CN104458842A - Ultrasensitive DNA electrochemical detection method, reagent and system - Google Patents
Ultrasensitive DNA electrochemical detection method, reagent and system Download PDFInfo
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Abstract
The invention provides an ultrasensitive DNA electrochemical detection method which comprises the following steps: fixing a DNA capture probe on a working electrode of a DNA electrochemical sensor; fixing MCH on the working electrode fixedly provided with the DNA capture probe; additionally arranging a to-be-detected DNA sample and a DNA signal probe to the working electrode for performing hybrid treatment; and detecting an electrochemical signal of the working electrode subjected to the hybrid treatment. According to the ultrasensitive DNA electrochemical detection method disclosed by the invention, the working electrode is closed by using MCH (6-mercapto-1-hexanol), a number of non-target DNA signals and background signals can be shielded, a sensor noise signal is reduced, and the sensitivity of the sensor is improved.
Description
Technical field
The invention belongs to electrochemica biological sensor technical field, be specifically related to a kind of overdelicate DNA electrochemical detection method, reagent and system.
Background technology
Biochip technology can realize quick, high flux screening and the test of cell, nucleic acid, protein, carbohydrate and other components.
Wherein, DNA electrochemica biological sensor, be study in biology sensor the earliest, a most widely used branch; The concentration information of ion, chemical substance is converted to the signals such as current potential, electric current or electric the moon/conductance by it, combines the high sensitivity of electricity analytical method and the specific recognition capability of bioactive molecule.Current DNA electrochemica biological sensor is widely used in fields such as clinical medical inspection and genetic engineering, drug mechanism and new medicament screen, food engineering and environmental monitorings.Such as, based on the technology adopting the electrochemica biological sensor of DNA to be detected analysans by high specific recognition component, at present in the DNA checkout and diagnosis of genetic disease, communicable disease (common hepatitis A, hepatitis B, AIDS virus, tubercle bacillus etc.), just gradually in the DNA detection method replacing the complicated operation of main flow, the quantitative fluorescent PCR of testing process length consuming time.
But in existing DNA electrochemica biological sensor, traditional three-electrode system that normal use working electrode is gold electrode detects.Gold is a kind of inert metal, is not easily oxidized, is hydrolyzed and other surface reactions.But this gold electrode makes needs polishing repeatedly, polishing, cleaning, a series of pre-treatment such as ultrasonic, and be difficult in continuous detecting maintain the stability of response and long-term reappearance, be everlasting and inevitably polluted in line analysis and corrode, be unfavorable for miniaturization and the commercialization of biology sensor.Simultaneously, common DNA electrochemica biological sensor is when detecting, because the object content participating in hybridization is too low, and be subject to the restriction of the sensitivity of method for sensing own, often be difficult to background signal and feeble signal to make a distinction, cause the super low loading DNA that conventional DNA electrochemica biological sensor can not be used in detection of complex biological sample.The accuracy of detection of current existing DNA electrochemical biosensor technology be difficult to reach clinical with scientific research on required accuracy of detection, must depend on and detect PCR and in advance sample is increased, re-use sensing technology and detect; The use of electrochemica biological sensor is subject to certain restrictions.
Summary of the invention
The object of the embodiment of the present invention is to overcome the deficiency in prior art detection sensitivity, provides a kind of overdelicate DNA electrochemical detection method, reagent and system.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A kind of overdelicate DNA electrochemical detection method, comprises the steps:
Fixed dna capture probe on the working electrode of DNA electrochemical sensor;
The working electrode of the described DNA of being fixed with capture probe fixes MCH;
DNA sample to be measured, DNA signal probe are added on described working electrode and carry out hybridization process;
Detect the electrochemical signals of the rear described working electrode of hybridization process.
Above-mentioned overdelicate DNA electrochemical detection method of the present invention, by using MCH (6-sulfydryl hexanol), working electrode is closed, signal and the background signal of some non-target DNAs can be shielded, realize the reduction to sensor noise signal, improve transducer sensitivity.
The present invention also proposes a kind of overdelicate DNA electrochemical detecting reagent further, comprise one end for combining with the working electrode that DNA detects, the other end is for contacting the molecular wire of signaling molecule on DNA signal probe.
This reagent is in the process detected, after being fixed on working electrode at DNA capture probe, then molecular wire is combined with MCH and jointly closes working electrode, after closing its one end for combining with the working electrode that DNA detects, the other end is for contacting the molecular wire of signaling molecule on DNA signal probe, the marking signal molecule of its activation signal DNA probe, make the electrochemical signals receiving target dna strand generation while ensureing sensor low noise as much as possible, improve sensor spirit.
The present invention also proposes a kind of overdelicate DNA electrochemical detection system further, comprises DNA electrochemical sensor, and above-mentioned overdelicate DNA electrochemical detecting reagent.
Said system of the present invention comprises and has at least one three electrode DNA electrochemical sensor and overdelicate DNA electrochemical detecting reagent; In the process of its detection and use, using sensor as the carrier detected, the use of binding reagents simultaneously, the three electrode working electrode of sensor is closed, by using MCH (6-sulfydryl hexanol), working electrode is closed, signal and the background signal of some non-target DNAs can be shielded, realize the reduction to sensor noise signal, improve transducer sensitivity.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is the structural representation that in the overdelicate DNA electrochemical detection method of the embodiment of the present invention, target DNA forms the signal probe chain of " L " shape lower end overlength;
Fig. 2 is the structural representation of the overdelicate DNA electrochemical sensor of the embodiment of the present invention;
Fig. 3 is three electrode material layer schematic diagram of the overdelicate DNA electrochemical sensor of the embodiment of the present invention;
Fig. 4 is the embodiment of the present invention overdelicate DNA Electrochemical Detection result schematic diagram.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention provides a kind of overdelicate DNA electrochemical sensor, and it is based on three-electrode system; Wherein, three electrode working electrode and auxiliary electrode all can adopt gold electrode, contrast electrode to adopt Ag/AgCl electrode.
Testing process of the present invention is carried out detection based on above-mentioned overdelicate DNA electrochemical sensor and is realized, and the step of concrete grammar is according to carrying out as follows:
Working electrode surface is fixed with DNA capture probe during detection, and the surface of working electrode also has by the fixing MCH (6-sulfydryl hexanol) of the mode of Au-S and molecular wire
its concrete form can be shown in Figure 1.
Certainly, known to those skilled in the art, and the biology sensor that existing DNA detects, above-mentioned DNA capture probe is according to target DNA MOLECULE DESIGN to be measured, can hybridize with target DNA complementary element, and its fixed form can adopt common mode to carry out.Such as, coupling agent (EDAC or EDAC and NHS) is utilized, the method that-OH group that gold electrode surfaces is formed and DNA probe 5'-hold phosphate group to be combined by covalent bond.
In the present invention, the MCH fixing in conjunction with the mode of above-mentioned Au-S and molecular wire, DNA capture probe fixed form on the working electrode of golden material adopts golden sulfide linkage (Au-S-C6-DNA) mode to fix, and after it is fixing, the form combined on the working electrode (s can be shown in Figure 1.Its fixing process can adopt first modifies sulfydryl on the 5' end of DNA capture probe, then the sulfydryl orientation on the 5'-end of DNA is combined in metal working electrode surface, can avoids DNA probe non-specific adsorption.The design of DNA signal probe can form complementation with the complementary strand thereof of target dna strand.
Above-mentioned overdelicate DNA electrochemical sensor of the present invention, by using MCH (6-sulfydryl hexanol) to close working electrode, realizes the reduction to sensor noise signal; By in closed process can also doped with one end for combining with the working electrode that DNA detects, the other end is for contacting the molecular wire of signaling molecule on DNA signal probe, the marking signal molecule of its activation signal DNA probe, make the electrochemical signals receiving target dna strand generation while ensureing sensor low noise as much as possible, improve transducer sensitivity.
Meanwhile, proposition is a kind of further in the present invention adopts above-mentioned overdelicate DNA electrochemical sensor to carry out the method for DNA detection, comprises the steps:
S100, working electrode DNA sample to be measured being added into DNA electrochemical sensor carries out first time hybridizes with DNA capture probe;
S200, is added on the working electrode of electrochemical sensor by the specific DNA signal probe that ferrocene marks, and carries out second time hybridization;
S300, three electrodes of the sensor after being hybridized by secondary are infiltrated in damping fluid, carry out detection electrochemical signals, obtain the testing result of DNA to be measured with alternating voltammetry (ACV) or differential pulse voltammetry.
And state on the invention in the process of enforcement, in order to promote degree of accuracy and the quality of detection, time in above-mentioned steps S200 by the design of the specific DNA signal probe of wherein ferrocene mark, make it possess two hybridized fragment regions, the first hybridising region and the second hybridising region can be comprised.
Wherein, the first hybridising region be used for and target dna strand complementary, form " sandwich " structure in conjunction with the common use of DNA capture probe and target dna strand.For another design one assist probes in second hybridising region the present invention, this assist probes also has two hybridising region, is respectively triple-crossing region and the 4th hybridising region; Wherein triple-crossing region can form complementary pairing with the second hybridising region, the 4th hybridising region can with the first hybridising region complementary pairing.The concrete using method of this assist probes, also can include following steps further after S200 step in above-mentioned testing process:
S210, adds assist probes on the working electrode of electrochemical sensor, carries out third time hybridization;
In this step assist probes be designed to can with DNA signal probe Complementary hybridization, because after secondary hybridization, if there is the target DNA molecule of specificity coupling, then in secondary hybridization, the first hybridising region of the specific DNA signal probe that the double-strand of DNA marks with capture probe and ferrocene is respectively combined, final formation deoxyribonucleotide strand " sandwich " structure.Certainly, according to structural design, the result of secondary hybridization is the second hybridising region of DNA signal probe is non-complementary state.
Now, assist probes is added further by continuing on working electrode, carry out three hybridization, make the second hybridising region of the DNA signal probe of " sandwich " structure and the triple-crossing region phase mutual cross of assist probes, and further signal probe side in " sandwich " structure is extended; After hybridization, the 4th hybridising region of this assist probes is not for matching state, it can the carrying out of cofree signal probe matches with working electrode further, be bonded to the first hybridising region of signal probe freely, vacate the second hybridising region of this pairing signal probe simultaneously; Just be cycled to repeat above-mentioned pairing process afterwards, the final hybridization forming the overlength that signal probe is connected mutually with assist probes sequence head and the tail extends; Thus the structure produced at the signal probe chain of " L " shape lower end overlength, as shown in Figure 1.
It is pointed out that the first hybridising region of DNA signal probe in the present invention can comprise two DNA hybridization sections, one of them section is used for hybridizing with target DNA complementary element, and another section is used for the 4th hybridising region of assist probes complementary; And reality is in implementation process, these two sections can be the same or different, or it is all passable to overlap; All can not affect the signal probe chain forming " L " shape lower end overlength after adding assist probes.
Finally, in the structural chain structure of the signal probe chain of this lower end overlength formed, there is multiple signal probe and assist probes, signaling molecule wherein all containing ferrocene derivatives formation on each signal probe, the signal being several times as much as usually signal probe single in " sandwich " structure can be produced, thus make the detectable concentration of sensor to target dna reach fM rank.
Certainly, in above-mentioned embodiment of the present invention, in order to strengthen binding signal further, by the existing specific DNA signal probe usually only having a ferrocene mark, when marking, the quantity of ferrocene mark increases to 4, further can the signal intensity of promotion signal probe.
In the detection method of above-mentioned assist probes of the present invention, by the end to end Crossing system to signal probe and assist probes, formed and promote at double with the signal probe quantity of target DNA crossbred, realize signal and amplify; Thus when the methods such as final alternating voltammetry or differential pulse voltammetry detect, its detection signal can increase greatly, thus reduces the disturbing effect of background signal, improves the sensitivity that super low loading DNA detects.
The present invention also proposes a kind of preparation method of above-mentioned overdelicate DNA electrochemical sensor further, and its process prepared comprises the steps:
S10, obtains substrate;
S20, substrate is prepared electrode material layer and forms three electrodes;
S30, is fixed to capture probe on three electrode working electrode;
S40, with the mixed solution of molecular wire and MCH, carries out infiltration to the working electrode being fixed with capture probe and closes.
Wherein, the preparation method of the above-mentioned overdelicate DNA electrochemical sensor of the present invention, substrate can adopt the flexible and transparent substrates such as the nontransparent substrate such as quartz glass substrate, ceramic bases and PET, PVC, PDMS all can, do not limit at this.
Further, in the step S20 preparing electrode material layer, for the present invention, most preferably working electrode and auxiliary electrode adopt Au electrode, the Au electrode material layer that then can generate in substrate according to designed three electrode shape, can adopt the modes such as magnetron sputtering, electron beam evaporation plating or vacuum ion plating to realize.
Simultaneously, based on combination stability and the quality requirements of electrode, the transition metal layer (as titanium layer) preferentially forming three electrode shapes in substrate can also be adopted before step S20 deposits Au electrode material layer, on the basis of this transition metal layer, carry out Au electrode material layer more afterwards; Further, because general contrast electrode selects Ag/AgCl, on the region of the Au layer that so now can deposit on contrast electrode, cover further by the mode using Ag/AgCl ink to carry out serigraphy and form Ag/AgCl contrast electrode.
And the formation of wherein three electrode shape in above process, can realize by means of mask plate, in the formation of such as Ag/AgCl contrast electrode, the serigraphy mask plate matched with contrast electrode can be adopted, on the Au layer that contrast electrode has precipitated, use the method for serigraphy, carry out serigraphy with Ag/AgCl ink, obtain the Ag/AgCl layer of 5 ~ 10um thickness; And then use insullac to print mask plate, formed after closing insulation course, obtain final Ag/AgCl electrode shape.
Be more preferably, in order to electrode detection is more accurate, in the preparation of electrode, transition metal layer is as the THICKNESS CONTROL 30 ~ 50nm of Ti layer, and gold (Au) layer thickness controls the scope of 80 ~ 150nm.
Meanwhile, in order to make sensor of the present invention more accurate, the conductor part that working electrode, auxiliary electrode, contrast electrode are drawn all uses insulation course to cover, to ensure the only having working portion of electrode can touch detection solution.Insulation course part is also carried out printing according to the mask plate of the profile design according to corresponding insulating layer region and is carried out.
And further, in order to increase the efficiency of the detection of sensor of the present invention, overdelicate DNA electrochemical sensor of the present invention can comprise 8 three electrode units altogether and independently form, shown in Fig. 2-3, can detect multiple sample in conjunction with multi-channel detection equipment and multiple probe system simultaneously.And each three electrode units all can independently not interfere with each other; Further in three electrode unit designs of the present invention, auxiliary electrode 1 is the circular arc of 4 ~ 5mm diameter, the circumference that arc length correspondence is 160 ~ 200 degree; Working electrode 2 is the round-shaped of 1 ~ 1.5mm diameter, is positioned at the circular arc of auxiliary electrode 1; Contrast electrode 3 is circular arcs of 4 ~ 5mm diameter, and arc length, to should the circumference of 30-60 degree, lays respectively at the both sides of working electrode with auxiliary electrode.
After prepared by three electrodes of above-mentioned steps S20, step S30 is immobilized capture probes on the working electrode (s, and its mode can preferably be fixed with reference to above-mentioned golden sulfide linkage (Au-S-C6-DNA) mode.First on the 5' end of DNA capture probe, modify sulfydryl, then the sulfydryl orientation on the 5'-end of DNA is combined in metal working electrode surface, DNA probe non-specific adsorption can be avoided; Or general covalent bond mode carries out all can.
After step S30, molecular wire and MCH are fixed by further S40, realize infiltrating the working electrode being fixed with capture probe closing.
MCH (6-sulfydryl hexanol) is combined by Au-S and is connected to the surface of working electrode in the present invention, thus makes-OH the end dissociative of MCH; Similar with MCH, the molecular structure of the present invention's molecular wire exemplarily
its sulfydryl end is also fixed to and working electrode makes the other end of linear molecule dissociate.
For this molecular wire function in the present invention, technician, in implementation process, if there are other structural molecule of identical function also can replace, does not limit at this.
Simultaneously, in this step S40, fix MCH and molecular wire at the same time, realize in the process closed to the working electrode being fixed with capture probe, preferred employing MCH and molecular wire are closed according to the ratio mixing of 1:1, while adopting control MCH and molecular wire Uniform Doped ratio, the quantity that final signal probe is combined is more conducive to crossbred and extends.
For making above-mentioned implementation detail of the present invention easy to understand more, and those skilled in the art clearly also can be understood, and more can give prominence to the sensitivity of testing result and the convenience of operation, be illustrated by the following examples:
Embodiment 1
In this embodiment 1, be target DNA to be measured with following DNA sequence dna:
5’-TACTTCAGCGGGGAACTAAAAGGTCTGAGGG-3’；
Simultaneously according to the following probe of method design of the optimum of above-mentioned detection of the present invention:
Capture probe: 5 '-HS-C6-TCCCCGCTGAAAGTA-3 '
Signal probe:
5’-TACCCGCAGAAGAAGCCCCTCAGACCTTTTAGT[Fc][Fc][Fc][Fc]-3’
Assist probes:
5’-GCTTCTTCTGCGGGTAACTAAAAGGTCTGAGGG-3’；
Then prepare electrode in accordance with the following steps, and detect:
S00, first uses CAD Software on Drawing sensor design figure, uses the method for cut to obtain multiple mask plate;
S10, select thickness to be 1mm, length is 10cm, and width is the rectangle quartz glass substrate of 7cm, and use ammoniacal liquor, ethanol and distilled water ultrasonic cleaning 3 minutes respectively, nitrogen dries up;
S21, after mask plate is put in substrate, the method using magnetron sputtering to prepare, under the electrode shape of mask plate guides, first sputters titanium (Ti) layer of one deck 30 ~ 50nm, gold (Au) layer of one deck 80 ~ 150nm is sputtered again, parital vacuum 5 × 10 on titanium layer
-5pa, sputtering pressure 0.7Pa, sputtering power 300W, substrate temperature is room temperature.
S22, uses the serigraphy mask plate matched with contrast electrode: on the electrode that step S21 is formed, and uses the method for serigraphy, carries out serigraphy, obtain the Ag/AgCl layer of 5 ~ 10um thickness with Ag/AgCl ink; And then use insullac to print mask plate, formed after closing insulation course, obtain final Ag/AgCl electrode shape.
S23, after prepared by above-mentioned three electrodes, by electrode successively with superoxol, sulfuric acid solution cleaning, has finally dried up for subsequent use with nitrogen.
S31, then film gold electrode surfaces uses the sulfuric acid of 0.1M to use cyclic voltammetry to scan between the scope of-0.2 ~ 1.2V, until scanning curve is stablized, with activated electrode;
S32, be added to 5 ' with the 1mM TCEP of 1 μ l and be modified with in the capture probe solution of sulfydryl, ambient temperatare puts 30min, in order to the disulfide bond that ruptures;
S33, packing after the capture probe solution I buffer after step S32 process is diluted to 1 μM, drops on working electrode, and after incubated at room 1-2 hour, with ultrapure water (18.2M Ω) cleaning, nitrogen dries up; Wherein, I buffer is: 10mM Tris pH7.1+10mM TCEP+1mM EDTA+0.1M NaCl.
S34, to be immersed in the mixed solution containing 1mM molecular wire and 1mMMCH 2 hours by the working electrode after step S33 process; Make working electrode combines and close upper MCH and molecular wire; Re-use, ultrapure water (18.2M Ω) cleans, and nitrogen dries up;
Then sample detection is carried out:
S100, the 1M Tris-HCLbuffer (PH7.4) 5 μ l being contained variable concentrations (the dilution gradients of 10 times) target dna drops on cleaned working electrode, 37 degrees Celsius of hybridization 1 hour, re-use ultrapure water (18.2M Ω) cleaning, nitrogen dried up.
S200, the 1 μM of signal probe newly prepared by 10 μ l and 1 μM of assist probes mixed liquor drop on cleaned working electrode, and after room temperature hybridizes 2 hours, use ultrapure water (18.2M Ω) cleaning, nitrogen dries up;
S300, uses the NaClO of 1M
4as Electrochemical Detection damping fluid, all infiltrated in damping fluid by three-electrode system, use alternating voltammetry (ACV), optimum configurations is 100Hz, carries out detection and obtain result in the interval of-0.3 ~ 0.5V.
Its result detected see shown in figure X, the concentration 0fM (negative control) that the standard specimen to be measured simultaneously adopted in corresponding the present invention carries out with 10 dilution gradients, 1fM, 10fM, 100fM, 1nM; First, under the standard specimen concentration to be measured of super low loading, still have very good detection signal peak curve, bent peak is single, without background signal interference, compare existing add survey sensitivity greatly promote; Further, the size of peak value itself is relevant with concentration, therefore can also carry out concentration calculating according to the curve of surveyed peak value.
And, from the collection of illustrative plates of above-described embodiment, if there is peak value between 0.1 ~ 0.3V, then show that target dna to be detected is combined at biosensor surface with capture probe, signal probe, assist probes, namely show this target dna to be detected, and the height of peak working load can obtain the concentration of target dna; If not there is peak value between 0.1 ~ 0.3V, then show that target cannot combine with probes such as capture probes or cannot detect in conjunction with the electrochemical signals of rear generation is too small, namely show not exist in the too low or solution of DNA concentration to be detected the DNA that native system will detect.
Certainly, based on the angle that the above embodiment of the present invention and business are applied, the present invention also proposes a kind of overdelicate DNA electrochemical detecting reagent further, above-mentioned signal probe and assist probes etc. that reagent can comprise MCH, molecular wire and design according to target DNA to be measured.Technician in use, carries out realizing in conjunction with the electrochemica biological sensor that a DNA detects with reference to said method of the present invention.
Simultaneously in order to the direct use of aspect product more, the present invention also directly proposes a kind of overdelicate DNA electrochemical detection system, directly include above-mentioned electrochemica biological sensor of the present invention and reagent, so technician in use directly can arrange in pairs or groups and use, without the need to separately seeking collocation again.Wherein, reagent and electrochemica biological sensor with reference to the description in above-described embodiment, can not repeat them here.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. an overdelicate DNA electrochemical detection method, is characterized in that, comprise the steps:
Fixed dna capture probe on the working electrode of DNA electrochemical sensor;
The working electrode of the described DNA of being fixed with capture probe fixes MCH;
DNA sample to be measured, DNA signal probe are added on described working electrode and carry out hybridization process;
Detect the electrochemical signals of the rear described working electrode of hybridization process.
2. overdelicate DNA electrochemical detection method as claimed in claim 1, it is characterized in that, after the working electrode of the described DNA of being fixed with capture probe fixes MCH step, described DNA sample to be measured, DNA signal probe are added into before working electrode carries out hybridization treatment step, also comprise:
Fixed member wire on the working electrode being fixed with MCH.
3. overdelicate DNA electrochemical detection method as claimed in claim 2, it is characterized in that, described molecular wire is
4. overdelicate DNA electrochemical detection method as claimed in claim 2 or claim 3, is characterized in that, described DNA sample to be measured, DNA signal probe is added into after working electrode carries out hybridization treatment step, also comprises:
DNA assist probes is added on working electrode and carries out hybridization process; Wherein,
Described DNA signal probe has for the first hybridising region and the second hybridising region with target DNA complementation to be measured;
Described DNA assist probes has triple-crossing region and the 4th hybridising region;
Described triple-crossing region and the complementation of the second hybridising region, described 4th hybridising region and the complementation of the first hybridising region.
5. an overdelicate DNA electrochemical detecting reagent, is characterized in that, comprise one end for combining with the working electrode that DNA detects, the other end is for contacting the molecular wire of signaling molecule on DNA signal probe.
6. overdelicate DNA electrochemical detecting reagent as claimed in claim 5, it is characterized in that, described molecular wire is
7. overdelicate DNA electrochemical detecting reagent as claimed in claim 5, is characterized in that, also comprise MCH.
8. the overdelicate DNA electrochemical detecting reagent as described in any one of claim 5 to 7, is characterized in that, also comprises DNA signal probe and DNA assist probes;
Described DNA signal probe has for the first hybridising region and the second hybridising region with target DNA complementation to be measured;
Described DNA assist probes has triple-crossing region and the 4th hybridising region;
Described triple-crossing region and the complementation of the second hybridising region, described 4th hybridising region and the complementation of the first hybridising region.
9. an overdelicate DNA electrochemical detection system, comprises DNA electrochemical sensor, it is characterized in that, also comprises the overdelicate DNA electrochemical detecting reagent described in any one of claim 5 to 7.
10. overdelicate DNA electrochemical detection system as claimed in claim 9, is characterized in that, described electrochemical sensor comprises at least one three electrode; Wherein,
Described three electrode working electrode comprises and is set in turn in suprabasil transition metal layer and Au layer;
Described three electrode auxiliary electrode comprises and is set in turn in suprabasil transition metal layer and Au layer;
Described three electrode contrast electrode comprises and is set in turn in suprabasil transition metal layer, Au layer and Ag/AgCl layer;
And described transition metal layer thickness is 30 ~ 50nm, the thickness of described Au layer is 80 ~ 150nm.
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