CN106434903B - Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof - Google Patents

Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof Download PDF

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CN106434903B
CN106434903B CN201610806532.3A CN201610806532A CN106434903B CN 106434903 B CN106434903 B CN 106434903B CN 201610806532 A CN201610806532 A CN 201610806532A CN 106434903 B CN106434903 B CN 106434903B
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夏建飞
徐姣艳
王宗花
龚世达
孙华
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Qingdao University
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Abstract

The present invention relates to technical field of electrochemical detection, specifically discloses a kind of for detecting tumor suppressor gene P53 modified electrode and preparation method thereof.For detecting the probe of P53 genes, it is made up of a single stranded DNA assist probes S1 and a single stranded DNA capture probe S2, the DNA sequence of the S1 is：5’‑Fc‑CTC TCA GTG ATT TTT TTA GTG AGA GAG‑(CH2)6SH 3 ', the S2 DNA sequence is：5’‑MB‑TCA CTG AGT CTT CCA GTG TGA TGA TCA CT‑3’.Method provided by the present invention prepares simple, manipulation is convenient, use cost is low, compared with the methods of allele function analysis technique separated in Capillary Electrophoresis, denaturing high-performance chromatography, denaturing gradient gel electrophoresis and yeast, the mode of electrochemica biological sensor has sample without pre-treatment, easily manipulation, reaction condition is simple, low cost and other advantages.

Description

Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof
Technical field
The present invention relates to technical field of electrochemical detection, and in particular to a kind of ratio for being used to detect tumor suppressor gene P53 Rate electrochemical DNA biosensor modified electrode and preparation method thereof.
Background technology
Tumor suppressor gene P53 is the tumor suppressor gene that one kind is referred to as " defender of genome ", is a series of isoforms The homologous gene of protein (P53 albumen).There is regulating cell division by a series of protein of the gene code, repair and wither The effect died.When cell damage, P53 albumen can monitor the extent of damage of cytogene.When gene impairing degree is smaller, P53 albumen can promote cell to carry out self-regeneration；And in defective gene unrepairable, P53 albumen then can inducing cell apoptosis. Therefore, when P53 gene mutations, the reparation apoptotic process of cell can be interfered, so that a part of damaged cell is not just Continue merisis under conditions of often, so as to lead oncogenic generation.
International cancer genome association is it has been proved that P53 genes are that frequency the most is mutated in the relevant gene of human cancer Numerous gene.In all malignant tumor tissues known to the mankind, there is the probability being mutated more than 50% in the gene, is so far Untill the mankind find with cancer associated highest gene.
The existing detection for P53 genes mainly has Capillary Electrophoresis, denaturing high-performance chromatography, denaturing gradient gel The methods of allele function analysis technique separated in electrophoresis and yeast, although these methods have high sensitivity, still The shortcomings such as complex operation, cost height be present；Instrument Meteorological is high simultaneously and consumptive material is expensive, also limit facing for these methods Bed application.
The content of the invention
For above-mentioned prior art, an object of the present invention is to provide a kind of probe for being used to detect P53 genes, purpose Two be to provide it is a kind of be used to detecting the application of the probes of P53 genes in the modified electrode for preparing detection P53 genes, purpose it Three are to provide a kind of preparation method for being used to detect the modified electrode of P53 genes, and the fourth purpose is to provide a kind of for detecting P53 The modified electrode of gene, the fifth purpose are to provide a kind of modified electrode and are preparing the electrochemical sensor for detecting P53 genes In application, the sixth purpose be to provide it is a kind of based on double hairpin structures be used for detect P53 genes ratio electrochemical DNA sensing The application process of device.
To achieve these goals, the present invention adopts the following technical scheme that：
One aspect of the present invention, there is provided it is a kind of to be used to detect the probes of P53 genes, by a single stranded DNA assist probes S1 and One single stranded DNA capture probe S2 composition, the DNA sequence of the S1 are：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG-(CH2)6- SH-3 ', the S2 DNA sequence is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT-3’。
Second aspect of the present invention, there is provided a kind of probe using above-mentioned detection P53 genes is preparing repairing for detection P53 genes The application adornd in electrode.
Third aspect present invention, there is provided a kind of preparation method for being used to detect the modified electrode of P53 genes, step are as follows：
(1) assist probes S1 is heated in the Tris-HCl buffer solutions containing three (2- carboxyethyls) phosphines, and annealing obtains single-stranded Assist probes S1 Tris-HCl buffer solutions；
(2) Tris-HCl that the gold electrode after polishing, being cleaned and dried is immersed in the assist probes S1 that step (1) obtains is buffered In liquid, gold electrode surfaces are arrived into assist probes S1 modifications by way of self assembly, obtain S1/GE modified electrodes；
(3) step (2) the electrode obtained is immersed in capture probe S2 Tris-HCl buffer solutions, heating water bath, makes capture Probe S2 is combined with assist probes S1, is formed double hairpin structures in gold electrode surfaces, is obtained S2/S1/GE modified electrodes；
It is preferred that：Polishing in the step (2), concretely comprising the following steps of being cleaned and dried：Gold electrode is polished with alumina powder Minute surface is polished to, then is cleaned by ultrasonic 20-60s through ethanol, redistilled water respectively, nitrogen is dried, and obtains cleaning gold electrode.
It is preferred that：90 DEG C of heating water bath 4-6 minutes in the step (1).
It is preferred that：The Tris-HCl buffer solutions containing three (2- carboxyethyls) phosphines is to be containing concentration in the step (1) 110-150mM trishydroxymethylaminomethane (Tris), 100-120mM NaCl, 1-10mM MgCl2, 9-11mM three (2- Carboxyethyl) phosphine mixed liquor, pH 7.4-7.8.
It is preferred that：The trihydroxy methyl ammonia that it is 110-150mM containing concentration that Tris-HCl buffer solutions, which are, in the step (2), (3) Methylmethane (Tris), 100-120mM NaCl, 1-10mM MgCl2, pH 7.4-7.8.
It is preferred that：It is to the condition of gold electrode surfaces by assist probes S1 modifications in the step (2)：Reaction temperature is 22- 25 DEG C of reaction time are 10-24 hours, the concentration of assist probes S1 buffer solutions for 40-50mM (i.e.：Assist probes S1 in buffer solution Concentration).
It is preferred that：Heating operation is specially in the step (3)：35-37 DEG C of heating 1-3 hour of water-bath, the capture probe Concentration be 35-50mM.
Fourth aspect present invention, there is provided a kind of modified electrode being prepared using above-mentioned preparation method and the modification Electrode is preparing the application in being used to detect the electrochemical sensor of P53 genes.
Fifth aspect present invention, there is provided a kind of method for detecting tumor suppressor gene P53, step are as follows：
(1) by above-mentioned S2/S1/GE modified electrodes in Tris-HCl buffer solutions after heating water bath, with calomel reference electrode Three-electrode system is formed to electrode with platinum filament, DPV detections are carried out in Tris-HCl buffer solutions, obtain the peak current of methylene blue I0(MB), the peak current of ferrocene is I0(Fc)；
(2) the P53 gene buffer solutions of above-mentioned S2/S1/GE modified electrodes and various concentrations are carried out under conditions of heating Tris-HCl wash buffers are used after identification, form three-electrode system with calomel reference electrode and platinum filament comparison electrode afterwards, DPV detections are carried out in Tris-HCl buffer solutions, with the value of obtained ferrocene peak current than the peak current of methylene blue：I=IFc/ IMB；Equation of linear regression is done to the logarithm of P53 mrna concentrations with I, obtains working curve；
(3) when being detected to actual sample, DPV inspections are carried out to the P53 genes in testing sample using same method Survey, try to achieve ratio of the ferrocene peak current to methylene blue peak current, the rear equation of linear regression that substitutes into produces P53 genes in sample Concentration.
It is preferred that：In the step (1), (2), Tris-HCl buffer solutions are the trihydroxy methyl amino first containing 110-150mM Alkane (Tris), 100-120mM NaCl, 1-10mM MgCl2Mixed liquor, its pH is 7.4-7.8.
It is preferred that：In step (1), the heat time is 15-30 minutes.
It is preferred that：In the step (2), P53 genes buffer solution is the trishydroxymethylaminomethane containing 110-150mM (Tris), 100-120mM NaCl, 1-10mM MgCl2And 10-8-10-12The mixed liquor of M P53 genes, its pH are 7.4- 7.8。
It is preferred that：In the step (2), heating is heating water bath, and reaction temperature is 35-37 DEG C, and the reaction time is 15-30 points Clock.
Beneficial effects of the present invention：
(1) method of detection P53 genes provided by the present invention is simple, and manipulation is convenient, and use cost is low, with capillary electricity The side such as allele function analysis technique separated in swimming, denaturing high-performance chromatography, denaturing gradient gel electrophoresis and yeast Method compares, and method of the invention has sample without pre-treatment, easily manipulation, and reaction condition is simple, low cost and other advantages.
(2) the ratio electrochemical methods application based on double hairpin structures is limited due to following technical barrier at present To the detection of P53 genes：First is not easy to build double hairpin structures in electrode surface.First, if assist probes are simply modified to electricity Pole surface and do not form loop-stem structure, then the electroactive material (ferrocene) of assist probes and electrode surface be at a distance of too far, so as to Cause electronics transfer can not occur in electrode surface, electric signal can not be detected.Its is secondary to pass through certain experiment condition and one The reagent induction capture probe and assist probes for determining concentration are combined, and otherwise can not be formed double hairpin structures, also can not just be carried out The measure of ratio.Second can not build enough double hairpin structures.In electrode surface, double optimal states of hairpin structure be with Electrode surface is vertical.If double hairpin structures are laid in electrode surface, the area shared by single double hairpin structures will increase, electricity Double hairpin structures of pole surface modification will be reduced, so as to influence testing result.Instant invention overcomes above-mentioned technical barrier, first Ratio electrochemical methods based on double hairpin structures is applied in the detection of P53 genes to realize high-sensitivity detection (test limit reaches 2.4 × 10-12M while) also have it is easy to operate, cost is low, rapidly and efficiently the advantages that.
(3) present invention utilizes the specific binding of capture probe and P53 genes, (specific based on base pair complementarity principle Principle：After capture probe is combined with P53 genes, because 19 pairs of complementary bases be present in P53 genes and capture probe, and assist probes 10 pairs of complementary bases are only existed with capture probe, cause the adhesion between P53 genes and capture probe to be much larger than assist probes Adhesion between capture probe.Therefore under the conditions of existing for P53 genes, capture probe can be combined with P53 genes and quilt Electrode surface is pulled away from so as to cause MB electric signal to weaken), the selectivity having had and sensitivity can be directly to actual samples In P53 genes carry out Accurate Determining.
(4) present invention has good power of regeneration, and the sensor after use (is referred specifically to：Utilize the modification of the present invention The electrochemical sensor of detection P53 genes prepared by electrode) or modified electrode in S2 cushioning liquid after soaked overnight, for P53 genes still have preferable detectability.
(5) there is presently no the introduction on the ratio electrochemical sensor based on double hairpin structures, modification of the invention Electrode, which is used for the advantages of sensor, mainly two：
Firstth, the defects of in general electrochemical sensor is difficult to overcome electrochemical method itself, i.e. electrochemical signals are unstable Fixed, receipts ectocine is big, poor repeatability etc..But due to ratio (the i.e. electric signal of ferrocene using modified electrode of the present invention Value of electrical signals of the value than methylene blue) electrochemica biological sensor detection is two signals, therefore when sensor is interfered When, the interference suffered by two signals is cancelled out each other, so as to ensure the accuracy of testing result.
Secondth, ratio electrochemical sensor is realized with DNA double hairpin structure so that prepared sensor has good Reproducibility.The advantages of this is not available for common ratio electrochemical sensor.If by the modified electrode after use again It is immersed in S2 cushioning liquid, electrode surface can rebuild double hairpin structures, the still inspection available for P53 genes again Survey.
Brief description of the drawings
Fig. 1 is the schematic diagram of embodiment 1；
Fig. 2 is the linear relationship chart that embodiment 3 detects P53 genes.
Embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Instrument and reagent used in experiment are：(1) instrument：(Shanghai Chen Hua instrument has CHI650 electrochemical workstations Limit company)；Saturated calomel electrode (SCE) is used as reference electrode, platinum electrode is to electrode；(2) reagent：Assist probes, catch Probe and P53 genes (Shanghai Sheng Gong bioengineering limited company) are obtained, other reagents are to analyze pure, experimental water two Secondary distilled water.
Embodiment 1
A kind of preparation method of modified electrode for the P53 genes based on double hairpin structures, as shown in figure 1, including with Lower step：
(1) a single stranded DNA assist probes S1 is designed, the DNA both ends base can be with capture probe chain S2 both ends alkali Base complementary pairing and the complementary pairing sequence for itself there are 10 bases longs, hair can be formed after gold electrode surfaces are fixed on Clamping structure；S1 chains 5 ' it is terminal modified have ferrocene label, the label can produce electrochemical signals；53 ' end sulphur of S1 chains Alcoholization so that S1 can arrive gold electrode surfaces by gold-sulfide linkage modification；Assist probes S1 DNA sequence is：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG-(CH2)6-SH-3’；
(2) a single stranded DNA capture probe S2 is designed, the DNA center section base can match somebody with somebody with P53 gene complementations It is right；Both ends base can form double hair clips with assist probes S1 both ends complementary pairing after being combined with assist probes S1 simultaneously Structure；S2 chains 5 ' it is terminal modified have methylene blue label, the label can produce electrochemical signals；Assist probes S2's DNA sequence is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT-3’；
(3) it is gold electrode is clear through ethanol, redistilled water ultrasound into minute surface, then respectively with alumina powder sanding and polishing Wash, nitrogen is dried, and obtains cleaning gold electrode；
(4) assist probes S1 opened disulfide bonds in the Tris-HCl buffer solutions containing three (2- carboxyethyls) phosphines, after by its 90 DEG C of water-bath is heated 6 minutes, and single-stranded assist probes S1 buffer solutions are obtained with annealing；The Tris-HCl of described three (2- carboxyethyls) phosphine Buffer solution is to contain 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, 9~11mM tri- (2- carboxyethyls) The mixed liquor of phosphine, its pH are 7.4.
(5) gold electrode that step (2) is handled well is immersed in into step (3) to obtain in assist probes S1 buffer solutions, the auxiliary The concentration of probe S1 buffer solutions is 40mM, reaction temperature be 22 DEG C of reaction time be 24 hours, by assist probes S1 modifications to gold Electrode surface, obtain S1/GE modified electrodes.
(6) step (5) the electrode obtained is immersed in capture probe S2 buffer solutions, the concentration of the capture probe S2 is 50mM, heating water bath, reaction temperature are 37 DEG C, and the reaction time is 1 hour, capture probe S2 is combined with assist probes S1, Gold electrode surfaces form double hair frame structures, obtain S2/S1/GE modified electrodes；
Embodiment 2
A kind of preparation method of modified electrode for the P53 genes based on double hairpin structures, as shown in figure 1, including with Lower step：
(1) a single stranded DNA assist probes S1 is designed, the DNA both ends base can be with capture probe chain S2 both ends alkali Base complementary pairing and the complementary pairing sequence for itself there are 10 bases longs, hair can be formed after gold electrode surfaces are fixed on Clamping structure；S1 chains 5 ' it is terminal modified have ferrocene label, the label can produce electrochemical signals；53 ' end sulphur of S1 chains Alcoholization so that S1 arrives gold electrode surfaces by gold-sulfide linkage modification；Wherein, assist probes S1 DNA sequence is：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG-(CH2)6-SH-3’；
(2) a single stranded DNA capture probe S2 is designed, the DNA center section base can match somebody with somebody with P53 gene complementations It is right；Both ends base can form double hair clips with assist probes S1 both ends complementary pairing after being combined with assist probes S1 simultaneously Structure；S2 chains 5 ' it is terminal modified have methylene blue label, the label can produce electrochemical signals；Assist probes S2's DNA sequence is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT-3’；
(3) by gold electrode with alumina powder sanding and polishing into minute surface, then respectively through ethanol, redistilled water be cleaned by ultrasonic 60s, nitrogen are dried, and obtain cleaning gold electrode；
(4) assist probes S1 opened disulfide bonds in the Tris-HCl buffer solutions containing three (2- carboxyethyls) phosphines, after by its 90 DEG C of water-bath is heated 4 minutes, and single-stranded assist probes S1 buffer solutions are obtained with annealing；The Tris-HCl of described three (2- carboxyethyls) phosphine Buffer solution is to contain 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, 9~11mM tri- (2- carboxyethyls) The mixed liquor of phosphine, its pH are 7.8.
(5) gold electrode that step (2) is handled well is immersed in into step (3) to obtain in assist probes S1 buffer solutions, the auxiliary The concentration of probe S1 buffer solutions is 50mM, reaction temperature be 25 DEG C of reaction time be 22 hours, by assist probes S1 modifications to gold Electrode surface,
(6) step (4) the electrode obtained is immersed in capture probe S2 buffer solutions, the concentration of the capture probe S2 is 35mM, heating water bath, reaction temperature are 35 DEG C, and the reaction time is 3 hours, capture probe S2 is combined with assist probes S1, Gold electrode surfaces form double hairpin structures, obtain S2/S1/GE modified electrodes.
Embodiment 3
The application of modified electrode prepared by embodiment 1 in P53 genes are detected, step are as follows：
The modified electrode of embodiment 1 is immersed in Tris-HCl buffer solutions, the Tris-HCl buffer solutions be containing 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl mixed liquor, its pH are 7.4, and heating water bath 15 divides Clock.Then three-electrode system is formed with calomel reference electrode and platinum filament comparison electrode, DPV inspections is carried out in Tris-HCl buffer solutions Survey, obtain the peak current I of methylene blue0(MB), the peak current of ferrocene is I0(Fc), repeatedly average.
A series of p53 genes cushioning liquid are configured, the wherein concentration of cushioning liquid and pH value is kept constant, p53 genes Concentration is adjusted to 10 respectively-8, 5 × 10-9, 10-9, 5 × 10-10, 10-10, 5 × 10-11, 10-11, 10-12M, then by the electricity of gained Pole is immersed in p53 cushioning liquid, and 35 DEG C of water-bath is heated 15 minutes, and the electrochemistry that each sample is determined with three-electrode method is believed Number, and linear fit (i.e. I is carried out to the p53 genes of measureFc/IMBWith the linear relationship of the logarithm of p53 mrna concentrations).
DPV detections are carried out to the P53 genes in actual sample using same method using constructed three-electrode system, asked Oxidation of Ferrocene electric current to the ratio of methylene blue oxidation current, substitute into equation of linear regression and determine P53 genes in sample Concentration, to examine actual analysis ability of the present invention in biological sample or complex system, referring to Fig. 2 (six points in figure Concentration is 1 × 10-8, 5 × 10-9, 1 × 10-9, 5 × 10-10, 1 × 10-10, 5 × 10-11, 1 × 10-11M)。
The testing result of P53 genes is shown, various concentrations P53 gene peak current curves changed substantially, P53 genes It is the detection of the electrochemica biological sensor of the application to lysozyme into good linear relationship in 0.01~100nM in concentration Limit can as little as 2.4 × 10-12M, instrument cost is low, high sensitivity, rapidly and efficiently.
Embodiment 4
The application of modified electrode prepared by embodiment 1 in P53 genes are detected, step are as follows：
The modified electrode of embodiment 1 is immersed in Tris-HCl buffer solutions, the Tris-HCl buffer solutions are to contain 110- 150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl mixed liquor, its pH are 7.8, heating water bath 30 minutes.So Three-electrode system is formed with calomel reference electrode and platinum filament comparison electrode afterwards, DPV detections is carried out in Tris-HCl buffer solutions, obtains The peak current I of methylene blue0(MB), the peak current of ferrocene is I0(Fc), repeatedly average.
A series of p53 genes cushioning liquid are configured, the wherein concentration of cushioning liquid and pH value is kept constant, p53 genes Concentration is adjusted to 10 respectively-8, 5 × 10-9, 10-9, 5 × 10-10, 10-10, 5 × 10-11, 10-11, 10-12M, then by the electricity of gained Pole is immersed in p53 cushioning liquid, and 35 DEG C of water-bath is heated 30 minutes, and the electrochemistry that each sample is determined with three-electrode method is believed Number, and linear fit (i.e. I is carried out to the p53 genes of measureFc/IMBWith the linear relationship of the logarithm of p53 mrna concentrations).
DPV detections are carried out to the P53 genes in actual sample using same method using constructed three-electrode system, asked Oxidation of Ferrocene electric current to the ratio of methylene blue oxidation current, substitute into equation of linear regression and determine P53 genes in sample Concentration, to examine actual analysis ability of the present invention in biological sample or complex system.
The testing result of P53 genes is shown, various concentrations P53 gene peak current curves changed substantially, P53 genes It is the detection of the electrochemica biological sensor of the application to lysozyme into good linear relationship in 0.01~100nM in concentration Limit can as little as 2.4 × 10-12M, instrument cost is low, high sensitivity, rapidly and efficiently.
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention The limitation enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not Need to pay various modifications or deformation that creative work can make still within protection scope of the present invention.

Claims (13)

1. a kind of probe for being used to detect P53 genes, it is characterized in that：By a single stranded DNA assist probes S1 and a single stranded DNA Capture probe S2 is formed, and the DNA sequence of the S1 is：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG- (CH2)6- SH-3 ', the S2 DNA sequence is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT- 3’。
2. application of the probe for being used to detect P53 genes described in claim 1 in the modified electrode for preparing detection P53 genes.
3. a kind of preparation method for being used to detect the modified electrode of P53 genes, it is characterized in that：Step is as follows：
（1）Assist probes S1 is heated in the Tris-HCl buffer solutions containing three (2- carboxyethyls) phosphines, and annealing obtains single-stranded auxiliary Probe S1 Tris-HCl buffer solutions；
（2）Gold electrode after polishing, being cleaned and dried is immersed in step（1）Obtained assist probes S1 Tris-HCl buffer solutions In, gold electrode surfaces are arrived into assist probes S1 modifications by way of self assembly, obtain S1/GE modified electrodes；
（3）By step（2）The electrode obtained is immersed in capture probe S2 Tris-HCl buffer solutions, heating, make capture probe S2 with it is auxiliary Help probe S1 to combine, form double hairpin structures in gold electrode surfaces, obtain S2/S1/GE modified electrodes；
The step（1）In containing the Tris-HCl buffer solutions of three (2- carboxyethyls) phosphines be the trihydroxy methyl containing 110-150mM Aminomethane, 100-120mM NaCl, 1-10mM MgCl2, the mixed liquor of 9-11mM three (2- carboxyethyls) phosphines, pH is 7.4-7.8；The S1 and S2 is as described in the appended claim 1.
4. preparation method as claimed in claim 3, it is characterized in that：The step（1）Middle heating is specially：90 DEG C of heating water baths 4-6 minutes.
5. preparation method as claimed in claim 3, it is characterized in that：The step（2）Middle polishing, the specific steps being cleaned and dried For：By gold electrode with alumina powder sanding and polishing into minute surface, then it is cleaned by ultrasonic 20-60s through ethanol, redistilled water respectively, Nitrogen is dried, and obtains cleaning gold electrode.
6. preparation method as claimed in claim 3, it is characterized in that：The step（2）It is middle that assist probes S1 modifications is electric to gold The condition on pole surface is：Reaction temperature is 22-25 DEG C, and the reaction time is 10-24 hours, the concentration of assist probes S1 buffer solutions For 40-50mM.
7. preparation method as claimed in claim 3, it is characterized in that：The step（3）Middle heating is specially：35-37 DEG C of water-bath 1-3 hours are heated, the concentration of the capture probe S2 is 35-50mM.
8. the modified electrode that any described preparation methods of claim 3-7 are prepared is being prepared for detecting P53 genes Application in electrochemical sensor.
9. a kind of method for detecting caused by tumor suppressor p 53, it is characterized in that：Step is as follows：
（1）By S2/S1/GE modified electrodes in Tris-HCl buffer solutions after heating water bath, with calomel reference electrode and platinum filament pair Electrode forms three-electrode system, and DPV detections are carried out in Tris-HCl buffer solutions, obtain the peak current I of methylene blue0（MB）, two The peak current of luxuriant iron is I0（Fc）；
（2）Used after the P53 genes buffer solution of S2/S1/GE modified electrodes and various concentrations is identified under conditions of heating Tris-HCl wash buffers, three-electrode system is formed with calomel reference electrode and platinum filament comparison electrode afterwards, in Tris-HCl DPV detections are carried out in buffer solution, with the value of obtained ferrocene peak current than the peak current of methylene blue：I=IFc/IMB；With I pairs The logarithm of P53 mrna concentrations does equation of linear regression, obtains working curve；
（3）When being detected to actual sample, DPV detections are carried out to the P53 genes in testing sample using same method, asked Ferrocene peak current to the ratio of methylene blue peak current, the rear equation of linear regression that substitutes into produces the dense of P53 genes in sample Degree；
The step（1）、（2）In S2/S1/GE modified electrodes be to be prepared by any described preparation methods of claim 3-7 Obtain.
10. method as claimed in claim 9, it is characterized in that：The step（1）、（2）In, Tris-HCl buffer solutions be containing 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mM MgCl2Mixed liquor, its pH is 7.4-7.8.
11. method as claimed in claim 9, it is characterized in that：The step（1）In, the heating water bath time is 15-30 minutes.
12. method as claimed in claim 9, it is characterized in that：The step（2）In, P53 genes buffer solution is to contain 110- 150mM Tris-HCl, 100-120mM NaCl, 1-10mM MgCl2And 10-12-10-8The mixed liquor of MP53 genes, its PH is 7.4-7.8.
13. method as claimed in claim 9, it is characterized in that：The step（2）In, heating is heating water bath, and reaction temperature is 35-37 DEG C, the reaction time is 15-30 minutes.
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