CN103940880A - Electrochemical biosensor as well as preparation method, application and detection method - Google Patents
Electrochemical biosensor as well as preparation method, application and detection method Download PDFInfo
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- CN103940880A CN103940880A CN201410193708.3A CN201410193708A CN103940880A CN 103940880 A CN103940880 A CN 103940880A CN 201410193708 A CN201410193708 A CN 201410193708A CN 103940880 A CN103940880 A CN 103940880A
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Abstract
The invention relates to an electrochemical biosensor as well as a preparation method, application and a detection method. According to the preparation method, gold nanoparticles (AuNPs) are synthesized by virtue of a sodium citrate reduction method, a probe marked with ferrocene (Fc) and a probe capable of forming simulated DNA (deoxyribonucleic acid) enzyme are assembled to the surface of a gold electrode by virtue of a complementary pairing action of base among DNA sequences, so as to prepare the electrochemical biosensor capable of catalyzing the simulated enzyme and amplifying the gold nanoparticles based on the driving of the ferrocene. By utilizing the electrochemical biosensor, the sensitivity, the specificity and the stability of a target potassium ion are detected.
Description
Technical field
The present invention relates to biosensor technology field, be specifically related to build based on ferrocene drive analog D NA enzyme catalysis and the electrochemica biological sensor of golden nanometer particle amplification realize the Sensitive Detection to potassium ion.
Background technology
In recent years, there is the DNA enzyme of catalytic performance, because of its easy preparation, assembling and hydrolysis and heat-staple character, become the focus of research.In the research field of DNA enzyme, further find that G-tetrad horseradish peroxidase analog D NA enzyme has character like peroxidase, it can the reaction of catalysis taking hydrogen peroxide as substrate.Based on the catalytic property of analogue enztme, analog D NA enzyme has been widely used in amplification detection signal.Traditional enzyme (as horseradish peroxidase, glucose oxidase etc.) has been proved to be with electron mediator in addition good Electron Transfer.Electron mediator in relevant research but, about with analogue enztme still little in conjunction with the tactful report of electron mediator amplifying signal.
Summary of the invention
The object of the present invention is to provide a kind of analog D NA enzyme catalysis driving based on ferrocene and the electrochemica biological sensor of golden nanometer particle amplification to realize the detection to potassium ion, the present invention uses sodium citrate reducing process to synthesize golden nanometer particle (AuNPs), utilize the complementary pairing effect of base between DNA sequence dna, by mark ferrocene (Fc) probe with can form the probe assembling of analog D NA enzyme to the surface of gold electrode, prepare based on ferrocene drive analog D NA enzyme catalysis and the electrochemica biological sensor of golden nanometer particle amplification, this sensor has been realized target potassium ion sensitivity, specificity, the detection of stability.Concrete technical scheme is as follows:
A preparation method for electrochemica biological sensor, comprises the steps:
(1) DNA sequence dna is dissolved respectively in Tris-HCl buffer solution, and save backup;
(2) buffer solution that contains probe S1 is dripped and is coated onto on gold electrode, cultivate, the gold electrode of modifying by Au-S covalent bond effect self assembly S1;
(3) with step (2) simultaneously, probe S2 joined in the buffer solution that contains AuNPs and cultivate this mixed solution, preparation S2-AuNPs biological composite;
(4) gold electrode of S1 being modified is put in the buffer solution that contains S2-AuNPs biological composite and S3 and is cultivated, and utilizes the base complementrity pairing effect between DNA, prepares electrochemica biological sensor.
Further, between step (1) and (2), also comprise the polishing of gold electrode and/or cleaning step.
Further, described polishing and/or cleaning step comprise: gold electrode successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO
3: H
2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
Further, DNA sequence dna (sulfhydrylation: S1, S2 (one end is rich G sequence) in step (1), one end flag F c:S3), and be dissolved in respectively in 0.05M Tris-HCl (pH7.4) buffer solution, and save backup at 4 DEG C.
Further, in step (2) and (3), incubation time is 10h.
Further, the analog D NA enzyme catalysis and the golden nanometer particle amplification that in step (4), drive based on ferrocene are prepared electrochemica biological sensor.
A kind of electrochemica biological sensor, adopts preparation method as above-mentioned in claim to prepare.
A purposes for above-mentioned electrochemica biological sensor, for detection of potassium ion.
Above-mentioned electrochemica biological sensor detects a method for potassium ion, utilizes catalytic action that electron mediator the drives analog D AN enzyme detection to potassium ion.
Further, when the increase of potassium concentration, the amount of analog D NA enzyme increases thereupon, realizes variable concentrations potassium ion is quantitatively detected.
Compared with currently available technology, the preparation method of this biology sensor, use golden nanometer particle synthetic simple, consume energy low, cost is low, good biocompatibility, use preparation analog D NA enzyme simple, stable in properties carries out living things catalysis, and use electron mediator to drive the catalytic action of analog D NA enzyme, make analog D NA enzyme catalysis hydrogen peroxide matrix more effectively, Electrochemical Detection signal is amplified.Utilize in the cavity of the embedding G-tetrad configuration that potassium ion can be special, make the formation of analog D NA enzyme relevant to the concentration of potassium ion, testing result is satisfactory, the range of linearity detecting is wider, approximately within the scope of 200 μ M, there is sensitiveer detection from 5 μ M, and have that detectability is low, selectivity good, the feature of good stability.
Brief description of the drawings
Fig. 1 is the impedance diagram of each step electrode modification process electrode;
In figure:
A is the impedance diagram of naked gold electrode;
B is the impedance diagram of the gold electrode of probe S1 modification;
C is the impedance diagram of the gold electrode modified after probe S1 and the effect of S2-AuNPs biological composite;
D is the impedance diagram of the gold electrode of modification after probe S1 and S2-AuNPs biological composite and S3 hybridization;
E is after probe S1 and S2-AuNPs biological composite and S3 hybridization and forms the impedance diagram of the gold electrode of modifying after analog D AN enzyme;
Fig. 2 a be based on ferrocene drive analog D NA enzyme catalysis and the electrochemica biological sensor cyclic voltammogram of golden nanometer particle amplification;
Fig. 2 b is the cyclic voltammogram of the gold electrode of probe S1 modification;
Fig. 2 c is the cyclic voltammogram that forms the gold electrode of modifying after analog D NA enzyme after probe S1 and S2 hybridization.
Fig. 2 d is the cyclic voltammogram of the gold electrode of modification after probe S1 and S2-AuNPs biological composite and S3 hybridization;
Fig. 2 e is the cyclic voltammogram of the gold electrode of modification after probe S1 and S2-AuNPs biological composite and S4 hybridization;
Fig. 2 f is the cyclic voltammogram of the gold electrode of modification after probe S1 and S2 and S3 hybridization;
Fig. 3 a is analog D NA enzyme catalysis and the electrochemica biological sensor of the golden nanometer particle amplification cyclic voltammogram to the catalysis of variable concentrations hydrogen peroxide driving based on ferrocene;
Fig. 3 b is the typical curve of sensor to the catalysis of variable concentrations hydrogen peroxide for this reason;
Fig. 4 a is the affect figure of pH value on this experiment;
Fig. 4 b is the affect figure of hybridization temperature on experiment;
Fig. 4 c is that electrode is at the figure that affects on experiment containing incubation time in potassium ion solution;
Fig. 5 a is the cyclic voltammogram that the analog D NA enzyme catalysis that drives based on ferrocene and the electrochemica biological sensor of golden nanometer particle amplification detect variable concentrations potassium ion;
Fig. 5 b is the chronoamperogram that the analog D NA enzyme catalysis that drives based on ferrocene and the electrochemica biological sensor of golden nanometer particle amplification detect variable concentrations potassium ion;
Fig. 5 c typical curve that sensor detects variable concentrations potassium ion for this reason;
Fig. 6 selectivity comparison diagram that sensor detects potassium ion for this reason;
Fig. 7 is the schematic diagram of preparing of the analog D NA enzyme catalysis that drives based on ferrocene and the electrochemica biological sensor of golden nanometer particle amplification.
Embodiment
Describe the present invention with reference to the accompanying drawings below, it is a kind of preferred embodiment in numerous embodiments of the present invention.
The present invention relates to the synthetic method of golden nanometer particle, utilize the base complementrity effect between DNA molecular, at the surface-assembled electrochemica biological sensor of gold electrode, utilize electron mediator to drive the catalytic action of analog D AN enzyme to test the detection to potassium ion.Use sodium citrate reducing process to synthesize golden nanometer particle (AuNPs), utilize the complementary pairing effect of base between DNA sequence dna, by mark ferrocene (Fc) probe with can form the probe assembling of analog D NA enzyme to the surface of gold electrode, prepare based on ferrocene drive analog D NA enzyme catalysis and the electrochemica biological sensor of golden nanometer particle amplification, this sensor has been realized the detection to target potassium ion sensitivity, specificity, stability.Specific as follows:
The preparation of golden nanometer particle: utilize sodium citrate reducing process, first synthesize the AuNPs of size uniform, this nano particle has good bio-compatibility, can be in conjunction with many probe that design, and the detection that therefore nano particle of preparation is applied to biology sensor has good amplification.
The step of the analog D NA enzyme catalysis driving based on ferrocene and the preparation of the electrochemica biological sensor of golden nanometer particle amplification and application is as follows:
A, the DNA sequence dna (sulfhydrylation: S1, S2 (one end is rich G sequence), one end flag F c:S3) of buying is dissolved in respectively in 0.05MTris-HCl (pH7.4) buffer solution, and saves backup at 4 DEG C
B, by gold electrode first successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO
3: H
2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
C, the buffer solution that contains probe S1 is dripped and is coated onto on the clean gold electrode of surface treatment, cultivate 10h, the gold electrode of modifying by Au-S covalent bond effect self assembly S1.Meanwhile, probe S2 joined in the buffer solution that contains AuNPs and cultivate this mixed solution 6-10h, preparation S2-AuNPs biological composite.
D, the gold electrode that S1 is modified are put in the buffer solution that contains S2-AuNPs biological composite and S3 and are cultivated 3-5h, utilize the base complementrity pairing effect between DNA, the analog D NA enzyme catalysis driving based on ferrocene and the electrochemica biological sensor of golden nanometer particle amplification are successfully prepared.
E, because the formation of analog D NA enzyme is relevant to the concentration of potassium ion, along with the increase of potassium concentration, the amount of analog D NA enzyme can increase thereupon, therefore, this sensor can quantitatively detect variable concentrations potassium ion.
By reference to the accompanying drawings the present invention is exemplarily described above; obviously specific implementation of the present invention is not subject to the restrictions described above; as long as the various improvement that adopted method design of the present invention and technical scheme to carry out; or directly apply to other occasion without improvement, all within protection scope of the present invention.
Claims (10)
1. a preparation method for electrochemica biological sensor, is characterized in that, comprises the steps:
(1) DNA sequence dna is dissolved respectively in Tris-HCl buffer solution, and save backup;
(2) buffer solution that contains probe S1 is dripped and is coated onto on gold electrode, cultivate, the gold electrode of modifying by Au-S covalent bond effect self assembly S1;
(3) with step (2) simultaneously, probe S2 joined in the buffer solution that contains AuNPs and cultivate this mixed solution, preparation S2-AuNPs biological composite;
(4) gold electrode of S1 being modified is put in the buffer solution that contains S2-AuNPs biological composite and S3 and is cultivated, and utilizes the base complementrity pairing effect between DNA, prepares electrochemica biological sensor.
2. the preparation method of electrochemica biological sensor as claimed in claim 1, is characterized in that, between step (1) and (2), also comprises the polishing of gold electrode and/or cleaning step.
3. the preparation method of electrochemica biological sensor as claimed in claim 2, is characterized in that, described polishing and/or cleaning step comprise: gold electrode successively with 0.3 and the aluminium powder of 0.5mm carry out polishing, then put into successively HNO
3: H
2in O (v/v)=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3～5min.
4. the preparation method of the electrochemica biological sensor as described in any one in claim 1-3, it is characterized in that, DNA sequence dna (sulfhydrylation: S1, S2 (one end is rich G sequence) in step (1), one end flag F c:S3), and be dissolved in respectively in 0.05M Tris-HCl (pH7.4) buffer solution, and save backup at 4 DEG C.
5. the preparation method of the electrochemica biological sensor as described in any one in claim 1-4, is characterized in that, in step (2) and (3), incubation time is 10h.
6. the preparation method of the electrochemica biological sensor as described in any one in claim 1-5, it is characterized in that, the analog D NA enzyme catalysis and the golden nanometer particle amplification that in step (4), drive based on ferrocene are prepared electrochemica biological sensor.
7. an electrochemica biological sensor, is characterized in that, adopts preparation method as described in claim 1-6 to prepare.
8. a purposes for electrochemica biological sensor as claimed in claim 7, is characterized in that, for detection of potassium ion.
9. electrochemica biological sensor detects the method for potassium ion as claimed in claim 8, it is characterized in that, utilizes catalytic action that electron mediator the drives analog D AN enzyme detection to potassium ion.
10. electrochemica biological sensor detects the method for potassium ion as claimed in claim 9, it is characterized in that, when the increase of potassium concentration, the amount of analog D NA enzyme increases thereupon, realizes variable concentrations potassium ion is quantitatively detected.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675687A (en) * | 2016-03-23 | 2016-06-15 | 安徽师范大学 | Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase |
| CN113340881A (en) * | 2021-06-04 | 2021-09-03 | 安徽师范大学 | Target substance and redox dual-response aptamer sensor, preparation method and application thereof, and quantitative detection method of anabaena toxin |
Citations (2)
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| WO1996041173A1 (en) * | 1995-06-07 | 1996-12-19 | Martinex R & D Inc. | Nanoparticles imprinted with recognition sites for target molecules |
| CN101706504A (en) * | 2009-11-27 | 2010-05-12 | 东南大学 | Method for applying gold nanoparticles mimetic enzyme in biological detection |
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2014
- 2014-05-08 CN CN201410193708.3A patent/CN103940880B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996041173A1 (en) * | 1995-06-07 | 1996-12-19 | Martinex R & D Inc. | Nanoparticles imprinted with recognition sites for target molecules |
| CN101706504A (en) * | 2009-11-27 | 2010-05-12 | 东南大学 | Method for applying gold nanoparticles mimetic enzyme in biological detection |
Non-Patent Citations (3)
| Title |
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| GUANGFENG WANG 等: "Conformational switch for cisplatin with hemin/G-quadruplex DNAzyme supersandwich structure", 《BIOSENSORS AND BIOELECTRONICS》 * |
| JIE ZHAO 等: "Ultrasensitive electrochemical aptasensor for thrombin based on the amplification of aptamer–AuNPs–HRP conjugates", 《BIOSENSORS AND BIOELECTRONICS》 * |
| NING XIA等: "Label-free and sensitive strategy for microRNAs detection based on the formation of boronate ester bonds and the dual-amplification of gold nanoparticles", 《BIOSENSORS AND BIOELECTRONICS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675687A (en) * | 2016-03-23 | 2016-06-15 | 安徽师范大学 | Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase |
| CN105675687B (en) * | 2016-03-23 | 2018-04-13 | 安徽师范大学 | A kind of preparation method of electrochemica biological sensor and the method for detecting dnmt rna activity |
| CN113340881A (en) * | 2021-06-04 | 2021-09-03 | 安徽师范大学 | Target substance and redox dual-response aptamer sensor, preparation method and application thereof, and quantitative detection method of anabaena toxin |
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| CN103940880B (en) | 2016-09-14 |
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