CN103698374A - Electrochemical sensor of DNA (deoxyribonucleic acid) probe based on fluorescein and sulfydryl modification - Google Patents
Electrochemical sensor of DNA (deoxyribonucleic acid) probe based on fluorescein and sulfydryl modification Download PDFInfo
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- CN103698374A CN103698374A CN201310716662.4A CN201310716662A CN103698374A CN 103698374 A CN103698374 A CN 103698374A CN 201310716662 A CN201310716662 A CN 201310716662A CN 103698374 A CN103698374 A CN 103698374A
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Abstract
The invention discloses an electrochemical sensor of a DNA probe based on fluorescein and sulfydryl modification. The electrochemical sensor comprises an electrode and a DNA probe, wherein the electrode adopts a three-electrode system, and the two ends of the DNA probe are respectively modified with the fluorescein and the sulfydryl; gold nanoparticles are deposited on the surface of a gold electrode, and the sulfydryl modified on the DNA probe is combined with the gold nanoparticles through a gold-sulfur bond; the gold nanoparticles and the fluorescein modified on the DNA probe can enable the DNA probe to form an arch-shaped structure through the surface adsorption action. The electrochemical sensor provided by the invention has the advantages that the gold nanoparticles are modified on the surface of the gold electrode through an electro-deposition effect, the nanogold modification can greatly increase the fixed carrying capacity of probe ssDNA of the surface of the electrode, and the probe forms the arch-shaped structure through the surface adsorption action of the nanogold on the fluorescein. The connection has high electron transmission efficiency, the change of the DPV signal before and after hybridization can be improved, and the detection sensitivity is improved.
Description
Technical field
The present invention relates to electrochemica biological sensor technical field, particularly a kind of electrochemical sensor of the DNA probe based on fluorescein and sulfydryl modification.
Background technology
Recently, many electrochemical DNAs (E-DNA) sensor and similar galvanochemistry (E-AB) sensor based on fit biography emerge in multitude, and they are a kind of novel DNA sensors that the conformation change in dynamics develops based on DNA probe.Wherein DNA probe can be linearity or cyclic DNA.The general terminal modified sulfydryl of DNA probe, a terminal modified redox label.Effect by gold-sulfide linkage self-assembles to gold electrode surfaces.After target DNA hybridization, changed the configuration of DNA probe, the electronics transmission efficiency of redox label and electrode surface is changed, thereby changed electric signal, indication hybridization.This simple sensor is widely used in the research of DNA, enzyme, metallic ion and organic molecule etc.But the conformational change of some DNA probe is very complicated, and for some simple sensors, its sensitivity and selectivity also need further raising, the research that is therefore intended to further to improve sensor detection efficiency has broad prospects.
Summary of the invention
The electrochemical sensor that the object of this invention is to provide a kind of DNA probe based on fluorescein and sulfydryl modification, comprise electrode and DNA probe, described electrode adopts three-electrode system, and wherein gold electrode is working electrode, mercurous chloride electrode is contrast electrode, and platinum filament is to electrode; The two ends of described DNA probe are modified with respectively fluorescein and sulfydryl; The surface deposition of described gold electrode has gold nano grain; The sulfydryl of modifying on described DNA probe is combined by gold-sulfide linkage between gold nano grain; The fluorescein of modifying on gold nano grain and DNA probe, by adsorption effect, makes DNA probe form bow-shaped structural.
An electrochemical sensor for DNA probe based on fluorescein and sulfydryl modification as above, the concrete grammar of gold electrode and DNA probe assembling is as follows:
1) use clean-out system to clean gold electrode surfaces, then clean with intermediate water, nitrogen dries up, then at 0.1molL
-1h
2sO
4scan cycle volt-ampere in solution is until stable; Afterwards three-electrode system is inserted into 6mmolL
-1hAuCl
4(containing 0.1molL
-1kNO
3) carry out electro-deposition in solution, in solution also containing 0.1molL
-1kNO
3,-400mV (vs.SCE) deposits 300s, after having deposited, with the solution of intermediate water cleaning electrode remained on surface;
Described clean-out system is 30%H
2o
2/ 70%H
2sO
4, volume ratio.
2) the DNA probe solution of fluorescein and sulfydryl will be equipped with, concentration 29 μ molL
-1, to drip and be coated in the gold electrode surfaces that nanogold particle is modified, the place that then electrode is placed on to circulation of air is naturally dried and is spent the night; In this process, with a small beaker, cover on electrode, put a small beaker of filling water in it and keep the humidity in beaker cover; After completing, use intermediate water cleaning electrode, nitrogen dries up, and obtains the gold electrode that DNA probe is modified.
The electrochemical sensor of a kind of DNA probe based on fluorescein and sulfydryl modification of the present invention is by electro-deposition effect, gold nano grain on gold electrode surfaces is modified.This decorated by nano-gold has not only increased the supported quantity of electrode surface probe ssDNA greatly, and due to the adsorption effect of nm of gold to fluorescein, makes probe form bow-shaped structural.This connection has higher electronics transmission efficiency, can improve the change of hybridization front and back DPV signal, and then improves detection sensitivity.
Accompanying drawing explanation
Fig. 1 is the electron microscope photo scanning of naked gold electrode surfaces;
Fig. 2 is that naked gold electrode is at 3mmolL
-1in chlorauric acid solution, the electron microscope photo scanning after-200mV (vs.SCE) deposition 300s;
Fig. 3 is that naked gold electrode is at 6mmolL
-1in chlorauric acid solution, the electron microscope photo scanning after-400mV (vs.SCE) deposition 300s;
Fig. 4 is that naked gold electrode is at 6mmolL
-1in chlorauric acid solution, the electron microscope photo scanning after-400mV (vs.SCE) deposition 400s;
Fig. 5 is that electrode is at 0.1molL
-1h
2sO
4cyclic voltammetry curve in solution: naked gold electrode (a); Decorated by nano-gold electrode (c);
Fig. 6 is that the concentration of naked gold electrode ssDNA-B in hybridization solution is 15nmolL
-1dPV peak current signal intensity figure before and after time hybridization;
Fig. 7 is that the concentration of gold electrode ssDNA-B in hybridization solution of gold nano particle modification is 15nmolL
-1dPV peak current signal intensity figure before and after time hybridization;
Fig. 8 is that the concentration of naked gold electrode ssDNA-B in hybridization solution is 5nmolL
-1dPV peak current signal intensity figure before and after time hybridization;
Fig. 9 is that the concentration of gold electrode ssDNA-B in hybridization solution of gold nano particle modification is 5nmolL
-1dPV peak current signal intensity figure before and after time hybridization;
Figure 10 is the graph of a relation of ultraviolet absorption value and target dna concentration;
Figure 11 is the linear relationship of DPV signal reduced rate and target ssDNA-B concentration;
Figure 12 is the DPV signal of different target ssDNA, (a) the DPV signal of a stationary probe ssDNA-A; And 50nmolL (b)
-1the DPV signal of ssDNA-C hybridization; (b) with 50nmol L
-1the DPV signal of ssDNA-D hybridization; And 50nmolL (d)
-1the DPV signal of ssDNA-B hybridization; (e) the DPV signal of the decorated by nano-gold gold electrode of unmodified ssDNA-A.
Embodiment
Below by embodiment, the present invention will be further described.
An electrochemical sensor for DNA probe based on fluorescein and sulfydryl modification, comprises electrode and DNA probe, and described electrode adopts three-electrode system, and wherein gold electrode is working electrode, and mercurous chloride electrode is contrast electrode, and platinum filament is to electrode; The two ends of described DNA probe are modified with respectively fluorescein and sulfydryl; The surface deposition of described gold electrode has gold nano grain; The sulfydryl of modifying on described DNA probe is combined by gold-sulfide linkage between gold nano grain; The fluorescein of modifying on gold nano grain and DNA probe, by adsorption effect, makes DNA probe form bow-shaped structural.
An electrochemical sensor for DNA probe based on fluorescein and sulfydryl modification as above, the concrete grammar of gold electrode and DNA probe assembling is as follows:
1) use clean-out system to clean gold electrode surfaces, then clean with intermediate water, nitrogen dries up, then at 0.1molL
-1h
2sO
4scan cycle volt-ampere in solution is until stable; Afterwards three-electrode system is inserted into 6mmolL
-1hAuCl
4(containing 0.1molL
-1kNO
3) carry out electro-deposition in solution, in solution also containing 0.1molL
-1kNO
3,-400mV (vs.SCE) deposits 300s, after having deposited, with the solution of intermediate water cleaning electrode remained on surface;
Described clean-out system is piranha solution, wherein 30%H
2o
2/ 70%H
2sO
4, volume ratio.
2) the DNA probe solution of fluorescein and sulfydryl will be equipped with, concentration 29 μ molL
-1, to drip and be coated in the gold electrode surfaces that nanogold particle is modified, the place that then electrode is placed on to circulation of air is naturally dried and is spent the night; In this process, with a small beaker, cover on electrode, put a small beaker of filling water in it and keep the humidity in beaker cover; After completing, use intermediate water cleaning electrode, nitrogen dries up, and obtains the gold electrode that DNA probe is modified.
On gold electrode, the generative process of nanogold particle characterizes by SEM.By controlling deposition voltage, solution concentration and sedimentation time, can control size and the quantity of electro-deposition nm of gold.Fig. 1-4 are the scanning electron microscope of the gold electrode surfaces of naked gold electrode and gold nano particle modification.As shown in Figure 1, naked gold electrode surfaces is essentially smooth, there is no nano particle on its surface; When voltage is lower, when gold chloride concentration is rarer, electrodeposition rate is slow, and after deposition 180s, gold electrode surfaces only has some granules, as shown in Figure 2; At 6mmolL
-1in chlorauric acid solution, after-400mV (vs.SCE) deposition 300s, can see that gold electrode surfaces has covered a large amount of gold nano grains, between these gold nano grains, have many spaces, greatly increase gold electrode specific surface area, as shown in Figure 3.Along with the prolongation of sedimentation time, its effect is only that the size of deposited particles is increased, as shown in Figure 4.
Decorated by nano-gold electrode prepared by use this method and naked gold electrode are at 0.1molL
-1h
2sO
4middle making cyclic voltammetry curve, as shown in Figure 5, in figure, the peak current of naked gold electrode (a) is 0.24 μ A, the peak current of decorated by nano-gold electrode (c) is 1.4 μ A.The surface area of decorated by nano-gold electrode is 5.8 times of gold electrode, can significantly improve the surface area of gold electrode after visible electro-deposition nanogold particle.
Selecting following sequence is probe, 5 '-T
6-TAG GAA ACA CCA AAG ATG ATA TTT-T
6-3 ', upper fluorescein and sulfydryl are modified respectively in the two ends of probe sequence, sequence is as follows:
ssDNA-A:
5 '-F-T
6-TAG GAA ACA CCA AAG ATG ATA TTT-T
6-SH-3 ', wherein F represents fluorescein.
ssDNA-B:
5 '-AAA TAT CAT CTT TGG TGT TTC CTA-3 ' (target dna)
ssDNA-C:
5 '-TAC GAG TTG AGA GCA AGC AGA GTT-3 ' (incomplementarity DNA)
ssDNA-D:
5 '-AAA TAT CAT CTT TTG TGT TTC CTA-3 ' (single base mismatch DNA)
Above artificial sequence is provided by match Parkson, Beijing company limited.
Utilize the method for embodiment 1 that ssDNA-A sequence is modified respectively on naked gold electrode and decorated by nano-gold electrode, respectively the electrode of having modified is inserted into separately respectively in the PBS buffer solution that contains target dna again, under room temperature, hybridize 1h, then the electrode after hybridization is taken out, with intermediate water, clean, nitrogen dries up.Form respectively three-electrode system and carry out DPV Electrochemical Detection.Buffer system is phosphoric acid buffer system (PBS) (100mmolL
-1phosphate buffer solution, 1.5molL
-1naCl, 1mmolL
-1mg2+, pH=7.2).
Table 1DPV peak current signal
In hybridization solution, the target ssDNA-B concentration of coupling is 15nmolL completely
-1time, on naked gold electrode, DPV signal has reduced by 43%, and on the gold electrode that corresponding gold nano is modified, DPV signal has reduced by 77%.
When electrode surface only has immobilized probe ssDNA-A, the DPV peak current of naked gold electrode is approximately 0.13 μ A, and on the gold electrode of gold nano particle modification, the peak current of DPV has reached 0.77 μ A.After object chain hybridization, DNA forms two strands, due to double-stranded rigid backbone, causes fluorescein away from gold electrode surfaces, the gold electrode surfaces after modification now, and gold nano grain disappears for the sucking action of fluorescein.
When with the concentration of two kinds of electrode catheters hybridization target dnas be 5nmolL
-1time, on naked gold electrode, do not observe the variation of DPV peak value, and 50% the peak value of still having an appointment on gold electrode after modifying declines.
More than narration explanation: nanogold particle is the sensitivity that has strengthened sensor to the suction-operated of fluorescein.
Probe ssDNA-A is separately fixed on the gold electrode and naked gold electrode of decorated by nano-gold.Then electrode is inserted in target ssDNA-B solution and hybridizes, by ultraviolet spectrophotometry, detect the reduction of target ssDNA-B in the hybridization solution of hybridization front and back.When the target dna-B concentration matching well is completely 15nmolL
-1time, the efficiency of hybridizing on two kinds of electrodes is respectively 47% and 33%.The peak value of DPV changes the configuration that is due to each single stranded DNA and changes and cause, according to Principle of Statistics, calculates the variation of the peak current that each single stranded DNA causes, on the gold electrode of decorated by nano-gold and naked gold electrode, is respectively 2.8 * 10
-20a and 3.3 * 10
-21a.From this result, can reach a conclusion, gold nano grain compared with bigger serface, can increase the efficiency of hybridization, but the principal element that affects peak current is the change of electrode surface DNA configuration, one end used in experiment is connected to sulfydryl, the DNA that one end is connected to fluorescein can be attracted on gold nano grain and form bow-shaped structural, and being also affects DPV peak current signal on the gold electrode that gold nano modifies to change larger key factor.
The standard solution of target ssDNA-B is concentration 1.5 * 10-9molL
-1pBS solution 5mL.As shown in figure 10, the uv absorption intensity of solution presents linear change along with the variation of ssDNA-B concentration in solution.Linear equation: the Y=0.0007X+0.1768 (concentration 10 that X is DNA-B
-9molL
-1, Y is ultraviolet absorption value, n=9, R=0.9944).
On the gold electrode of decorated by nano-gold and naked gold electrode, the amount of Probe Hybridization DNA can be reacted by the change of ssDNA-B amount in hybridization solution.We are by measuring the 15nmolL of 5mL under 260nm
-1the reduction of DNA in hybridization solution is calculated in the variation of the ultraviolet absorption value of ssDNA-B.Detailed process is as follows:
The gold electrode of decorated by nano-gold
Concentration: 15nmolL before ssDNA-B hybridization
-1
Ultraviolet absorption value before hybridization: 0.1873
Ultraviolet absorption value after hybridization: 0.1831
Concentration: 8nmolL after ssDNA-B hybridization
-1
Hybridization efficiency: (15-8)/15=47%
The molal quantity that DNA is fixing: (15-8) * 10
-9* 5 * 10
-3=3.5 * 10
-11mol
DNA fixed qty: 3.5 * 10
-11* 6.02 * 10
23=2.1 * 10
13
DPV peak current change value: 5.9 * 10
-7a
The peak current change value of each DNA impact: 2.8 * 10
-20a
Naked gold electrode
Concentration: 15nmolL before ssDNA-B hybridization
-1
Ultraviolet absorption value before hybridization: 0.1873
Ultraviolet absorption value after hybridization: 0.1861
Concentration: 10nmolL after ssDNA-B hybridization
-1
Hybridization efficiency: (15-10)/15=33%
The molal quantity that DNA is fixing: (15-10) * 10
-9* 5 * 10
-3=2.5 * 10
-11mol
DNA fixed qty: 2.5 * 10
-11* 6.02 * 10
23=1.5 * 10
13
DNA fixed qty: 5 * 10
-8a
The peak current change value of each DNA impact: 3.3 * 10
-21a
Determining of the detectability of sensor:
By changing the concentration of target dna-B, the sensitivity of crossover process is studied.As shown in Figure 11, along with the increase of target ssDNA-B concentration, the reduced rate of DPV peak current increases.The target ssDNA-B of variable concentrations is carried out to three replicate determinations, average and make working curve.Figure 11 shows, the concentration of target ssDNA-B is 2.0 * 10
-9molL
-1-2.0 * 10
-8molL
-1in scope, the concentration of peak reduction rate and ssDNA-B is good linear relationship, and the DNA in this concentration range can quantitative measurement.The equation of linear regression of working curve is that (wherein y is peak current reduced rate % to y=3.0641x+35.897; X is ssDNA concentration, 10
-9molL
-1; N=11, related coefficient γ=0.9956).According to 3 times, to the calculated signals of noise, lowest detection is limited to 7.10 * 10
-10molL
-1(S/N=3).
The specific detection of sensor:
The specificity research of sensor as shown in figure 12.Do not modify on the decorated by nano-gold electrode of probe ssDNA-A and there is no DPV signal.When probe ssDNA-A is fixed on electrode surface, there is a very strong DPV current peak, with 50nmolL
-1the target dna of complete complementary-B hybridization, DPV signal can reduce by 91%.When with 50nmolL
-1during single mispairing ssDNA-D hybridization, signal reduces by 43%.With 50nmolL
-1during complete unmatched ssDNA-C hybridization, signal only reduces by 5%.This explanation is under identical concentration, and sensor of the present invention has good recognition capability for target dna.
Electrode after hybridization is heated to about 5min in 95 ℃ of boiling water baths, then shift rapidly, in ice-water bath, be cooled to room temperature.Under this condition is processed, the dsDNA sex change of unwinding is ssDNA, by recross, realizes recycling.The value of electrical signals obtaining after recross can arrive and return to original electrical signal 95%.
In the present invention, fluorescein is to be used as a kind of electrooxidation reduction probe to use, the method can be used for electrochemical DNA biosensor and galvanochemistry aptamer sensor, detects target and comprises DNA, protein or micromolecular compound etc., has high selectivity, high sensitivity, and the advantage such as fast detecting.
Claims (2)
1. an electrochemical sensor for the DNA probe based on fluorescein and sulfydryl modification, is characterized in that comprising electrode and DNA probe, and described electrode adopts three-electrode system, and wherein gold electrode is working electrode, and mercurous chloride electrode is contrast electrode, and platinum filament is to electrode; The two ends of described DNA probe are modified with respectively fluorescein and sulfydryl; The surface deposition of described gold electrode has gold nano grain; The sulfydryl of modifying on described DNA probe is combined by gold-sulfide linkage between gold nano grain; The fluorescein of modifying on gold nano grain and DNA probe, by adsorption effect, makes DNA probe form bow-shaped structural.
2. an electrochemical sensor for the DNA probe based on fluorescein and sulfydryl modification as claimed in claim 1, is characterized in that the concrete grammar of gold electrode and DNA probe assembling is as follows:
1) use clean-out system to clean gold electrode surfaces, then clean with intermediate water, nitrogen dries up, then at 0.1molL
-1h
2sO
4scan cycle volt-ampere in solution is until stable; Afterwards three-electrode system is inserted into 6mmolL
-1hAuCl
4(containing 0.1molL
-1kNO
3) carry out electro-deposition in solution, in solution also containing 0.1molL
-1kNO
3,-400mV (vs.SCE) deposits 300s, after having deposited, with the solution of intermediate water cleaning electrode remained on surface;
Described clean-out system is 30%H
2o
2/ 70%H
2sO
4
2) the DNA probe solution of fluorescein and sulfydryl will be equipped with, concentration 29 μ molL
-1, to drip and be coated in the gold electrode surfaces that nanogold particle is modified, the place that then electrode is placed on to circulation of air is naturally dried and is spent the night; In this process, with a small beaker, cover on electrode, put a small beaker of filling water in it and keep the humidity in beaker cover; After completing, use intermediate water cleaning electrode, nitrogen dries up, and obtains the gold electrode that DNA probe is modified.
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|---|---|---|---|
| CN201310716662.4A CN103698374A (en) | 2013-12-23 | 2013-12-23 | Electrochemical sensor of DNA (deoxyribonucleic acid) probe based on fluorescein and sulfydryl modification |
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| CN105420090A (en) * | 2015-11-20 | 2016-03-23 | 青岛大学附属医院 | Gene detection system based on nanogold DNA probe and method |
| CN105842453A (en) * | 2015-01-12 | 2016-08-10 | 南京理工大学 | DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme |
| CN107271518A (en) * | 2017-07-31 | 2017-10-20 | 首都师范大学 | A kind of amperometric electrochemical sensor and its preparation method and application |
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Application publication date: 20140402 |