CN105842453A - DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme - Google Patents
DNA detection method based on electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and enzyme Download PDFInfo
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Abstract
The invention discloses a DNA detection method based on an electrochemical sensor with three-stage amplification of terminal extension, gold nanoparticles and oxido-reductase. The method comprises: modifying the surface of a gold electrode with a layer of sulfydryl-modified capturing probe molecules for capturing an objective DNA molecule; after the objective DNA molecule is captured by the capturing probe, modifying the surface of the electrode with complementary pairing of signal-probe-modified gold nanoparticles and basic groups on the other end of the objective DNA molecule; in the catalytic action of a terminal extension enzyme, extending biotin-labeled deoxyribonucleotide to 3' terminal of the signal probe; during the extension process, introducing biotin for specific binding to avidin-labeled horse radish peroxidase; and modifying the surface of the electrode with horse radish peroxidase. High-sensitivity and high-specificity detection of the objective DNA molecule is realized through detection of change of an electrochemical signal generated from an electrolyte catalyzed by horse radish peroxidase. The method is used for detection of a DNA sample in the fields, such as molecular recognition, clinical diagnosis, environmental monitoring, and food security.
Description
Technical field
The invention belongs to electrochemica biological sensor research field, relate to a kind of method urging three grades of amplification electrochemical sensors detection DNA based on end extension, golden nanometer particle and enzyme.
Background technology
DNA detection is widely applied at aspects such as molecular recognition, clinical diagnosis, environmental monitoring, food safetys.And the concentration of the DNA of required detection is the lowest in actual sample, designs and study and there is high sensitivity and selective sensor just becomes the emphasis of research.In order to improve the sensitivity of DNA detection, various signals amplify strategy and also arise at the historic moment, and amplify strategy including nucleic acid amplification signal, and nano material signal amplifies strategy and enzyme catalysis signal amplifies strategy etc..
At present conventional nucleic acid amplification technologies has polymerase chain reaction, rolling-circle replication amplification and some other isothermal amplification.But polymerase chain reaction technology needs the change of strict temperature control, and needs the reaction kit of rate of exchange complexity, relatively costly；And rolling-circle replication amplification procedure is also required to add reaction masterplate, thus add the complexity of reaction system, also improve cost.In order to avoid these problems, a kind of new nucleic acid amplification technologies spatial induction end elongation technology arises at the historic moment, this end extend enzyme catalytic action under without masterplate nucleic acid chains 3 ' end extend nucleic acid long-chains amplifying technique be widely used.Nano material, because its distinctive dimensional properties, is used widely in signal amplifies strategy.Golden nanometer particle has that high surface volume ratio, preparation technology be more ripe, good stability, good biocompatibility, it is prone to the advantages such as functionalization (can be by gold-sulfide linkage especially by DNA modification on golden nanometer particle surface), is used widely as signal amplified material in the detection of DNA.The catalytic action of enzyme can be catalyzed the reaction of multiple substrate in the case of introducing an enzyme molecule and make signal be amplified, and also plays very important role in signal amplifies strategy.
Summary of the invention
The invention aims to the highly sensitive and DNA fragmentation of highly selective detection trace, including the DNA fragmentation in complete complementary, single base mispairing, two base mispairings, three base mispairings, complete non-complementary sequence and serum sample, it is provided that a kind of extend based on end amplify, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme catalysis.
The technical solution realizing the object of the invention is: a kind of based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, comprises the steps:
Step one, the preparation of Au-DNA solution: be slowly added to the marking sulfhydryl signal probe solution that 5 ' ends are labeled in solution of gold nanoparticles, be aged after reaction, be then centrifuged for, wash, be centrifuged, resuspended；
Step 2, the preparation of electrochemical sensor: the cleaning of (a) gold electrode: with NaBH4Polish after solution soaking, then ultrasonic cleaning；(b) self-cleaning surface: at the capture probe solution of gold electrode surfaces self assembly 3 ' end labeling SH groups under room temperature, then with HS-(CH2)11Soak under-EG2-OH room temperature and close, obtain self-cleaning surface；The modification of (c) Au-DNA: after target dna is added drop-wise to self-cleaning surface reaction 1 hour, drip Au-DNA solution；D () end extends: under end extends the effect of enzyme, 3 ' ends of the signal probe modifying gold electrode surfaces in step (c) extend biotin labeled nucleic acid long-chain, add the horseradish peroxidase reaction 15min of Avidin labelling, introduce horseradish peroxidase by biotin-avidin interaction and prepare electrochemical sensor；
Step 3, electrochemical signals detect: electrochemical sensor preparation completed is placed in three-electrode system and detects with cyclic voltammetry and time current curve method, obtains electrochemical signals.
The resuspended solvent used in step one is PBST solution, and wherein the particle diameter of golden nanometer particle is 30nm, and its solution concentration is 1nmol/L；The solution concentration of marking sulfhydryl signal probe is 3.3 μm ol/L.
In step 2 (b), the concentration of capture probe is 0.5 μm ol/L, and the self assembly time is more than 4 hours；HS-(CH2)11-EG2-OH concentration is 0.5mmol/L, and soak time is more than 4 hours.
In step 2 (c), the concentration of Au-DNA solution is 0.5nmol/L, and the modification time is 1 hour.
In step 2 (d), end extends the concentration of enzyme is 1U, and biotin labeled nucleotide (substrate) is biotin labeled adenine deoxyribonucleotide, and concentration is 5.7 μm ol/L, and extension of time is 1 hour.
Electrochemical Detection system in step 3 is three-electrode system, working electrode is gold electrode, and reference electrode is Ag/AgCl electrode, is platinum electrode to electrode, wherein, electrolyte is 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution, cyclic voltammetry high potential is 0.7V, and electronegative potential is 0V, and scanning speed is 0.1V/s；The scanning current potential of time current curve method is 0.1V, and sweep time is 100s.
Described in step one and two, DNA sequence is as follows: the marking sulfhydryl signal probe sequence that 5 ' described ends are labeled is: 5 '-GAA ACC CTA TGT ATG CTC-SH-3 '；The sequence capture probe of 3 ' described end labeling SH groups is: 5 '-SH-T TTT TTT TTT GTA TGA ATT ATA ATC AAA-3 '；nullDescribed target dna sequence is as follows: fully-complementary sequence GAG CAT ACA TAG GGT TTC TCT TGG TTT CTT TGA TTA TAA TTC ATA C、Single base mispairing sequence 5 '-GAG CAT ACA TAG GGT TTC TCT TGG TTT CTT TGA TTA TGA TTC ATA C-3 '、Two base mispairing sequence 5 '-GAG CAT ACA TAG GGT TTC TAT TGG TTT CTT TGA TTA TGA TTC ATA C-3 '、Three base mispairing sequence 5 '-GAG CAT GCA TAG GGT TTC TAT TGG TTT CTT TGA TTA TGA TTC ATA C-3 ' or completely non-complementary sequence 5 '-ATCATC GAT ATC GTT CGA TAT CGT TAT CGA TTA TCG TTA TCA TCG A-3 '.
The present invention compared with prior art, has the following characteristics that
1, detection sensitivity is high: the present invention is less than 10fM to the Monitoring lower-cut of DNA, is in higher level of sensitivity in existing DNA detection sensor.
2, the wide ranges of detection: the present invention is more than 8 orders of magnitude to the detection range width of DNA, is in wider detection width in existing DNA sensor.
3, detection system is simple: the detection method that the present invention uses is simple Electrochemical Detection, and the color not requirement to sample is highly sensitive, and is prone to simplify and miniaturization.
4, practical application is strong: the DNA sensor of the present invention still has higher detection signal in the serum sample of simulation, illustrates that this sensor application in actual sample is strong, has the highest clinical diagnosis aspect application prospect.
Accompanying drawing explanation
Fig. 1 is the process schematic of present invention method based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors detection DNA of enzyme.
Fig. 2 is that the Au-DNA in the embodiment of the present invention 1 prepares result, and figure a is the absorbance curve before modifying signal probe, and b is the absorbance curve after modifying signal probe.
Fig. 3 is the condition optimizing result figure (wherein A is embodiment 2, and B is embodiment 3, and C is embodiment 4) in embodiment of the present invention 2-4.
Fig. 4 is the map of current to variable concentrations target dna in the embodiment of the present invention 5.
Fig. 5 is scheming complete complementary and comparison DNA fragmentation detection current vs in the embodiment of the present invention 6.
Fig. 6 is that in the embodiment of the present invention 7, in buffer solution and serum, target dna detection current vs schemes.
Detailed description of the invention
Below by embodiment, the present invention is further illustrated, its purpose is to be best understood from present disclosure, but for embodiment be not limiting as protection scope of the present invention:
Step as shown in Figure 1, builds based on end extension, golden nanometer particle and three grades of amplification electrochemical sensor detection system of enzyme, and carries out Electrochemical Detection.
(1) foundation of electrochemical sensor detection DNA system
A, gold electrode clean: 1) NaBH4Solution soaking is (toward NaBH4Solid is initially charged the dehydrated alcohol of certain volume, adds isopyknic distilled water, after mixing, electrode is immersed in wherein, soak 15min, rinse out NaBH with distilled water4Solution) 2) polish and (on mill cloth, go up a certain amount of Al2O3Powder, then plus a small amount of water, vertically polish on mill cloth 3min by electrode, clean with ultrapure water) 3) ultrasonic cleaning (first cleans 4-5min with EtOH Sonicate, again with distilled water ultrasonic cleaning 4-5min, rinse with distilled water) 4) Electrochemical Scanning (H with 0.5mol/L2SO4For electrolyte, Ag/AgCl electrode as to electrode, carries out Electrochemical Scanning to metal working electrode as reference electrode, platinum electrode.The most slowly sweep, then add 2V potential electrolysis 5s, then add-0.35V potential electrolysis 10s, the most quickly scanning 2 times, cleaning electrode and change H2SO4The most slowly sweeping 1 time, observe the cyclic voltammogram of scanning, be completely superposed if for the last time sweeping two curves obtained slowly, and oxidation peak has four, reduction current is 40 times of minimum current, then judge that electrode clean is clean) the electrode N that cleans up2Dry up, do not have the electrode of wash clean then to need repeated washing step.
Prepared by b, self-cleaning surface: 1) obtain assembling liquid with capture probe to the desired concn of 5 × PBS dilution 5 ' end marking sulfhydryl, cleaning up and using N2The electrode surface dried up drips 3 μ L and assembles liquid, assembling liquid is made to cover in gold electrode surfaces, guarantee that assembling liquid completely attaches to gold electrode surfaces, and drop does not contains bubble, electrode is added a cover 1.5ml centrifuge tube to reduce the evaporation assembling liquid in course of reaction and to avoid impurity to enter assembling liquid, at room temperature reaction more than 4h.2) HS-(CH is diluted with dehydrated alcohol2)11-EG2-OH, to desired concn, is divided in 2ml centrifuge tube, 100 μ L/ pipes, the electrode PBS solution assembled is rinsed 10s, and uses N2Dry up, then electrode surface is immersed in the confining liquid prepared, it is ensured that electrode surface completely attaches to confining liquid does not has bubble, has twined centrifuge tube and electrode with diaphragm seal, reduces the evaporation of confining liquid, under room temperature, react more than 4h.
C, target dna hybridize: obtain hybridization solution to desired concn, by HS-(CH with 5 × PBS dilution target DNA fragments2)11Electrode PBS after-EG2-OH processes rinses 10s, and uses N2Dry up, then the hybridization solution of dilution is dripped 3 μ L at dry electrode surface, electrode is added a cover 1.5ml centrifuge tube, reduce in course of reaction and assemble the evaporation of liquid and avoid impurity to enter assembling liquid, at room temperature react 1h.
D, the modification of signal probe: the electrode PBS solution after hybridization is rinsed 10s, and uses N2Drying up, the electrode surface after drying up drips 3 μ L Au-DNA solution, after adding a cover 1.5ml centrifuge tube, reacts 1h under room temperature.
E, end extend: the electrode PBS solution after hybridization is rinsed 10s, and uses N2Drying up, the electrode surface after drying up drips 3 μ L ends and extends liquid (it is 1U that end extends the concentration of enzyme, and substrate is the biotin labeling adenine deoxyribonucleotide of concentration 5.7 μm ol/L), after adding a cover 1.5ml centrifuge tube, reacts 1h under room temperature.Guarantee that extending liquid completely attaches to gold electrode surfaces.
F, horseradish peroxidase-labeled: the electrode PBS solution after will extend over is rinsed, and uses N2Drying up, the electrode surface after drying up drips 3 μ L biotin labeling horseradish peroxidase solution, after adding a cover 1.5ml centrifuge tube, reacts 15min under room temperature.
(2) detection of electrochemical signals
The electrode PBS solution of good for labelling horseradish peroxidase being rinsed, with commercially available 3,3', 5,5'-tetramethyl biphenyl amine aqueous solutions for electrolyte, detect with three-electrode system, reference electrode is Ag/AgCl electrode, is platinum electrode to electrode.The high potential of cyclic voltammogram is 0.7V, and electronegative potential is 0V, and scanning speed is 0.1V/s；The experiment current potential of time current curve is 0.1V, and test period is 100s.
The preparation of embodiment 1:Au-DNA and result thereof
Use of the present invention based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, prepare Au-DNA operation as follows: the solution of gold nanoparticles centrifugal concentrating of 1000mL is about 1nmol/L to 300 μ L, its final concentration；It is slowly added to the signal probe solution of 5 ' the end marking sulfhydryls that 20 μ L concentration are 50 μm ol/L, its final concentration of 3.3 μm ol/L；Add equal-volume 0.02mol/L PBS (PH=7,0.2mol/L NaCl) after reacting 16 hours with 350rpm speed oscillation, be aged 40 hours；It is centrifuged 15 minutes with 10000rpm rotating speed, adds 0.01mol/L PBS (PH=7,0.1mol/L NaCl), it is centrifuged 15 minutes with 10000rpm rotating speed, respectively with PBS (0.01M, 0.25M NaCl, PH=7), PBST (0.01M, 0.25M NaCl, 0.1% tween, PH=7), PBSB (0.01M, 0.25M NaCl, 1% bSA, PH=7) resuspended, prepare Au-DNA solution.The Au-DNA solution of preparation is carried out ultraviolet detection, investigate its particle diameter, its result is as shown in Figure 2, a is the absorbance curve before modifying marking sulfhydryl signal probe, b is the absorbance curve after modifying marking sulfhydryl signal probe, show that the particle diameter of golden nanometer particle does not change a lot before and after modifying signal probe, namely in modification, control agglomeration well；The Au-DNA solution of preparation is carried out study on the stability, after adding the saturated sodium-chloride of 2 times of volumes, the solution of gold nanoparticles modified is not had to be become purple from redness, and Au-DNA solution keeps red constant, illustrate that Au-DNA solution has more preferable stability in high level salt solution, also illustrate that the preparation of Au-DNA solution is successful.
Embodiment 2: the different packing densities impact on electrochemical signals testing result.
Use of the present invention extend based on end, golden nanometer particle and the method for three grades of amplification electrochemical sensors of enzyme detection DNA, using complete complementary DNA sequence as target fragment, all operations step is all as it has been described above, wherein confining liquid HS-(CH2)11The concentration of-EG2-OH is 0.5mmol/L, the concentration of target dna is 1 μm ol/L, packing density is followed successively by 10,5,1,0.5,0.2,0.1 μm ol/L, analyze the impact on detection electrochemical signals of the different packing densities, shown in its analysis result such as accompanying drawing 3 (A), before 0.5 μm ol/L, along with the reduction signal to noise ratio of packing density strengthens, after 0.5 μm ol/L, along with the reduction signal to noise ratio of packing density weakens, therefore the more excellent condition of packing density is 0.5 μm ol/L.
Embodiment 3: different HS-(CH2)11-EG2-OH closes the concentration impact on Electrochemical Detection result.
Use of the present invention based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, using complete complementary DNA sequence as target fragment, described in all operations step all embodiments described above 2, wherein packing density is 0.5 μm ol/L, the concentration of target dna is 1 μm ol/L, confining liquid HS-(CH2)11The concentration of-EG2-OH is followed successively by 2,1.5,1,0.5,0.1,0mmol/L analyze different HS-(CH2)11The impact on the detection electrochemical signals of-EG2-OH concentration, shown in its analysis result such as accompanying drawing 3 (B), before 0.5mmol/L, along with HS-(CH2)11The reduction signal to noise ratio of-EG2-OH concentration strengthens, after 0.5mmol/L, along with HS-(CH2)11The reduction signal to noise ratio of-EG2-OH concentration weakens, therefore HS-(CH2)11The more excellent condition of-EG2-OH concentration is 0.5mmol/L.
Embodiment 4: the different Au-DNA dispersion liquid impacts on Electrochemical Detection result.
Use of the present invention based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, using complete complementary DNA sequence as target fragment, described in all operations step all embodiments described above 2, wherein packing density is 0.5 μm ol/L, HS-(CH2)11It is 0.5mmol/L that-EG2-OH closes concentration, the concentration of target dna is 1 μm ol/L, Au-DNA dispersion liquid is followed successively by: PBS, PBSB, PBST, analyze the impact on detection electrochemical signals of the different dispersion liquids, shown in its analysis result such as accompanying drawing 3 (C), when the dispersion liquid of Au-DNA is that background concentration during PBST is less, signal to noise ratio is best, and therefore the more excellent dispersion condition of Au-DNA is PBST.
Embodiment 5: the methods based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors detection DNA of the enzyme detection characteristic to variable concentrations target DNA fragments.
Use of the present invention based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, using complete complementary DNA sequence as target fragment, described in all operations step all embodiments described above 2, wherein packing density is 0.5 μM, HS-(CH2)11It is 0.5mmol/L that-EG2-OH closes concentration, Au-DNA dispersion liquid is PBST, the concentration of target dna to be measured is followed successively by: 1 μm ol/L, 100nmol/L, 10nmol/L, 1nmol/L, 100pmol/L, 10pmol/L, 1pmol/L, 100fmol/L, 10fmol/L, analyze the electrochemical signals response characteristic of variable concentrations DNA fragmentation to be measured, analysis result is as shown in Figure 4, in this detection range, electrochemical signals is with the increase exponentially relation of DNA fragmentation concentration, and detection limit is less than 10fmol/L.
Embodiment 6: methods based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors detection DNA of enzyme are to the complementary and electrochemical signals contrast of comparison DNA fragmentation.
Using the method detecting DNA based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors of enzyme of the present invention, described in all operations step all embodiments described above 2, wherein packing density is 0.5 μm ol/L, HS-(CH2)11It is 0.5mmol/L that-EG2-OH closes concentration, Au-DNA dispersion liquid is PBST, the concentration of target dna to be measured is 1nmol/L, target DNA fragments is followed successively by a complete complementary, the single base mispairing of b, two base mispairings of c, tri-base mispairings of d and e non-complementary sequence, analyzing the specificity of electrochemical sensor, analysis result is as it is shown in figure 5, as seen from the figure, the method of the electrochemical sensor detection DNA of the present invention can distinguish the mispairing of single base, has high specificity.
Embodiment 7: methods based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors detection DNA of enzyme are to the electrochemical signals contrast of DNA fragmentation in buffer and serum.
Use of the present invention based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, using complete complementary DNA sequence as target fragment, described in all operations step all embodiments described above 2, wherein packing density is 0.5 μm ol/L, HS-(CH2)11It is 0.5mmol/L that-EG2-OH closes concentration, Au-DNA dispersion liquid is PBST, the concentration of target dna to be measured is 1nmol/L, the dispersion liquid of target dna is PBS buffer solution and serum, and analysis electrochemical sensor is to the Detection results of target dna in serum, and analysis result is as shown in Figure 6, although it is the best that the current signal of detection does not has in buffer, but still is able to the target dna detecting in serum well, it is shown that well practice prospect.
Claims (7)
1. the method detecting DNA based on end extension, golden nanometer particle and three grades of amplification electrochemical sensors of enzyme, it is characterised in that comprise the steps:
Step one, the preparation of Au-DNA solution: be slowly added to the marking sulfhydryl signal probe solution that 5 ' ends are labeled in solution of gold nanoparticles, be aged after reaction, be then centrifuged for, wash, be centrifuged, resuspended；
Step 2, the preparation of electrochemical sensor: the cleaning of (a) gold electrode: with NaBH4Polish after solution soaking, then ultrasonic cleaning；(b) self-cleaning surface: at the capture probe solution of gold electrode surfaces self assembly 3 ' end labeling SH groups under room temperature, then with HS-(CH2)11Soak under-EG2-OH room temperature and close, obtain self-cleaning surface；The modification of (c) Au-DNA: after target dna is added drop-wise to self-cleaning surface reaction 1 hour, drip Au-DNA solution；D () end extends: under end extends the effect of enzyme, 3 ' ends of the signal probe modifying gold electrode surfaces in step (c) extend biotin labeled nucleic acid long-chain, add the horseradish peroxidase reaction 15min of Avidin labelling, introduce horseradish peroxidase by biotin-avidin interaction and prepare electrochemical sensor；
Step 3, electrochemical signals detect: electrochemical sensor preparation completed is placed in three-electrode system and detects with cyclic voltammetry and time current curve method, obtains electrochemical signals.
2. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterised in that the resuspended solvent used in step one is PBST solution, and wherein the particle diameter of golden nanometer particle is 30
Nm, its solution concentration is 1
nmol/L；The marking sulfhydryl signal probe solution concentration that 5 ' ends are labeled is 3.3 μm ol/L.
3. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterised in that in step 2 (b), the concentration of capture probe is 0.5 μm ol/L, and the self assembly time is more than 4 hours；HS-(CH2)11-EG2-OH concentration is 0.5 mmol/L, and soak time is more than 4 hours.
4. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterised in that in step 2 (c), the concentration of Au-DNA solution is 0.5 nmol/L, and the modification time is 1 hour.
5. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterized in that, in step 2 (d), end extends the concentration of enzyme is 1 U, biotin labeled nucleotide is biotin labeled adenine deoxyribonucleotide, concentration is 5.7 μm ol/L, and extension of time is 1 hour.
6. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterised in that the Electrochemical Detection system in step 3 is three-electrode system, working electrode is gold electrode, and reference electrode is Ag/AgCl electrode, is platinum electrode to electrode, wherein, electrolyte is 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution, cyclic voltammetry high potential is 0.7 V, and electronegative potential is 0 V, and scanning speed is 0.1 V/s；The scanning current potential of time current curve method is 0.1 V, and sweep time is 100 s.
7. as claimed in claim 1 based on end extension, golden nanometer particle and the method for three grades of amplification electrochemical sensors detection DNA of enzyme, it is characterized in that, described in step one and step 2, DNA sequence is as follows: the marking sulfhydryl signal probe sequence that 5 ' ends are labeled is: 5 '-GAA ACC CTA TGT ATG CTC-SH-3 '；The sequence capture probe of 3 ' end labeling SH groups is: 5 '-SH-T TTT TTT TTT GTA TGA ATT ATA
ATC AAA-3’；Target dna sequence is as follows: fully-complementary sequence GAG CAT ACA TAG GGT TTC TCT TGG TTT CTT TGA TTA TAA TTC ATA C, single base mispairing sequence 5 '-GAG
CAT ACA TAG GGT TTC TCT TGG TTT CTT TGA TTA TGA TTC ATA C-3 ', two base mispairing sequence 5 '-GAG
CAT ACA TAG GGT TTC TAT TGG TTT CTT TGA TTA TGA TTC ATA C-3 ', three base mispairing sequence 5 '-GAG
CAT GCA TAG GGT TTC TAT TGG TTT CTT TGA TTA TGA TTC ATA C-3 ' or completely non-complementary sequence 5 '-ATCATC
GAT ATC GTT CGA TAT CGT TAT CGA TTA TCG TTA TCA TCG A-3’。
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106978473A (en) * | 2016-01-19 | 2017-07-25 | 南京理工大学 | It is a kind of that the method for enzyme structure supermolecule DNA nets and its application in DNA detections are extended based on end |
| CN108717076A (en) * | 2018-05-18 | 2018-10-30 | 山东农业大学 | A kind of electrochemica biological sensor and preparation method thereof of detection zeatin |
| CN109085220A (en) * | 2018-06-18 | 2018-12-25 | 南京理工大学 | Detect the aptamer electrochemica biological sensor and preparation method of malachite green |
| CN109306351A (en) * | 2017-07-28 | 2019-02-05 | 上海海洋大学 | The detection method that a kind of nanometer bio probe and terminal enzyme (DNA) mediate |
| CN109655505A (en) * | 2019-02-15 | 2019-04-19 | 云南中烟工业有限责任公司 | A kind of method of electrochemistry amplification detection mercury ion |
| CN110592191A (en) * | 2019-09-18 | 2019-12-20 | 南京邮电大学 | Method for visually detecting nucleic acid based on enzyme catalysis circulation and molybdenum disulfide adsorption mediation |
| CN112852923A (en) * | 2021-02-03 | 2021-05-28 | 杭州电子科技大学 | Two-stage amplification method based on DNA biosensor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130220809A1 (en) * | 2010-07-14 | 2013-08-29 | The Curators Of The University Of Missouri | Nanopore-facilitated single molecule detection of nucleic acid |
| CN103698374A (en) * | 2013-12-23 | 2014-04-02 | 邢士超 | Electrochemical sensor of DNA (deoxyribonucleic acid) probe based on fluorescein and sulfydryl modification |
-
2015
- 2015-01-12 CN CN201510015158.0A patent/CN105842453B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130220809A1 (en) * | 2010-07-14 | 2013-08-29 | The Curators Of The University Of Missouri | Nanopore-facilitated single molecule detection of nucleic acid |
| CN103698374A (en) * | 2013-12-23 | 2014-04-02 | 邢士超 | Electrochemical sensor of DNA (deoxyribonucleic acid) probe based on fluorescein and sulfydryl modification |
Non-Patent Citations (2)
| Title |
|---|
| JIONG ZHANG ET AL: "Sequence-Specific Detection of Femtomolar DNA via a Chronocoulometric DNA Sensor (CDS): Effects of Nanoparticle-Mediated Amplification and Nanoscale Control of DNA Assembly at Electrodes", 《J. AM. CHEM. SOC》 * |
| YING WAN ET AL: "A surface-initiated enzymatic polymerization strategy for electrochemical DNA sensors", 《BIOSENSORS AND BIOELECTRONICS》 * |
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| CN106978473B (en) * | 2016-01-19 | 2020-09-11 | 南京理工大学 | Method for constructing supramolecular DNA network based on terminal elongase and application of supramolecular DNA network in DNA detection |
| CN109306351A (en) * | 2017-07-28 | 2019-02-05 | 上海海洋大学 | The detection method that a kind of nanometer bio probe and terminal enzyme (DNA) mediate |
| CN108717076A (en) * | 2018-05-18 | 2018-10-30 | 山东农业大学 | A kind of electrochemica biological sensor and preparation method thereof of detection zeatin |
| CN109085220A (en) * | 2018-06-18 | 2018-12-25 | 南京理工大学 | Detect the aptamer electrochemica biological sensor and preparation method of malachite green |
| CN109655505A (en) * | 2019-02-15 | 2019-04-19 | 云南中烟工业有限责任公司 | A kind of method of electrochemistry amplification detection mercury ion |
| CN109655505B (en) * | 2019-02-15 | 2022-04-19 | 云南中烟工业有限责任公司 | Method for detecting mercury ions through electrochemical amplification |
| CN110592191A (en) * | 2019-09-18 | 2019-12-20 | 南京邮电大学 | Method for visually detecting nucleic acid based on enzyme catalysis circulation and molybdenum disulfide adsorption mediation |
| CN112852923A (en) * | 2021-02-03 | 2021-05-28 | 杭州电子科技大学 | Two-stage amplification method based on DNA biosensor |
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