CN100337112C - Manufacturing method of immunity electrode for detection transferrins - Google Patents
Manufacturing method of immunity electrode for detection transferrins Download PDFInfo
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- CN100337112C CN100337112C CNB2005100339774A CN200510033977A CN100337112C CN 100337112 C CN100337112 C CN 100337112C CN B2005100339774 A CNB2005100339774 A CN B2005100339774A CN 200510033977 A CN200510033977 A CN 200510033977A CN 100337112 C CN100337112 C CN 100337112C
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- gold electrode
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- transferrins
- amido
- benzenethiol
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Abstract
The present invention discloses a preparing method of immune electrodes used for transferrin detection, which comprises the steps of the preprocessing of gold electrodes, the formation of 2-amino benzenedithiol oligomer layers, the coupling operation of the gold electrodes and glutaraldehyde, the covalent immobilization of immune reagents of the gold electrodes, etc. The detecting sensitivity of the immune electrodes prepared by the method on the ferrous transferrin can reach the low limit of 0.04 ng/ml.
Description
Technical field:
The invention discloses a kind of preparation method of immunoelectrode, particularly utilize numerator self-assembly technique preparation to be used to detect the method for the immunoelectrode of transferrins.
Background technology:
G.Shen in 2000 and co-worker thereof have reported that at " Analyst " (125 volume 1595-1599 page or leaf) a kind of ampere based on paraffin-graphite-transferrins biological components embedding analyzes immunosensor.They at first are dissolved in paraffin in the ether, add dag then, add the potpourri of transferrins and bovine serum albumin(BSA) again, after the ether volatilization, be pressed in the pvc pipe, make immunoelectrode, the result is limited to 2.36 μ g/ml to the detection of iron content transferrins.Because this method uses mixing of paraffin and graphite and the embedding of transferrins makes immunoelectrode, can't control the optimum response combination of each materials of construction of immunoelectrode on molecular level, thereby limit the more highly sensitive detection of immunoelectrode realization.
Summary of the invention:
Purpose of the present invention is exactly to disclose a kind of method of utilizing numerator self-assembly technique to prepare highly sensitive detection transferrins immunoelectrode.
The preparation method of the immunoelectrode of detection transferrins of the present invention realizes with following technical scheme.At first prepare the gold electrode substrate of surfacing polishing, self assembly last layer 2-amido benzenethiol oligomer in the gold electrode substrate is used glutaraldehyde coupling 2-amido benzenethiol oligomer, at last self assembly one deck transferrins antiserum on glutaraldehyde then then.
The immunoelectrode of detection transferrins of the present invention, because preparation is identification and combination between the molecule on the single molecules level, make immunoelectrode have highly smooth intermolecular interface, electrode surface molecular activity point is neat outwards to be arranged, thereby can combine with the detected material height, the result significantly is increased to 0.04ng/ml to the detection sensitivity of iron content transferrins.
Description of drawings:
Fig. 1 is the preparation principle figure that is used to detect the immunoelectrode of transferrins of the present invention.
Specific embodiment:
High sensitivity is the final requirement of immunoelectrode, below in conjunction with accompanying drawing the preparation that is used for the high sensitivity immunoelectrode of transferrins detection of the present invention is described in further detail.The present invention can select instrument as described below and reagent:
1,970CRT fluorospectrophotometer (Shanghai analytical instrument head factory); UV-1100 ultraviolet-visible pectrophotometer (Beijing Rayleigh Analytical Instrument Co.,Ltd); CHI 660A electrochemical workstation (Shanghai occasion China instrument company); Autolab serial nr.AUT71374 (Eco Chemied B.V.Urecht, The Netherlands), GPES 4.8 current potential process softwares; The accurate pH meter (Shanghai thunder magnetic instrument plant) of PHS-3C type; The DL-120 supersonic wave cleaning machine; 100,000/balance (German Sartorius); Three-electrode system: gold electrode (diameter 1mm) is a working electrode, and saturated calomel electrode is a contrast electrode, and platinum electrode is an auxiliary electrode.
2, iron content transferrins (holo-Tf), purity＞98%, U.S. Sigma company produces; Apotransferrin (apo-Tf), U.S. Sigma company produces; Transferrins antiserum (anti-Tf) was tired 1: 40, and biochemical reagents company in Shanghai produces; Glutaraldehyde (Gludaradehyde, GA) (25% aqueous solution); 2-amido benzenethiol (O-ABT), purity 99%, Zhejiang Shou ﹠ Fu Chemical Co., Ltd.
3, phosphate buffer (PBS) (pH7.4): 8mMNa
2HPO
4-2mMNaH
2PO
4Fe (CN)
6 3-Or Fe (CN)
6 2-The solution Fe (CN) that contains 1mM
6 3-Or Fe (CN)
6 2-PBS solution (pH7.4); Glycocoll, all the other reagent are analysis except that indicating pure, and experimental water is a redistilled water.
The concrete preparation process of immunoelectrode of the present invention is as follows:
1, the pre-service of gold electrode
The gold electrode Al of＜0.05 μ m
2O
3The powder polishing, ultrasonic cleaning 15min in secondary water and ethanol uses 0.5MH at last then
2SO
4Scanning repeatedly in-0.4～1.2V scope is up to the voltammetric signal that obtains repeatability.
These processing procedures have guaranteed that immunoelectrode has a substrate that even structure is smooth.
2, the formation of 2-amido benzenethiol oligomer layer
2-amido benzenethiol solution 99.99%N with 0.5M
2Deoxygenation 5min, clean gold electrode is placed in the 2-amido benzenethiol solution of 10mL0.5M, temperature is 26～32 ℃, after soaking 24h, again gold electrode surfaces is cleaned with ethanol and secondary water, then gold electrode is immersed in the pH7.4PBS solution, scan round is carried out polymerization 25 times between-0.8～0.6V.
3, gold electrode and glutaraldehyde coupling:
The solution that will contain 15% (w/w) glutaraldehyde transfers to pH1.84 with 5%HCl, uses 99.99%N
2Deoxygenation 5min. inserts 24h in the coupling solution with gold electrode, and temperature of reaction is 26～32 ℃, cleans with secondary water more afterwards.
4, the immunoreagent Covalent Immobilization of gold electrode:
Under 4 ℃, the gold electrode that the coupled layer of above-mentioned 2-amido benzenethiol and glutaraldehyde is modified is soaked in that to contain 89ng/mL transferrins antiserum (anti-Tf), pH be 12h in 7.4 the PBS solution, the gold electrode that will embed then behind the antibody is soaked in 5% (v/v) ethylamine solution, till the unreacted carbonyl in complete blocking electrode surface, with after the flushing of secondary water, make immunoelectrode again.
The check of immunoelectrode:
To contain and insert three electrodes in the 5ml electrolytic cell of a certain amount of apotransferrin or iron content transferrins, record the variation of electric current and time with potential step method, draw straight line after data being fitted according to principle of least square method with the Metlab language program then, obtain the capacitance in the linear equation.The result shows: utilize the molecule self assembly, regulate molecular conecentration and built-up time, remove non-specific assembling molecule, thereby make molecule be implemented in the best combination ratio on the single molecules level, reactive group ending proper alignment, active site ratio height, thus realize that immunoelectrode sensitivity significantly improves.The present invention detects apotransferrin first, and obtained so far to the minimum so far detectability of transferrins: apotransferrin is 0.15ng/ml; The iron content transferrins is 0.04ng/ml.Reached the purpose that increases substantially the immunoelectrode detectability, the immunoelectrode that utilizes numerator self-assembly technique to make has solved the problem that how to improve immunoelectrode sensitivity.
Claims (1)
1, a kind of preparation method who is used to detect the immunoelectrode of transferrins, it is characterized in that: described preparation method is undertaken by following concrete steps:
(1) pre-service of gold electrode:
With the alundum (Al powder polishing of gold electrode with＜0.05 μ m, ultrasonic cleaning 15min in secondary water and ethanol scans in-0.4～1.2V scope repeatedly with 0.5M sulfuric acid at last then, up to the voltammetric signal that obtains repeatability;
(2) formation of 2-amido benzenethiol oligomer layer:
2-amido benzenethiol solution 99.99%N with 0.5M
2Deoxygenation 5min, clean gold electrode is placed in the above-mentioned 2-amido benzenethiol solution of 10mL0.5M and soaks 24h, soaking temperature is 26～32 ℃, then gold electrode surfaces is cleaned with ethanol and secondary water, gold electrode is immersed in the phosphate-buffered liquor of pH7.4, scan round is carried out polymerization 25 times between-0.8～0.6V again;
(3) gold electrode and glutaraldehyde coupling:
To contain 15% (w/w) glutaraldehyde solution, transfer to pH1.84, use 99.99%N again with 5% hydrochloric acid
2Get coupling solution behind the deoxygenation 5min., will insert 24h in the above-mentioned coupling solution through the gold electrode after step (2) is handled, temperature of reaction is 26～32 ℃, carries out oligomer and modifies, and cleans with secondary water again;
(4) the immunoreagent Covalent Immobilization of gold electrode:
Under 4 ℃, the gold electrode that the coupled layer of 2-amido benzenethiol and glutaraldehyde is modified be soaked in contain that the 89ng/mL transferrins is sero-fast, pH is 12h in 7.4 the phosphate-buffered liquor, the gold electrode that will embed antibody then is soaked in 5% (v/v) ethylamine solution, till the unreacted carbonyl in complete blocking electrode surface, with the flushing of secondary water, make immunoelectrode again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100339774A CN100337112C (en) | 2005-04-06 | 2005-04-06 | Manufacturing method of immunity electrode for detection transferrins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100339774A CN100337112C (en) | 2005-04-06 | 2005-04-06 | Manufacturing method of immunity electrode for detection transferrins |
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| Publication Number | Publication Date |
|---|---|
| CN1693887A CN1693887A (en) | 2005-11-09 |
| CN100337112C true CN100337112C (en) | 2007-09-12 |
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| CNB2005100339774A Expired - Fee Related CN100337112C (en) | 2005-04-06 | 2005-04-06 | Manufacturing method of immunity electrode for detection transferrins |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1101253C (en) * | 1998-08-25 | 2003-02-12 | 中国科学院长春应用化学研究所 | Single-layer and multilayer molecular assembly system of polyoxometallate |
| US20030119208A1 (en) * | 2001-12-04 | 2003-06-26 | Yoon Hyun Chul | Electrochemical immunosensor and kit and method for detecting biochemical anylyte using the sensor |
-
2005
- 2005-04-06 CN CNB2005100339774A patent/CN100337112C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1101253C (en) * | 1998-08-25 | 2003-02-12 | 中国科学院长春应用化学研究所 | Single-layer and multilayer molecular assembly system of polyoxometallate |
| US20030119208A1 (en) * | 2001-12-04 | 2003-06-26 | Yoon Hyun Chul | Electrochemical immunosensor and kit and method for detecting biochemical anylyte using the sensor |
Non-Patent Citations (2)
| Title |
|---|
| Covalent coupling Immunoglobulin G to self-assembledmonolayers at a method for immobilizing theinterfacial-recognition layer of a surface plasma resonanceimmunosensor Darren M D,Biosensors and Bioelectronics,Vol.13 No.11 1998 * |
| 电流型转铁蛋白免疫传感器的研究 刘成霞，曹轩，雷存喜，沈国励，俞汝勤，分析化学研究报告，第32卷第9期 2004 * |
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| CN1693887A (en) | 2005-11-09 |
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