine self assembled monolayer place glutaraldehyde water solution, after reaction is finished, washing, drip the phosphate buffer solution that contains schistosome antigen in gold electrode surfaces, after reaction is finished, drip BSA solution and seal unreacted glutaraldehyde, clean gold electrode, make sensor of the present invention.
3. the described electrochemical sensor of claim 1 is used to detect schistosome antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100360651A CN101158686A (en) | 2007-11-07 | 2007-11-07 | Electrochemical sensor used for detecting blood fluke, preparation method and applications |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100360651A CN101158686A (en) | 2007-11-07 | 2007-11-07 | Electrochemical sensor used for detecting blood fluke, preparation method and applications |
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| Publication Number | Publication Date |
|---|---|
| CN101158686A true CN101158686A (en) | 2008-04-09 |
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| CNA2007100360651A Pending CN101158686A (en) | 2007-11-07 | 2007-11-07 | Electrochemical sensor used for detecting blood fluke, preparation method and applications |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197054A (en) * | 2013-04-07 | 2013-07-10 | 中国科学院上海应用物理研究所 | Antigens jointly assembled sensing interface |
| CN105891465A (en) * | 2014-11-11 | 2016-08-24 | 中南大学 | Method for diagnosis of circulating antigen of schistosomiasis japonica |
-
2007
- 2007-11-07 CN CNA2007100360651A patent/CN101158686A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197054A (en) * | 2013-04-07 | 2013-07-10 | 中国科学院上海应用物理研究所 | Antigens jointly assembled sensing interface |
| CN105891465A (en) * | 2014-11-11 | 2016-08-24 | 中南大学 | Method for diagnosis of circulating antigen of schistosomiasis japonica |
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Open date: 20080409 |