CN105973963A - Construction method for hairpin DNA supported dual-signal molecular sensing interface and application of sensing interface - Google Patents
Construction method for hairpin DNA supported dual-signal molecular sensing interface and application of sensing interface Download PDFInfo
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- 238000010276 construction Methods 0.000 title claims abstract description 11
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 106
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 239000000523 sample Substances 0.000 claims abstract description 33
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229960000907 methylthioninium chloride Drugs 0.000 claims abstract description 29
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000010931 gold Substances 0.000 claims abstract description 21
- 229910052737 gold Inorganic materials 0.000 claims abstract description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000439 tumor marker Substances 0.000 claims abstract description 5
- 230000009977 dual effect Effects 0.000 claims description 35
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims description 21
- 102100034256 Mucin-1 Human genes 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 230000012447 hatching Effects 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000007689 inspection Methods 0.000 claims description 5
- 238000004062 sedimentation Methods 0.000 claims description 5
- 241000416536 Euproctis pseudoconspersa Species 0.000 claims description 4
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 3
- 230000008961 swelling Effects 0.000 claims 3
- 108091008104 nucleic acid aptamers Proteins 0.000 claims 2
- 108091023037 Aptamer Proteins 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 4
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- 238000002474 experimental method Methods 0.000 description 5
- 102000008070 Interferon-gamma Human genes 0.000 description 4
- 108010074328 Interferon-gamma Proteins 0.000 description 4
- 102000016943 Muramidase Human genes 0.000 description 4
- 108010014251 Muramidase Proteins 0.000 description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 4
- 238000004070 electrodeposition Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 229940044627 gamma-interferon Drugs 0.000 description 4
- 229960000274 lysozyme Drugs 0.000 description 4
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- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 2
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- 150000007523 nucleic acids Chemical class 0.000 description 2
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- 238000004365 square wave voltammetry Methods 0.000 description 2
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- 230000005907 cancer growth Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- -1 cyclopentadienyl ferrum Chemical compound 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention discloses a construction method for a hairpin DNA supported dual-signal molecular sensing interface and an application of the sensing interface; the construction method for the sensing interface comprises that a tumor marker aptamer I containing a methylene blue probe and a tumor marker aptamer II containing a ferrocene probe are co-incubated and are modified on the surface of an electrode having the surface modified with nano gold. The hairpin DNA supported dual-signal molecular sensing interface obtained by the method has good stability and low cost, can respectively detect two kinds of tumor markers, also can simultaneously detect the two kinds of tumor markers in a same interface, and has the advantages of simpler detection, low detection limit, high sensitivity and strong specificity.
Description
Technical field
The present invention relates to a kind of hair clip DNA and support the construction method of dual signal molecule sensing interface and with described
The electrochemical method of two kinds of tumor markerses of sensing interface synchronous detecting；Belong to electrochemica biological sensor field.
Background technology
Cancer base antigens c EA is a kind of proteoglycan complex, can be widely present in the Digestive of entoderm origin
System cancer, is a broad spectrum activity tumor markers, tentatively judges the existence of tumor according to the content of CEA in serum,
Estimating curative effect, estimates growth of cancer cells situation；MUC1 mucin is the glycoprotein of a kind of macromolecule, it
Canceration surface epithelial cell Height Anomalies is expressed, and is a kind of common tumor markers, and the serum levels of MUC1 is also
Relevant to the grade malignancy of tumor.Therefore both tumor markerses are detected the detective to cancerous cell and inspection
Survey has highly important effect.But in existing detection method, the sensitivity inspection of detection and concentration range are also
Have much room for improvement, and it is low to be distributed detection efficiency, in order to preferably assess advancing of disease situation, to both marks
The detection of will thing is particularly important, particularly when two kinds of tumor markerses can be detected simultaneously, will be greatly improved
Detection efficiency and accuracy.
Chinese patent (Application No. 201410181470.2) discloses one and detects two kinds of acute leukemia simultaneously
The preparation method of the electrochemical sensor of mark, specifically discloses lysozyme reporter probe and gamma interferon report
Accuse probe to stand in the solution containing TCEP respectively, with opened disulfide bond fit with lysozyme respectively and γ-
Interferon is fit formation duplex structure；Gold electrode is carried out pretreatment, the gold electrode handled well is immersed in pretreatment
After lysozyme reporter probe-fit double-strand and gamma interferon reporter probe-fit double-strand mixed liquor in, room temperature
Stand overnight, clean with redistilled water and cleanout fluid afterwards；Then electrode is immersed in the solution containing MCH
In, with enclosed-electrode, clean with redistilled water and cleanout fluid afterwards；Electrode after above-mentioned steps being processed is made
For working electrode, and reference electrode, electrode is connected on chem workstation, acute to obtain can be used for two kinds
Electrochemical sensor is detected while leukemia mark lysozyme and gamma interferon.But its sensor built
There is also following obvious shortcoming with method: 1, two kinds of probes respectively with corresponding fit formation double-strand time exist
Competitive reaction；The two kinds of hairpin structures formed are connected on electrode there is again competitive reaction, complex steps, sensing
Device character is unstable；2, receive molecule on electrode and there are four kinds of forms: two kinds of probe molecules and two kinds of hair clips
Structural molecule, quantity and the ratio of the effective hairpin structure every time connected are uncertain, its poor repeatability, and
And two kinds of probe molecules form interference to detection on electrode；3, use four kinds of nucleic acid molecules chains, cause sensing
The cost of device is high.
Summary of the invention
The defect existed for the method for existing detection tumor markers, it is an object of the invention to be to provide one
Plant side simple to operate, reproducible, that low cost structure hair clip DNA supports dual signal molecule sensing interface
Method, the sensing interface good stability of structure, signal are strong, specificity is high, highly sensitive, and may be used for detection
One or simultaneously two kinds of tumor markers of detection.
Second object of the present invention is to be to provide a kind of hair clip DNA to support dual signal molecule sensing interface
Application, this biosensor may be respectively used for detecting two kinds of tumor markerses, it is also possible on same interface
Two kinds of tumor markerses of detection simultaneously, and have that detection is simpler, detection limit is low, highly sensitive, high specificity
Feature.
In order to realize above-mentioned technical purpose, the invention provides a kind of hair clip DNA and support dual signal molecule sensing
The construction method at interface, the method comprises the following steps:
(1) at electrode surface by sedimentation modified nano gold；
(2) the fit I of tumor markers containing methylene blue probe and the tumor markers containing ferrocene probe are fitted
Body II is at a temperature of 25～40 DEG C, and in light protected environment, mixing hatches 2～4h in the solution, obtains hatching mixed
Close solution；
Described hatches in mixed solution containing the fit I of tumor markers of methylene blue probe and the probe Han ferrocene
Tumor markers fit II concentration be respectively 1～10 μM and 1～10 μM；
(3) in light protected environment, by surface finish nano gold electrode be placed in described in hatch in mixed solution, incubate
After educating 14～16h, rinse with water；
(4) electrode of the surface finish nano gold after (3) being processed again is placed in MCH solution closing, i.e.
Obtain hair clip DNA and support dual signal molecule sensing interface.
Preferably scheme, electrode described in (1) is placed in the chlorauric acid solution that concentration is 2～5mM, carries out perseverance
Potential deposition, current potential is-0.4V to-0.1V, and sedimentation time is 150～200s, obtains surface finish nano gold electricity
Pole.Depositing after one layer of nanogold particle on electrode, electrode specific surface increases, and is on the one hand conducive to combining fit,
On the other hand can be greatly enhanced signal intensity, possess higher detection limit.
Preferably scheme, hair clip DNA supports dual signal molecule sensing interface and includes containing methylene blue (MB)
The fit I of tumor markers of probe and the fit II of tumor markers containing ferrocene (Fc) probe.
More preferably scheme, the fit I of tumor markers containing methylene blue probe and the tumor containing ferrocene probe
Mark is fit, and II modifies the electrode surface at surface finish nano gold after hatching jointly.
Further preferred scheme, tumor markers is fit, and I is that CEA is fit.
Further preferred scheme, tumor markers is fit, and II is that MUC1 is fit.
The hair clip DNA that the method for the present invention builds supports dual signal molecule sensing interface and uses fit acting on
Corresponding albumen, has higher specificity and discernment, and the sensitivity of detection is higher；Two kinds are simultaneously introduced it
Signaling molecule, respectively two kinds of different tumor markers of detection, do not interfere with each other during detection simultaneously, and detection limit is low, effect
Rate is high.
The hair clip DNA that present invention also offers described method structure supports answering of dual signal molecule sensing interface
With, it is applied to detect fit I pair of tumor markers by described hair clip DNA support dual signal molecule sensing interface
The tumor markers I answered, or the tumor markers II that the detection fit II of tumor markers is corresponding, or simultaneously
The tumor markers I II corresponding tumor-marker fit with tumor markers that the detection fit I of tumor markers is corresponding
Thing II.Sensor of the invention can detect CEA and MUC1 respectively, and can also detect CEA simultaneously
And MUC1, and the variable quantity of the most corresponding two kinds of unlike signal molecules of detection of two kinds of tumor markers, special
The opposite sex, independence and stability are higher.
Hair clip DNA support dual signal molecule sensing interface is used for detecting tumor markers I by preferably scheme,
Before and after adding according to tumor markers I, the variable quantity of methylene blue electrochemical signals detects tumor markers I's
Content；
Or, it is used for detecting tumor markers II by hair clip DNA support dual signal molecule sensing interface, according to
Before and after tumor markers II addition, the variable quantity of ferrocene electrochemical signals detects the content of tumor markers II；
Or, hair clip DNA support dual signal molecule sensing interface is used for detecting simultaneously tumor markers I and
Tumor markers II, tumor markers I and tumor markers II are simultaneously introduced, and add according to tumor markers I
Front and back the variable quantity of methylene blue electrochemical signals detects the content of tumor markers I, according to tumor markers
Before and after II addition, the variable quantity of ferrocene electrochemical signals detects the content of tumor markers II.
More preferably scheme, tumor markers I is CEA.
More preferably scheme, tumor markers II is MUC1.
The fit I of tumor markers containing methylene blue probe of the present invention and the tumor markers containing ferrocene probe
Fit II can be obtained by existing conventional method screening technique.Or directly by being commercially available, such as life
Work biological engineering Shanghai (share) company limited.The tumor markers containing methylene blue probe that the present invention uses
Fit I and the tumor markers fit II one end containing ferrocene probe are modified with sulfydryl, and the main sulfydryl that passes through is with electric
The nanometer gold bonding on surface, pole, is fixed on electrode surface by the most fit.
The molecular probe that the present invention uses, Fc is reducing substances, measures square wave, at 0.35V in PBS
Left and right has the peak of obvious Fc；According to the variable quantity of square wave current, can be with the amount of detection by quantitative MUC1.
MB is also reducing substances, measures square wave, have obvious methylene blue at about-0.27V in PBS
Peak；According to the variable quantity of square wave current, can be with the amount of detection by quantitative CEA.
Hinge structure, the Advantageous Effects that technical solution of the present invention is brought:
1) construction method of the hair clip DNA support dual signal molecule sensing interface of the present invention it is critical only that two
Kind containing the tumor markers of different molecular probe is fit hatch hybridization after, then the electricity modified to surface finish nano gold
Surface, pole.The method can be hatched two kinds of tumor markers aptamer concentrations in crossover process by simple adjustment and be come real
Existing two kinds of fit modification ratios at electrode surface of tumor markers, two kinds fit hybridizes, and there is not competition
Relation, forms an entirety after two kinds of molecule hybridization, is connected on electrode the most there is not competition, and its controllability is strong,
And the sensing interface trim stable components built.Overcome two kinds of tumors of detection simultaneously of the prior art completely
Mark electrochemical sensor existence and stability is poor, the defect of poor repeatability.
2) the hair clip DNA built by the method for the present invention supports dual signal molecule sensing interface, and two kinds contain
The tumor markers of different molecular probe is fit by jointly hatching hybridization, then modifies the electrode modifying gold on surface
Surface, receives the molecule on electrode and there are two kinds of forms: individually detect the fit of a kind of material and support dual signal
Fit, two kinds of fit quantitative proportions are certain, and mark can be carried out by the molecule being connected on electrode
Detection, does not interferes with material, and detection limit is low, and sensitivity is higher.
3) the hair clip DNA support dual signal molecule sensing interface construction method of the present invention is simple, reproducible,
And only with two kinds of nucleic acid molecules chains, cost is the lowest.
4) probe molecule that the hair clip DNA support dual signal molecule sensing interface of the present invention is modified is the most fit,
Can be used directly to detect mark.
5) the hair clip DNA support dual signal molecule sensing interface of the present invention is fit by modifying tumor markers
(aptamer) act on tumor markers, examine according to the size of the electrochemical signals of molecular probe simultaneously
Survey tumor markers content, have highly sensitive, detection limit is low, the feature of high specificity.
6) the hair clip DNA of the present invention supports dual signal molecule sensing interface and has modified two kinds of different tumors simultaneously
Mark is fit and introduces two kinds of signaling molecules, and may be used for detecting a kind of tumor markers can also be simultaneously
Detect two kinds of tumor markerses, it is achieved that detect based on while the tumor markers of two kinds of same interface, there is inspection
Survey is simpler, detection limit is lower, highly sensitive, the feature of high specificity, and two kinds of tumor-markers of detection simultaneously
During thing, not interfereing with each other, detection limit is low, and efficiency is high.
7) the hair clip DNA support dual signal molecule sensing interface of the present invention introduces fit, good stability, and
Fit and corresponding tumor markers can form stable polymer, highly sensitive, detection limit is low.And fit phase
Being compared to antibody, its processing procedure is the simplest, and is easily-synthesized, and easily modifies signaling molecule, it is possible to be easier to inspection
Survey it and go out required electrochemical signals.
8) two kinds of fit sequences that hair clip DNA of the present invention support dual signal molecule sensing interface introduces are variable,
Can be generalized to after fit replacing sequence detect other corresponding marks, such as polypeptide, DNA, RNA,
Sensor can be more widely used, there is certain universality.
Accompanying drawing explanation
[Fig. 1] is for building the process schematic of sensing interface；
[Fig. 2] is that the cv of electrode characterizes before and after electro-deposition
[Fig. 3] is that the EIS of sensing interface building process characterizes；
[Fig. 4] is the SWV figure after the sensing interface surface built adds the MUC1 of a series of variable concentrations；
[Fig. 5] is the linear relationship chart of MUC1 in certain concentration range.
Detailed description of the invention
Following example are intended to further illustrate present invention rather than limit the protection of the claims in the present invention
Scope.
(1) method of the biosensor built on electrode, step is as follows:
Under two can be arrived at 25-40 DEG C with the 10 of partial complementarity μM DNA1-MB and 10 μMs of DNA2-Fc
In dark, 3h is hatched in mixing, obtains DNA mixed liquor.
Electrode carries out potentiostatic electrodeposition in the chlorauric acid solution of 0.5mM, and current potential is-0.25V, sedimentation time
For 180s, surface obtains one layer of nanogold particle, and this layer of nanometer gold can fixed adaptation in a subsequent step
Body, simultaneously works as the effect that signal amplifies.At room temperature, DNA mixture electrode being modified 10 μMs is black
Hatch 15h in the dark, then rinse with secondary water, remove the DNA of residual not have to modify, then by
Electrode is placed in MCH, and the purpose adding MCH is for site unreacted on enclosed-electrode and to make
The DNA modified is rising structure.Owing to methylene blue is shown in that light easily decomposes, so all of operating process is all
Carry out under dark condition.
As follows to the quantitative analysis step of CEA:
(2) prepare the CEA of variable concentrations respectively, be sequentially added into the biosensor of above structure
On, owing to DNA1-MB Partial Fragment is the aptamer of CEA, it can form stable gathering with CEA
Close structure.So that the hairpin structure of DNA1-MB is opened so that MB distance electrode is farther, according to
The variable quantity of DNA-MB upper methylene blue signal carrys out quantitative analysis CEA.
As follows to the quantitative analysis step of MUC1:
(3) it is respectively configured the MUC1 of variable concentrations, is sequentially added into the biosensor of above structure
On, owing to DNA2-Fc is the aptamer of MUC1, it can form stable paradigmatic structure with MUC1
Thus dropping in solution so that on electrode, Fc signal reduces.Variable quantity according to ferrocene signal comes quantitatively
Analyze MUC1.
(4) as follows to the step of the synchronous detecting of CEA and MUC1:
Methylene blue and the ferrocene signal of the sensor that detection prepares are initial signal, are simultaneously introduced different dense
CEA and MUC1 of degree, carrys out detection by quantitative CEA by the variable quantity of methylene blue signal, passes through ferrocene
The variable quantity of signal carrys out detection by quantitative MUC1.
After electro-deposition nanometer gold, the reduction peak of the gold of 0.8-1V occurs as can be seen from Figure 2, and than naked gold electrode
Big, show that nanometer gold successful deposition has arrived on electrode.
As can be seen from Figure 3 along with electrode surface build the most, its impedance increase, surface biological sensor
Success builds.
Gradually increasing from Fig. 4 along with the amount of the tumor markers MUC1 added, its response signal is gradually increased.
In certain concentration range, MUC1 concentration is the biggest as can be seen from Figure 5, and the variable quantity of its electric current is more
Greatly, can tend to balance after reaching a certain concentration, in low strength range, have well its linear relationship.
Comparative example 1
The specific experiment of matched group is as follows: according to the side of disclosed patent (Application No. 201410181470.2)
Method, by two kinds of probes respectively in the solution containing TCEP stand, with opened disulfide bond and respectively with respective phase
The fit formation duplex structure answered；Gold electrode is carried out pretreatment, the gold electrode handled well is soaked after the pre-treatment
Two kinds of probes-fit double-strand mixed liquor in, room temperature stands overnight, afterwards with redistilled water and cleanout fluid
Clean；Then electrode is immersed in the solution containing MCH, with enclosed-electrode, afterwards with redistilled water and
Cleanout fluid cleans；Using above-mentioned steps process after electrode as working electrode, and reference electrode, to electrode connect
On chem workstation, to obtain can be used for the electrochemical sensor that two kinds of marks detect simultaneously.It is added without appointing
What detectable substance, detects methylene blue and the initial signal of ferrocene of the sensor prepared with square wave voltammetry, and
It is calculated the ratio of two kinds of signal magnitude, carries out 5 parallel laboratory tests respectively, obtain the relative mark of 5 experiments
Quasi-deviation.
The specific experiment of experimental group group is as follows: according to methods described herein, by the tumor mark containing methylene blue probe
Will thing is fit I and the fit II of tumor markers containing ferrocene probe is at a temperature of 25～40 DEG C, in light protected environment
In, mixing hatches 2～4h in the solution, obtains the fit I of the tumor markers containing methylene blue probe and containing two
The mixed solution of the fit II of tumor markers of cyclopentadienyl ferrum probe；Gold electrode is carried out pretreatment, and electro-deposition one
Layer nanometer gold, at room temperature, electrode hatches 15h in modifying DNA mixture dark, rinses with secondary water；
Then electrode is immersed in the solution containing MCH, with enclosed-electrode, cleans with redistilled water afterwards；Will
Above-mentioned steps process after electrode as working electrode, and reference electrode, electrode is connected on chem workstation,
To obtain can be used for the electrochemical sensor that two kinds of marks detect simultaneously.It is added without any detectable substance, uses square wave
The methylene blue of the sensor that voltammetry detection prepares and the initial signal of ferrocene, and it is calculated two kinds of signals
The ratio of size, carries out 5 parallel laboratory tests respectively, obtains the relative standard deviation of 5 experiments.
| Experiment number | 1 | 2 | 3 | 4 | 5 | RSD (%) |
| (matched group) IMB/IFc | 1.18 | 0.91 | 1.13 | 1.08 | 0.99 | 10.25 |
| (experimental group) IMB/IFc | 1.64 | 1.6 | 1.62 | 1.59 | 1.63 | 1.28 |
From experimental result can, the RSD of matched group and experimental group is respectively 10.25%, 1.28%, by
This understands, and the sensing interface trim composition that this method builds is more stable, and repeatability is higher.
Claims (8)
1. hair clip DNA supports the construction method of dual signal molecule sensing interface, it is characterised in that: include following step
Rapid:
(1) at electrode surface by sedimentation modified nano gold；
(2) the fit I of tumor markers containing methylene blue probe and the fit II of tumor markers containing ferrocene probe
At a temperature of 25～40 DEG C, in light protected environment, mixing hatches 2～4h in the solution, obtains hatching mixed solution；
Described hatching contains the fit I of tumor markers of methylene blue probe and swelling containing ferrocene probe in mixed solution
II concentration that tumor markers is fit is respectively 1～10 μM and 1～10 μM；
(3) in light protected environment, by surface finish nano gold electrode be placed in described in hatch in mixed solution, hatch
After 14～16h, rinse with water；
(4) electrode of the surface finish nano gold after (3) being processed again is placed in MCH solution closing, must send out
Folder DNA supports dual signal molecule sensing interface.
Hair clip DNA the most according to claim 1 supports the construction method of dual signal molecule sensing interface, its
It is characterised by: electrode described in (1) is placed in the chlorauric acid solution that concentration is 2～5mM, carries out constant potential and sink
Long-pending, current potential is-0.4V to-0.1V, and sedimentation time is 150～200s, obtains surface finish nano gold electrode.
Hair clip DNA the most according to claim 1 supports the construction method of dual signal molecule sensing interface, its
It is characterised by: described hair clip DNA supports dual signal molecule sensing interface and includes swelling containing methylene blue probe
Tumor markers is fit I and the fit II of tumor markers containing ferrocene probe.
Methylene blue the most according to claim 3 and ferrocene dual signal molecular nucleic acid aptamer sensor, it is special
Levy and be: the described fit I of the tumor markers containing methylene blue probe and the tumor-marker containing ferrocene probe
Thing is fit, and II modifies the electrode surface at surface finish nano gold after hatching jointly.
Methylene blue the most according to claim 4 and ferrocene dual signal molecular nucleic acid aptamer sensor, it is special
Levy and be: described tumor markers is fit, and I is that CEA is fit；Described tumor markers is fit, and II is
MUC1 is fit.
6. the hair clip DNA that method described in any one of Claims 1 to 5 builds supports dual signal molecule sensing interface
Application, it is characterised in that: it is applied to detect tumor markers I corresponding to the fit I of tumor markers, or inspection
Survey tumor markers II corresponding to the fit II of tumor markers, or the detection fit I of tumor markers is corresponding simultaneously
Tumor markers I II corresponding tumor markers II fit with tumor markers.
Hair clip DNA the most according to claim 6 supports the application of dual signal molecule sensing interface, and its feature exists
In:
It is used for detecting tumor markers I, according to tumor-marker by hair clip DNA support dual signal molecule sensing interface
Before and after thing I addition, the variable quantity of methylene blue electrochemical signals detects the content of tumor markers I；
Or, it is used for detecting tumor markers II by hair clip DNA support dual signal molecule sensing interface, according to swollen
Before and after tumor markers II addition, the variable quantity of ferrocene electrochemical signals detects the content of tumor markers II；
Or, it is used for detecting tumor markers I simultaneously and swelling by hair clip DNA support dual signal molecule sensing interface
Tumor markers II, tumor markers I and tumor markers II are simultaneously introduced, before adding according to tumor markers I
The variable quantity of rear methylene blue electrochemical signals detects the content of tumor markers I, according to tumor markers II
Before and after addition, the variable quantity of ferrocene electrochemical signals detects the content of tumor markers II.
Hair clip DNA the most according to claim 7 supports the application of dual signal molecule sensing interface, and its feature exists
In: described tumor markers I is CEA；Described tumor markers II is MUC1.
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