CN104165913A - Electrochemical sensor for detecting osteopontin, and construction method thereof - Google Patents
Electrochemical sensor for detecting osteopontin, and construction method thereof Download PDFInfo
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- CN104165913A CN104165913A CN201410210922.5A CN201410210922A CN104165913A CN 104165913 A CN104165913 A CN 104165913A CN 201410210922 A CN201410210922 A CN 201410210922A CN 104165913 A CN104165913 A CN 104165913A
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Abstract
The invention relates to an electrochemical sensor for detecting osteopontin, and a construction method thereof. The sensor is a three-electrode system with a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode and a modified gold electrode as a working electrode. Dextran amine used as a common three-dimensional material connection agent has the following two advantages: 1, protein denaturation and nonspecific binding are reduced; and 2, a composite three-dimensional sensing interface formed by connecting the amino group of the dextran amine with antibody directed polypeptides has good antibody binding capacity and many fixed sites. The novel electrochemical biosensor is made by combining the respective advantages of an electrochemical technology and a three-dimensional material in order to realize the highly-specific quantitative detection of the osteopontin.
Description
Technical field
The present invention utilizes a kind of electrochemical sensor and construction method thereof that detects osteopontin.
Background technology
1979, the reported first such as Senger are a kind of comprise RGD integrin land and the phosphorylation glycoprotein relevant with vicious transformation and with the relation of tumour, be referred to as to transform correlativity phosphoric acid albumen.Franzen etc. isolated a kind of phosphoric acid albumen from bone matrix and tooth afterwards, characteristic with transform correlativity phosphoric acid protein similar, people are its called after Osteopontin (OPN), the transfer that it can modulate tumor.After secretion, OPN can participate in the activity of many tumours, for example: tumor vessel generates, invades, adheres to, moves, escapes identification/destruction immune system, inhibition tumor cell apoptosis.Its normal expression level in heart and blood vessel is very low, still, if continue to increase its expression, will cause the diseases such as cancer of the stomach, breast cancer, cancer of the esophagus, kidney, the straight cancer of colon and uterus cervical carcinoma.
In recent years, OPN is because can be used as the diagnosis marker of some diseases and the concern that therapeutic purpose has caused Many researchers.Therefore, some detect the method for OPN and are constantly reported, up to now, ELISA method is to detect the most frequently used method of OPN, but due to ELISA method, need to expend a large amount of time and sample and sensitivity not high, therefore, find out a kind of novelty, sensitive, method detects OPN and is very important easily.
Electrochemical sensor has the advantages such as selectivity is high, detection limit is low.In electrochemical immunosensor, immune complex (antibody) is fixed on electrode conventionally indirectly.Some special molecules are often used as coupling agent and come connecting electrode and antibody, and dextran amine, as one of a kind of modal coupling agent, is widely used in the fixing of protein.It is a kind of amino that is rich in, by 1, the 6 linear macromolecule polymkeric substance being connected to form between glucose molecule.Dextran amine has two large advantages: the first, and it not only can reduce the probability of protein denaturation, and can avoid the non-specific binding of part and sensor surface as far as possible, is effective barrier between biomolecule and gold substrate; The second, the complex three-dimensional sensing interface that the directed polypeptide of the amino of dextran amine and antibody is connected to form has good antibody binding ability and more fixedly site.Yet the carboxyl of protein and the direct reaction of the amino of dextran amine may destroy the active region of protein, therefore, many scientists are striving to find a kind of molecule that can be combined in specifically protein stabilization region.The Fc end of antibody molecule is its stable region, and Fab end is conjugated antigen stably.Carbonell etc. have found six new aggressiveness small peptide HWRGWV, can be incorporated into the Fc end of IgG.And the affinity of this small peptide binding is lower than protein, and this feature becomes the advantage that this small peptide is applied to other system.At present, dextran amine and small peptide HWRGWV have been applied in the research in a plurality of fields, and have obtained good effect.
Summary of the invention
One of object of the present invention is to provide a kind of electrochemical sensor that detects osteopontin, this sensor contains the three-dimensional system that the particular molecule such as dextran amine and small peptide form, and modified and build electrochemical immunosensor on gold electrode and detect OPN, and as the using method of OPN electrochemical immunosensor.
Two of object of the present invention is to provide the construction method of this sensor.
A kind of electrochemical sensor that detects osteopontin, three-electrode system, platinum electrode is as to electrode, saturated calomel electrode is as contrast electrode, after modifying, gold electrode is as working electrode, after it is characterized in that described modification, gold electrode has three-dimensional structure, first by golden mercapto key, in gold electrode surfaces, modify ω-sulfydryl undecanoic acid (MUA) and 6-sulfydryl hexanol (MCH), recycling is amino is connected three-dimensional material dextran amine with the cross-linking reaction of carboxyl, the carboxyl of small peptide is connected with the amino of dextran amine, osteopontin antibody is connected with small peptide by Fc end, obtain modifying rear gold electrode, the sequence of described small peptide is: Acetylated-HWRGWVA.
Build a method for the electrochemical sensor of above-mentioned detection osteopontin, it is characterized in that the construction method of sensor is:
A. the formation of 3-d modelling substrate:
A-1. the gold electrode of handling well is immersed in the mixed solution of ω-sulfydryl undecanoic acid (MUA) and 6-sulfydryl hexanol (MCH) and spends the night; Then with absolute ethyl alcohol and ultrapure water, rinse well and dry up stand-by respectively
A-2. step a-1 gained gold electrode is soaked 20 minutes in the mixed liquor of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), then this gold electrode is placed in the dextran amine solution that concentration is 0.1mg/mL and is hatched 30 minutes; Finally with monoethanolamine, carry out shrouding;
A-3. the gold electrode to step a-2 gained is immersed in the solution of 0.5mg/mL small peptide, reaction 12-15 hour; Form the 3-d modelling substrate of sensor;
B. step a gained gold electrode is immersed in the osteopontin antibody solution of 10 μ g/mL and jointly hatches 2 h, with fixing Anti-OPN, finally obtain modifying rear gold electrode;
C. after step b gained being modified, gold electrode forms three electrode sensors together with saturated calomel electrode with platinum electrode, is the electrochemical sensor that detects osteopontin.
The disposal route of the gold electrode of above-mentioned step a-1 is specially: with ultrapure water, gold electrode is fully rinsed to clean state, and dry up; Then, use respectively 3000,5000 object sand paperings, and containing different grain size l μ m successively, 0.3 μ m, polishing electrode in the alumina mortar of 0.05 μ m, until electrode surface is the seamless shape of minute surface, after clean with ultrapure water and alcohol flushing, then, to electrode surface, drip 20 μ L piranha, i.e. 98% H
2sO
4: 30% H
2o
2=3:1 solution; After 5 minutes, with ultrapure water, clean up; Finally, electrode is put into 0.5 M H
2sO
4in, with cyclic voltammetry scanning (0 V-+1.6V) to signal stabilization), with ultrapure water rinse well, nitrogen dries up rear stand-by.
Advantage and disadvantage of the present invention is as described below: the present invention utilizes the gold electrode that a kind of NEW TYPE OF COMPOSITE three-dimensional material is modified to build a kind of electrochemical sensor that detects osteopontin.Dextran amine is as one of a kind of modal coupling agent, be widely used for fixing protein, dextran amine has two large advantages: first, it not only can reduce the probability of protein denaturation, and can avoid the non-specific binding of part and sensor surface as far as possible, be effective barrier between biomolecule and gold substrate; The second, the complex three-dimensional sensing interface that the directed polypeptide of the amino of dextran amine and antibody is connected to form has good antibody binding ability and more fixedly site.With one section of small peptide, connect dextran amine and antibody again, thereby while having avoided the carboxyl of protein and the amino of dextran amine directly to react, destroy the active region of protein.The advantage of combined with electrochemical technology of the present invention and three-dimensional material, prepares a kind of novel electrochemica biological sensor, by AC impedance method, OPN is carried out to high specific and quantitatively detects.
Accompanying drawing explanation
Fig. 1 is under top condition, adds the AC impedance figure of the OPN solution of variable concentrations.
Embodiment
After now specific embodiments of the invention being described in.
embodiment 1
In the present embodiment, the construction method of 3-d modelling sensor and step are as follows:
1, the formation of 3-d modelling substrate:
A. the pre-service of gold electrode: first, with ultrapure water, electrode is fully rinsed to clean state, and dry up electrode surface with ear washing bulb; Then, the electrode of polishing on 3000,5000 object sand paper respectively, and containing different grain size (l μ m successively, 0.3 μ m, 0.05 μ m) polishing electrode in alumina mortar, until electrode surface is the seamless shape of minute surface, after rinsing well with ultrapure water, use successively again each ultrasonic cleaning of ethanol and ultrapure water 5 minutes, then, to electrode surface, drip 20 μ L piranha (98% H
2sO
4: 30% H
2o
2=3:1) solution, cleaned up with ultrapure water after 5 minutes.Finally, electrode is put into 0.5 M H
2sO
4in, with cyclic voltammetry scan (0 V-+1.6V) to signal stabilization (approximately 40 circle), with ultrapure water rinse well, nitrogen dries up rear stand-by.
B. the formation of self assembled monolayer: the electrode of handling well is immersed in immediately in ω-sulfydryl undecanoic acid (MUA) & 6-sulfydryl hexanol (MCH) mixed solution and is spent the night.Then with absolute ethyl alcohol and ultrapure water, rinse well respectively and with nitrogen, dry up rear for next step.
C. prepare the electrode that three-dimensional material is modified:
(1) electrode of modified is soaked 20 minutes in the mixed liquor of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) & N-hydroxy-succinamide (NHS), in order to activate the carboxyl of MUA end.Activation finishes, and electrode is placed in dextran amine solution and is hatched 30 minutes.In order to stop other non-specific molecules in conjunction with the carboxyl of activation, with monoethanolamine, the electrode of above-mentioned processing is carried out to shrouding.
(2) to the electrode surface after shrouding, drip the solution that contains small peptide Acetylated-HWRGWVA, at least react 12 hours.The carboxyl of small peptide Acetylated-HWRGWVA and the amino of dextran amine form covalency amido link and are combined in electrode surface, form the 3-d modelling substrate of OPN detecting sensor.
2, the detection of the fixing and OPN of antibody
Certain density OPN antibody (Anti-OPN) and the above-mentioned electrode of having modified three-dimensional material are hatched jointly, with fixing Anti-OPN, and utilize the specific reaction of antigen-antibody, detect target protein (OPN).After being modified by above-mentioned structure gold electrode as working electrode, platinum electrode as to electrode, saturated calomel electrode as contrast electrode, form traditional three-electrode system; During detection, AC impedance value when three-electrode system is placed in to potassium ferricyanide solution and measures variable concentrations OPN.
Electrochemical Detection OPN:
Adopt OPN concentration to be respectively 2.27 * 10
-9, 3.41 * 10
-9, 5.68 * 10
-9, 9.08 * 10
-9, 13.62 * 10
-9, 20.43 * 10
-9m/L.
Test condition: using build modify after gold electrode as working electrode, platinum electrode as to electrode, saturated calomel electrode as contrast electrode, form traditional three-electrode system; During detection, AC impedance value when three-electrode system is placed in to potassium ferricyanide solution and measures variable concentrations OPN.
Referring to accompanying drawing, Fig. 1 is under top condition, adds the AC impedance figure of the OPN solution of variable concentrations.
As seen from the figure, along with increasing of OPN concentration, impedance (Ω) also presents ascendant trend.
OPN electrochemical immunosensor prepared by the inventive method has unmarked, higher sensitivity, the good advantages such as selectivity.Not only for OPN detects, provide a kind of good method, also the detection for other tumor markers provides a good platform, and promotes the development of early diagnosis of tumor and treatment.
Claims (3)
1. an electrochemical sensor that detects osteopontin, three-electrode system, platinum electrode is as to electrode, saturated calomel electrode is as contrast electrode, after modifying, gold electrode is as working electrode, after it is characterized in that described modification, gold electrode has three-dimensional structure, first by golden mercapto key, in gold electrode surfaces, modify ω-sulfydryl undecanoic acid (MUA) and 6-sulfydryl hexanol (MCH), recycling is amino is connected three-dimensional material dextran amine with the cross-linking reaction of carboxyl, the carboxyl of small peptide is connected with the amino of dextran amine, osteopontin antibody is connected with small peptide by Fc end, obtain modifying rear gold electrode, the sequence of described small peptide is: Acetylated-HWRGWVA.
2. build a method for the electrochemical sensor of detection osteopontin according to claim 1, it is characterized in that the construction method of sensor is:
A. the formation of 3-d modelling substrate:
A-1. the gold electrode of handling well is immersed in the mixed solution of ω-sulfydryl undecanoic acid (MUA) and 6-sulfydryl hexanol (MCH) and spends the night; Then with absolute ethyl alcohol and ultrapure water, rinse well and dry up stand-by respectively
A-2. step a-1 gained gold electrode is soaked 20 minutes in the mixed liquor of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS), then this gold electrode is placed in the dextran amine solution that concentration is 0.1mg/mL and is hatched 30 minutes; Finally with monoethanolamine, carry out shrouding;
A-3. the gold electrode to step a-2 gained is immersed in the solution of 0.5mg/mL small peptide, reaction 12-15 hour; Form the 3-d modelling substrate of sensor;
B. step a gained gold electrode is immersed in the osteopontin antibody solution of 10 μ g/mL and jointly hatches 2 h, with fixing Anti-OPN, finally obtain modifying rear gold electrode;
C. after step b gained being modified, gold electrode forms three electrode sensors together with saturated calomel electrode with platinum electrode, is the electrochemical sensor that detects osteopontin.
3. method according to claim 2, is characterized in that the disposal route of the gold electrode of described step a-1 is specially: with ultrapure water, gold electrode is fully rinsed to clean state, and dry up; Then, use respectively 3000,5000 object sand paperings, and containing different grain size l μ m successively, 0.3 μ m, polishing electrode in the alumina mortar of 0.05 μ m, until electrode surface is the seamless shape of minute surface, after clean with ultrapure water and alcohol flushing, then, to electrode surface, drip 20 μ L piranha, i.e. 98% H
2sO
4: 30% H
2o
2=3:1 solution; After 5 minutes, with ultrapure water, clean up; Finally, electrode is put into 0.5 M H
2sO
4in, with cyclic voltammetry scanning (0 V-+1.6V) to signal stabilization), with ultrapure water rinse well, nitrogen dries up rear stand-by.
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Cited By (10)
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| CN104459123A (en) * | 2014-12-04 | 2015-03-25 | 济南大学 | Preparation method and application of avian leukemia virus antigen immunosensor |
| CN104502593A (en) * | 2015-01-25 | 2015-04-08 | 济南大学 | Preparation method of electrochemical mark-free immunosensor of gastric cancer tumor marker |
| CN104569435A (en) * | 2015-01-25 | 2015-04-29 | 济南大学 | Preparation method of unlabelled photoelectrochemical alpha fetoprotein immunosensor |
| CN104698165A (en) * | 2015-03-20 | 2015-06-10 | 济南大学 | Preparation method for platinum-base alloy-labeled three-channel sandwich type immunosensor and application |
| CN110003272A (en) * | 2019-03-11 | 2019-07-12 | 南京理工大学 | Functionalization quaternary alkylphosphonium salt self-assembled material and its biosensor |
| CN110057898A (en) * | 2019-04-15 | 2019-07-26 | 华南师范大学 | A kind of electrochemical detection method of high concentration denaturation bovine serum albumin(BSA) |
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| CN101066999A (en) * | 2006-03-17 | 2007-11-07 | 上海中信国健药业有限公司 | Recombinant anti-OPN monoclonal antibody and its prepn and use |
| CN101787393A (en) * | 2009-12-24 | 2010-07-28 | 上海吉凯基因化学技术有限公司 | Kit for rapidly diagnosing three transcripts of osteopontin (OPN) and use method thereof |
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Patent Citations (3)
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| CN101066999A (en) * | 2006-03-17 | 2007-11-07 | 上海中信国健药业有限公司 | Recombinant anti-OPN monoclonal antibody and its prepn and use |
| US20110180408A1 (en) * | 2007-11-27 | 2011-07-28 | Youssouf Badal | Enhanced method for detecting and/or quantifying an analyte in a sample |
| CN101787393A (en) * | 2009-12-24 | 2010-07-28 | 上海吉凯基因化学技术有限公司 | Kit for rapidly diagnosing three transcripts of osteopontin (OPN) and use method thereof |
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| CN104459123A (en) * | 2014-12-04 | 2015-03-25 | 济南大学 | Preparation method and application of avian leukemia virus antigen immunosensor |
| CN104459123B (en) * | 2014-12-04 | 2015-08-19 | 济南大学 | A kind of preparation method of avian leukosis virus antigen immune sensor and application |
| CN104502593A (en) * | 2015-01-25 | 2015-04-08 | 济南大学 | Preparation method of electrochemical mark-free immunosensor of gastric cancer tumor marker |
| CN104569435A (en) * | 2015-01-25 | 2015-04-29 | 济南大学 | Preparation method of unlabelled photoelectrochemical alpha fetoprotein immunosensor |
| CN104569435B (en) * | 2015-01-25 | 2015-10-21 | 济南大学 | A kind of preparation method of unmarked Optical Electro-Chemistry alpha-fetoprotein immunosensor |
| CN104502593B (en) * | 2015-01-25 | 2015-10-21 | 济南大学 | The preparation method of the unmarked immunosensor of a kind of galvanochemistry stomach neoplasms tumor markers |
| CN104698165A (en) * | 2015-03-20 | 2015-06-10 | 济南大学 | Preparation method for platinum-base alloy-labeled three-channel sandwich type immunosensor and application |
| CN104698165B (en) * | 2015-03-20 | 2015-12-02 | 济南大学 | A kind of preparation method of triple channel sandwich type immunosensor of platinum base alloy mark and application |
| CN111868529A (en) * | 2018-04-25 | 2020-10-30 | 松下知识产权经营株式会社 | Sensor substrate, method for manufacturing sensor substrate, and detection device |
| CN110003272A (en) * | 2019-03-11 | 2019-07-12 | 南京理工大学 | Functionalization quaternary alkylphosphonium salt self-assembled material and its biosensor |
| CN110003272B (en) * | 2019-03-11 | 2021-05-07 | 南京理工大学 | Functional quaternary phosphonium salt self-assembly material and biosensor thereof |
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| CN110057898B (en) * | 2019-04-15 | 2022-01-28 | 华南师范大学 | Electrochemical detection method of high-concentration denatured bovine serum albumin |
| CN111380933A (en) * | 2020-03-23 | 2020-07-07 | 山东农业大学 | Electrochemical immunosensor for detecting bombyx mori nuclear polyhedrosis virus and detection method thereof |
| CN111812183A (en) * | 2020-07-27 | 2020-10-23 | 山东理工大学 | Preparation method and application of intramolecular photo-electrochemical sensor |
| CN112684182A (en) * | 2020-12-15 | 2021-04-20 | 上海大学 | Immunosensor system for detecting PD-L1 in non-disease diagnosis |
| CN112684182B (en) * | 2020-12-15 | 2022-06-28 | 上海大学 | Immunosensor system for detecting PD-L1 in non-disease diagnosis |
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