CN106434903A - Ratio electrochemical DNA sensor-modified electrode for detecting gene P53 and preparation method of modified electrode - Google Patents

Ratio electrochemical DNA sensor-modified electrode for detecting gene P53 and preparation method of modified electrode Download PDF

Info

Publication number
CN106434903A
CN106434903A CN201610806532.3A CN201610806532A CN106434903A CN 106434903 A CN106434903 A CN 106434903A CN 201610806532 A CN201610806532 A CN 201610806532A CN 106434903 A CN106434903 A CN 106434903A
Authority
CN
China
Prior art keywords
electrode
gene
tris
modified electrode
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201610806532.3A
Other languages
Chinese (zh)
Other versions
CN106434903B (en
Inventor
夏建飞
徐姣艳
王宗花
龚世达
孙华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao University
Original Assignee
Qingdao University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qingdao University filed Critical Qingdao University
Priority to CN201610806532.3A priority Critical patent/CN106434903B/en
Publication of CN106434903A publication Critical patent/CN106434903A/en
Application granted granted Critical
Publication of CN106434903B publication Critical patent/CN106434903B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Electrochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of electrochemical detection, and particularly discloses a modified electrode for detecting a tumor suppressor gene P53 and a preparation method of the modified electrode. A probe for detecting the gene P53 is composed of a single-stranded DNA auxiliary probe S1 and a single-stranded DNA capturing probe S2, wherein the DNA strand sequence of the S1 is 5'-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG-(CH2)6-SH-3', and the DNA strand sequence of the S2 is 5'-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT-3'. According to the method, preparation is easy, control is convenient, the use cost is low, and compared with the methods such as capillary electrophoresis, denaturing high performance liquid chromatography, denaturing gel gradient electrophoresis and a yeast-separated allele function analysis technique, the electrochemical biosensor mode has the advantages that samples do not need to be pretreated, control is easy, the reaction conditions are simple, and the cost is low.

Description

Ratio electrochemical DNA biosensor modified electrode of detection 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 detecting tumor suppressor gene P53 Rate electrochemical DNA biosensor modified electrode and preparation method thereof.
Background technology
Tumor suppressor gene P53 is the antioncogene that one kind is referred to as " defender of genome ", is a series of isoforms The homologous geness of protein (P53 albumen).By a series of protein of the gene code, there is regulating cell division, repair and wither The effect that dies.When cell damage, P53 albumen can monitor the extent of damage of cytogene.In gene impairing degree compared with hour, 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 mutation, 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 gene is to be mutated frequency the most in the relevant gene of human cancer Numerous gene.In all malignant tumor tissues known to the mankind, there is the probability of mutation more than 50% in the gene, be so far Till the mankind find with cancer associated highest gene.
The existing detection for P53 gene mainly has capillary electrophoresis, denaturing high-performance chromatography, denaturing gradient gel The method such as detached allele function analysis technique in electrophoresis and yeast, although these methods have high sensitivity, but There are complex operation, the shortcomings such as cost height；While instrument Meteorological is high and consumptive material is expensive, facing for these methods is also limit Bed application.
Content of the invention
For above-mentioned prior art, an object of the present invention is to provide a kind of probe for detecting P53 gene, purpose Two be provide a kind of for detect the probe of P53 gene prepare detection P53 gene modified electrode in application, purpose it Three is to provide a kind of preparation method for detecting the modified electrode of P53 gene, and the four of purpose is to provide one kind for detecting P53 The modified electrode of gene, the five of purpose is to provide a kind of modified electrode to prepare the electrochemical sensor for detecting P53 gene In application, the six of purpose is to provide a kind of to sense for the ratio electrochemical DNA for detecting P53 gene based on double hairpin structures The application process of device.
To achieve these goals, the present invention is adopted the following technical scheme that：
One aspect of the present invention, provides a kind of probe for detecting P53 gene, by a single stranded DNA assist probes S1 and One single stranded DNA capture probe S2 constitutes, and the DNA sequence of the S1 is：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG-(CH2)6The DNA sequence of-SH-3 ', the S2 is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT-3’.
Second aspect present invention, provides a kind of probe using above-mentioned detection P53 gene and is preparing repairing for detection P53 gene Application in decorations electrode.
Third aspect present invention, provides a kind of preparation method for detecting the modified electrode of P53 gene, and step is as follows：
(1) assist probes S1 is heated in the Tris-HCl buffer containing three (2- carboxyethyl) phosphine, and annealing obtains single-stranded The Tris-HCl buffer of assist probes S1；
(2) gold electrode after polishing, cleaning-drying is immersed in the Tris-HCl buffering of the assist probes S1 that step (1) is obtained In liquid, by way of self assembly, assist probes S1 is modified to gold electrode surfaces, obtain S1/GE modified electrode；
(3) step (2) the electrode obtained is immersed in the Tris-HCl buffer of capture probe S2, heating in 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 electrode；
It is preferred that：Polishing in step (2), the concretely comprising the following steps of cleaning-drying：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 dries, obtain cleaning gold electrode.
It is preferred that：90 DEG C of heating in water bath 4-6 minutes in step (1).
It is preferred that：In step (1), the Tris-HCl buffer containing three (2- carboxyethyl) phosphine is to be containing concentration The trishydroxymethylaminomethane (Tris) of 110-150mM, the MgCl of NaCl, the 1-10mM of 100-120mM2, the three (2- of 9-11mM Carboxyethyl) phosphine mixed liquor, pH be 7.4-7.8.
It is preferred that：In step (2), (3), Tris-HCl buffer is the trihydroxy methyl ammonia containing concentration for 110-150mM Methylmethane (Tris), the MgCl of NaCl, the 1-10mM of 100-120mM2, pH is 7.4-7.8.
It is preferred that：In step (2), assist probes S1 modification to the condition of gold electrode surfaces is：Reaction temperature is 22- 25 DEG C of response time are 10-24 hour, and the concentration of assist probes S1 buffer is for 40-50mM (i.e.：Assist probes S1 in buffer Concentration).
It is preferred that：In step (3), heating operation is specially：35-37 DEG C of heating 1-3 hour of water-bath, the capture probe Concentration be 35-50mM.
Fourth aspect present invention, provides a kind of modified electrode for preparing using above-mentioned preparation method and the modification Electrode prepare for detect P53 gene electrochemical sensor in application.
Fifth aspect present invention, provides a kind of method of detection tumor suppressor gene P53, and step is as follows：
(1) by above-mentioned S2/S1/GE modified electrode after heating in water bath in Tris-HCl buffer, with calomel reference electrode With platinum filament, three-electrode system being constituted to electrode, DPV detection is carried out in Tris-HCl buffer, obtains the peak current of methylene blue I0(MB), the peak current of ferrocene is I0(Fc)；
(2) the P53 gene buffer by above-mentioned S2/S1/GE modified electrode with variable concentrations is carried out under conditions of heating Tris-HCl wash buffer is used after identification, constitutes three-electrode system with calomel reference electrode and platinum filament comparison electrode afterwards, Carry out DPV detection in Tris-HCl buffer, with the ferrocene peak current that obtains than the peak current of methylene blue value：I=IFc/ IMB；Equation of linear regression is done to the logarithm of P53 mrna concentration with I, obtains working curve；
(3) when detecting to actual sample, DPV inspection is carried out to the P53 gene in testing sample using same method Survey, ratio of the ferrocene peak current to methylene blue peak current is tried to achieve, substitute into equation of linear regression afterwards and obtain final product P53 gene in sample Concentration.
It is preferred that：In step (1), (2), Tris-HCl buffer is the trihydroxy methyl amino first containing 110-150mM Alkane (Tris), the MgCl of NaCl, the 1-10mM of 100-120mM2Mixed liquor, its pH be 7.4-7.8.
It is preferred that：In step (1), heat time heating time is 15-30 minute.
It is preferred that：In step (2), P53 gene buffer is the trishydroxymethylaminomethane containing 110-150mM (Tris), the MgCl of NaCl, the 1-10mM of 100-120mM2And 10-8-10-12The mixed liquor of the P53 gene of M, its pH is 7.4- 7.8.
It is preferred that：In step (2), heating is heating in water bath, and reaction temperature is that 35-37 DEG C, the response time divides for 15-30 Clock.
Beneficial effects of the present invention：
(1) method of detection P53 gene provided by the present invention is simple, and manipulation is convenient, and use cost is low, with capillary tube electricity The side such as detached allele function analysis technique in swimming, denaturing high-performance chromatography, denaturing gradient gel electrophoresises and yeast Method compares, and the method for the present invention has sample without the need for pre-treatment, easily manipulates, and reaction condition is simple, low cost and other advantages.
(2) at present due to following technical barrier, the ratio electrochemical methods application based on double hairpin structures is limited Detection to P53 gene：First is difficult to build double hairpin structures in electrode surface.First, if assist probes are modification 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 electron transfer occur in electrode surface, it is impossible to detect the signal of telecommunication.Which is secondary by certain experiment condition and one Determine the reagent induction capture probe of concentration and assist probes combine, double hairpin structures cannot be otherwise formed, also cannot just be carried out The measure of ratio.Second cannot build enough double hairpin structures.In electrode surface, the optimal state of double hairpin structures be with Electrode surface is vertical.If double hairpin structures are laid in electrode surface, the area shared by single pair of hairpin structure will increase, electricity Double hairpin structures of pole surface modification will be reduced, so as to affect testing result.Instant invention overcomes above-mentioned technical barrier, first To be applied in the detection of P53 gene realizing high-sensitivity detection based on the ratio electrochemical methods of double hairpin structures (test limit reaches 2.4 × 10-12M while) also have easy to operate, low cost, rapidly and efficiently the advantages of.
(3) present invention utilizes the specific binding of capture probe and P53 gene, (concrete based on base pair complementarity principle Principle：After capture probe is combined with P53 gene, due to P53 gene and capture probe 19 pairs of complementary bases of presence, and assist probes 10 pairs of complementary bases are only existed with capture probe, cause the adhesion between P53 gene and capture probe much larger than assist probes Adhesion between capture probe.Therefore, under conditions of the presence of P53 gene, capture probe can be combined with P53 gene and quilt Electrode surface is pulled away from so as to cause the signal of telecommunication of MB to weaken), with good selectivity and sensitivity, can be directly to actual sample In P53 gene carry out Accurate Determining.
(4) present invention has good regeneration capacity, and the sensor after use (is referred specifically to：Modification using the present invention Electrode prepare detection P53 gene electrochemical sensor) or modified electrode after soaked overnight in the buffer solution of S2, for P53 gene still has preferable power of test.
(5) introduction with regard to the ratio electrochemical sensor based on double hairpin structures, the modification of the present invention there is presently no Electrode is used for the advantage of sensor mainly two：
Firstth, general electrochemical sensor is difficult to overcome the defect of electrochemical method itself, i.e. electrochemical signals shakiness Fixed, receive ectocine greatly, poor repeatability etc..But due to ratio (the i.e. signal of telecommunication of ferrocene using modified electrode of the present invention Value is than the value of electrical signals of methylene blue) electrochemica biological sensor detection be 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 so that prepared sensor has good with DNA double hairpin structure Reproducibility.This is the advantage not available for common ratio electrochemical sensor.If by the modified electrode after use again It is immersed in the buffer solution of S2, electrode surface can rebuild double hairpin structures again, still can be used for the inspection of P53 gene Survey.
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 gene.
Specific embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Instrument and reagent used in experiment be：(1) instrument：(Shanghai occasion China instrument has CHI650 electrochemical workstation Limit company)；Using saturated calomel electrode (SCE) be reference electrode, platinum electrode be to electrode；(2) reagent：Assist probes, catch Obtain probe and P53 gene (Shanghai Sheng Gong biological engineering limited company), other reagents be analysis pure, experimental water be two Secondary distilled water.
Embodiment 1
A kind of preparation method of the modified electrode of the P53 gene for 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 two ends base can be with capture probe chain S2 two ends alkali Base complementary pairing and itself there is the complementary pairing sequence of 10 bases longs, can be formed after gold electrode surfaces are fixed on and send out Clamping structure；S1 chain 5 ' terminal modified have ferrocene label, the label can produce electrochemical signals；53 ' end sulfur of S1 chain Alcoholization so that S1 can pass through gold-sulfide linkage and modify to gold electrode surfaces；The DNA sequence of assist probes S1 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, and the DNA mid portion base can be joined with P53 gene complementation Right；While two ends base can combined the double hair clips of rear formation with assist probes S1 two ends complementary pairing with assist probes S1 Structure；S2 chain 5 ' 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) gold electrode alumina powder sanding and polishing is become minute surface, then clear through ethanol, redistilled water ultrasound respectively Wash, nitrogen dries, obtain cleaning gold electrode；
(4) assist probes S1 containing three (2- carboxyethyl) phosphine Tris-HCl buffer in opened disulfide bond, after by its 90 DEG C of water-bath is heated 6 minutes, obtains single-stranded assist probes S1 buffer to anneal；The Tris-HCl of three (the 2- carboxyethyl) phosphine Buffer be containing 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, 9～11mM tri- (2- carboxyethyl) The mixed liquor of phosphine, its pH is 7.4.
(5) gold electrode for handling step (2) well is immersed in step (3) and obtains in assist probes S1 buffer, the auxiliary The concentration of probe S1 buffer is 40mM, and it is 24 hours in 22 DEG C of response time that reaction temperature is, assist probes S1 is modified to gold Electrode surface, obtains S1/GE modified electrode.
(6) step (5) the electrode obtained is immersed in capture probe S2 buffer, the concentration of the capture probe S2 is 50mM, heating in water bath, reaction temperature is 37 DEG C, and the response time is 1 hour, so that capture probe S2 is combined with assist probes S1, Gold electrode surfaces form double frame structures, obtain S2/S1/GE modified electrode；
Embodiment 2
A kind of preparation method of the modified electrode of the P53 gene for 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 two ends base can be with capture probe chain S2 two ends alkali Base complementary pairing and itself there is the complementary pairing sequence of 10 bases longs, can be formed after gold electrode surfaces are fixed on and send out Clamping structure；S1 chain 5 ' terminal modified have ferrocene label, the label can produce electrochemical signals；53 ' end sulfur of S1 chain Alcoholization so that S1 is modified to gold electrode surfaces by gold-sulfide linkage；Wherein, the DNA sequence of assist probes S1 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, and the DNA mid portion base can be joined with P53 gene complementation Right；While two ends base can combined the double hair clips of rear formation with assist probes S1 two ends complementary pairing with assist probes S1 Structure；S2 chain 5 ' 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) gold electrode alumina powder sanding and polishing is become minute surface, then is cleaned by ultrasonic through ethanol, redistilled water respectively 60s, nitrogen dries, and obtains cleaning gold electrode；
(4) assist probes S1 containing three (2- carboxyethyl) phosphine Tris-HCl buffer in opened disulfide bond, after by its 90 DEG C of water-bath is heated 4 minutes, obtains single-stranded assist probes S1 buffer to anneal；The Tris-HCl of three (the 2- carboxyethyl) phosphine Buffer be containing 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, 9～11mM tri- (2- carboxyethyl) The mixed liquor of phosphine, its pH is 7.8.
(5) gold electrode for handling step (2) well is immersed in step (3) and obtains in assist probes S1 buffer, the auxiliary The concentration of probe S1 buffer is 50mM, and it is 22 hours in 25 DEG C of response time that reaction temperature is, assist probes S1 is modified to gold Electrode surface,
(6) step (4) the electrode obtained is immersed in capture probe S2 buffer, the concentration of the capture probe S2 is 35mM, heating in water bath, reaction temperature is 35 DEG C, and the response time is 3 hours, so that capture probe S2 is combined with assist probes S1, Gold electrode surfaces form double hairpin structures, obtain S2/S1/GE modified electrode.
Embodiment 3
The application of modified electrode prepared by embodiment 1 in detection P53 gene, step is as follows：
The modified electrode of embodiment 1 is immersed in Tris-HCl buffer, the Tris-HCl buffer be containing The mixed liquor of 110-150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, its pH is 7.4,15 points of heating in water bath Clock.Then three-electrode system being constituted with calomel reference electrode and platinum filament comparison electrode, carries out DPV inspection in Tris-HCl buffer Survey, obtain the peak current I of methylene blue0(MB), the peak current of ferrocene is I0(Fc), repeatedly average.
A series of p53 gene buffer solution are configured, the wherein concentration of buffer solution and pH value keeps constant, p53 gene 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 the buffer solution of p53, and 35 DEG C of water-bath is heated 15 minutes, determines the electrochemistry letter of each sample with three-electrode method Number, and linear fit (i.e. I is carried out to the p53 gene for determiningFc/IMBLinear relationship with the logarithm of p53 mrna concentration).
Using constructed three-electrode system, DPV detection is carried out to the P53 gene in actual sample using same method, ask Ratio of the Oxidation of Ferrocene electric current to methylene blue oxidation current, substitute into equation of linear regression and determine P53 gene in sample Concentration, to check actual analysis ability of the present invention in biological sample or complex system, referring to Fig. 2 (six points of 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 gene is shown, variable concentrations P53 gene peak current curve is all changed substantially, P53 gene It is in 0.01～100nM, to become good linear relationship in concentration, the detection of the electrochemica biological sensor of the application to lysozyme Limit can as little as 2.4 × 10-12M, instrument cost is low, sensitivity height, rapidly and efficiently.
Embodiment 4
The application of modified electrode prepared by embodiment 1 in detection P53 gene, step is as follows：
1 modified electrode of embodiment is immersed in Tris-HCl buffer, the Tris-HCl buffer be containing 110- The mixed liquor of 150mM Tris-HCl, 100-120mM NaCl, 1-10mMMgCl, its pH is 7.8, heating in water bath 30 minutes.So Three-electrode system being constituted with calomel reference electrode and platinum filament comparison electrode afterwards, DPV detection is carried out in Tris-HCl buffer, obtain The peak current I of methylene blue0(MB), the peak current of ferrocene is I0(Fc), repeatedly average.
A series of p53 gene buffer solution are configured, the wherein concentration of buffer solution and pH value keeps constant, p53 gene 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 the buffer solution of p53, and 35 DEG C of water-bath is heated 30 minutes, determines the electrochemistry letter of each sample with three-electrode method Number, and linear fit (i.e. I is carried out to the p53 gene for determiningFc/IMBLinear relationship with the logarithm of p53 mrna concentration).
Using constructed three-electrode system, DPV detection is carried out to the P53 gene in actual sample using same method, ask Ratio of the Oxidation of Ferrocene electric current to methylene blue oxidation current, substitute into equation of linear regression and determine P53 gene in sample Concentration, to check actual analysis ability of the present invention in biological sample or complex system.
The testing result of P53 gene is shown, variable concentrations P53 gene peak current curve is all changed substantially, P53 gene It is in 0.01～100nM, to become good linear relationship in concentration, the detection of the electrochemica biological sensor of the application to lysozyme Limit can as little as 2.4 × 10-12M, instrument cost is low, sensitivity height, rapidly and efficiently.
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not model is protected to the present invention The restriction that encloses, one of ordinary skill in the art are should be understood that on the basis of technical scheme, and those skilled in the art are not The various modifications that makes by needing to pay creative work or deformation are still within protection scope of the present invention.

Claims (10)

1. a kind of probe for detecting P53 gene, is characterized in that：By a single stranded DNA assist probes S1 and a single stranded DNA Capture probe S2 constitutes, and the DNA sequence of the S1 is：5’-Fc-CTC TCA GTG ATT TTT TTA GTG AGA GAG- (CH2)6The DNA sequence of-SH-3 ', the S2 is：5’-MB-TCA CTG AGT CTT CCA GTG TGA TGA TCA CT- 3’.
2. for detecting application of the probe of P53 gene in the modified electrode for preparing detection P53 gene described in claim 1.
3. a kind of preparation method for detecting the modified electrode of P53 gene, is characterized in that：Step is as follows：
(1) assist probes S1 is heated in the Tris-HCl buffer containing three (2- carboxyethyl) phosphine, and annealing obtains single-stranded auxiliary The Tris-HCl buffer of probe S1；
(2) gold electrode after polishing, cleaning-drying is immersed in the Tris-HCl buffer of the assist probes S1 that step (1) is obtained In, by way of self assembly, assist probes S1 is modified to gold electrode surfaces, obtain S1/GE modified electrode；
(3) step (2) the electrode obtained is immersed in the Tris-HCl buffer of capture probe S2, heating, make capture probe S2 with auxiliary Helping probe S1 to combine, double hairpin structures is formed in gold electrode surfaces, obtain S2/S1/GE modified electrode；
In step (1), the Tris-HCl buffer containing three (2- carboxyethyl) phosphine is the trihydroxy methyl containing 110-150mM Aminomethane, the MgCl of NaCl, the 1-10mM of 100-120mM2, the mixed liquor of three (2- carboxyethyl) phosphine of 9-11mM, 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, is characterized in that：90 DEG C of heating in water bath 4-6 minutes in step (1).
5. preparation method as claimed in claim 3, is characterized in that：Polishing, the concrete steps of cleaning-drying in step (2) For：Gold electrode alumina powder sanding and polishing is become minute surface, then is cleaned by ultrasonic 20-60s through ethanol, redistilled water respectively, Nitrogen dries, and obtains cleaning gold electrode；It is preferred that：By the condition of assist probes S1 modification to gold electrode surfaces in step (2) For：It is 10-24 hour in 22-25 DEG C of response time that reaction temperature is, the concentration of assist probes S1 buffer is 40-50mM.
6. preparation method as claimed in claim 3, is characterized in that：In step (3), heating is specially：35-37 DEG C of water-bath Heating 1-3 hour, the concentration of the capture probe S2 is 35-50mM.
7. the modified electrode that the arbitrary described preparation method of claim 3-6 is prepared；It is preferred that：Prepared by the modified electrode For detecting the application in the electrochemical sensor of P53 gene.
8. a kind of method of detection caused by tumor suppressor p 53, is characterized in that：Step is as follows：
(1) by S2/S1/GE modified electrode after heating in water bath in Tris-HCl buffer, with calomel reference electrode and platinum filament pair Electrode constitutes three-electrode system, carries out DPV detection, obtain the peak current I of methylene blue in Tris-HCl buffer0(MB), two The peak current of luxuriant ferrum is I0(Fc)；
(2) use after being identified the P53 gene buffer of S2/S1/GE modified electrode and variable concentrations under conditions of heating Tris-HCl wash buffer, constitutes three-electrode system with calomel reference electrode and platinum filament comparison electrode, afterwards in Tris-HCl Carry out DPV detection in buffer, with the ferrocene f peak current that obtains than the peak current of methylene blue value：I=IFc/IMB；With I Equation of linear regression is done to the logarithm of P53 mrna concentration, obtains working curve；
(3) when detecting to actual sample, DPV detection is carried out to the P53 gene in testing sample using same method, is asked Ratio of the ferrocene peak current to methylene blue peak current, substitute into equation of linear regression afterwards and obtain final product the dense of P53 gene in sample Degree；
S2/S1/GE modified electrode in step (1), (2) is to be prepared by the arbitrary described preparation method of claim 3-6 Obtain.
9. preparation method as claimed in claim 8, is characterized in that：In step (1), (2), Tris-HCl buffer be containing There is the MgCl of NaCl, the 1-10mM of Tris-HCl, the 100-120mM of 110-150mM2Mixed liquor, its pH be 7.4-7.8；Excellent Choosing：In step (1), the heating in water bath time is 15-30 minute.
10. preparation method as claimed in claim 8, is characterized in that：In step (2), P53 gene buffer be containing The MgCl of NaCl, the 1-10mM of Tris-HCl, the 100-120mM of 110-150mM2And 10-8-10-12The mixing of MP53 gene Liquid, its pH is 7.4-7.8；It is preferred that：In step (2), heating is heating in water bath, and reaction temperature is 35-37 DEG C, response time For 15-30 minute.
CN201610806532.3A 2016-09-07 2016-09-07 Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof Active CN106434903B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610806532.3A CN106434903B (en) 2016-09-07 2016-09-07 Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610806532.3A CN106434903B (en) 2016-09-07 2016-09-07 Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof

Publications (2)

Publication Number Publication Date
CN106434903A true CN106434903A (en) 2017-02-22
CN106434903B CN106434903B (en) 2017-12-19

Family

ID=58164634

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610806532.3A Active CN106434903B (en) 2016-09-07 2016-09-07 Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof

Country Status (1)

Country Link
CN (1) CN106434903B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108445067A (en) * 2018-05-04 2018-08-24 安徽师范大学 A kind of signal amplification RNA nano biological sensors without enzyme of dual signal, preparation method and applications
CN109706221A (en) * 2018-09-21 2019-05-03 湘潭大学 A method of the resonant light scattering probe combination CHA technology quantitative detection mutant DNA based on GQDs
CN115165983A (en) * 2022-06-29 2022-10-11 上海市计量测试技术研究院 Inverted stem-loop ratio type electrochemical DNA biosensor based on poly-adenine and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103940872A (en) * 2014-04-30 2014-07-23 青岛大学 Preparation method and application of electrochemical sensor capable of simultaneously detecting two acute leukemia markers
CN104502437A (en) * 2015-01-08 2015-04-08 青岛大学 Label-free electrochemical transducer for multi-signal amplification and detection of label-free electrochemical transducer on nucleic acid
CN105223250A (en) * 2015-09-23 2016-01-06 南京邮电大学 Two stem circular DNA electrochemical sensor of polyA overseas Chinese federation and Synthesis and applications thereof
CN105510411A (en) * 2015-09-14 2016-04-20 陕西师范大学 Method for single cancer cell detection based on cell-microelectrode interaction

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103940872A (en) * 2014-04-30 2014-07-23 青岛大学 Preparation method and application of electrochemical sensor capable of simultaneously detecting two acute leukemia markers
CN104502437A (en) * 2015-01-08 2015-04-08 青岛大学 Label-free electrochemical transducer for multi-signal amplification and detection of label-free electrochemical transducer on nucleic acid
CN105510411A (en) * 2015-09-14 2016-04-20 陕西师范大学 Method for single cancer cell detection based on cell-microelectrode interaction
CN105223250A (en) * 2015-09-23 2016-01-06 南京邮电大学 Two stem circular DNA electrochemical sensor of polyA overseas Chinese federation and Synthesis and applications thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JIANFEI XIA ET AL.: "Single electrode biosensor for simultaneous determination of interferon gamma and lysozyme", 《BIOSENSORS AND BIOELECTRONICS》 *
ROBERT M. UMEK ET AL.: "Electronic Detection of Nucleic Acids", 《JOURNAL OF MOLECULAR DIAGNOSTICS》 *
ZONGHUA WANG ET AL.: "Lable-free quadruple signal amplification strategy for sensitive electrochemical p53 gene biosensing", 《BIOSENSORS AND BIOELECTRONICS》 *
刘萍等: "非标记型p53抑癌基因电化学DNA生物传感器的研究", 《分析测试学报》 *
罗贤文等: "基于金纳米粒子组装电化学DNA传感器检测p53抑癌基因的研究", 《分析化学》 *
钱孝清: "双检测电流型适体传感器的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108445067A (en) * 2018-05-04 2018-08-24 安徽师范大学 A kind of signal amplification RNA nano biological sensors without enzyme of dual signal, preparation method and applications
CN108445067B (en) * 2018-05-04 2020-07-03 安徽师范大学 Double-signal enzyme-free signal amplification RNA nano biosensor, preparation method and application thereof
CN109706221A (en) * 2018-09-21 2019-05-03 湘潭大学 A method of the resonant light scattering probe combination CHA technology quantitative detection mutant DNA based on GQDs
CN109706221B (en) * 2018-09-21 2022-05-31 湘潭大学 Method for quantitatively detecting mutant DNA by combining resonance light scattering probe based on GQDs with CHA technology
CN115165983A (en) * 2022-06-29 2022-10-11 上海市计量测试技术研究院 Inverted stem-loop ratio type electrochemical DNA biosensor based on poly-adenine and application thereof
CN115165983B (en) * 2022-06-29 2023-10-24 上海市计量测试技术研究院 Reverse stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof

Also Published As

Publication number Publication date
CN106434903B (en) 2017-12-19

Similar Documents

Publication Publication Date Title
CN106525940A (en) Electrochemical method of detecting single-chain target DNA concentration based on G-quadruplex-heme compound and polymeric chain type amplification reaction
CN102262118A (en) Bioelectrochemical sensor for detecting tumor markers and preparation method thereof
CN106434903B (en) Detect ratio electrochemical DNA biosensor modified electrode of P53gene and preparation method thereof
CN104990966B (en) A kind of for electrochemica biological sensor detecting lysozyme and preparation method thereof
Yu et al. A ratiometric electrochemical aptasensor for sensitive detection of protein based on aptamer–target–aptamer sandwich structure
Wu et al. Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry
CN107064258B (en) The method of the electrochemical aptamer sensor measurement HER2 of electric signal and its self assembly amplified signal is generated based on DNA
CN108007992A (en) Detect the preparation method of the poly ion liquid@3D nanogold cluster modified electrodes of hemoglobin
CN108051492A (en) A kind of method based on controllable self assembly forceps constructions Electrochemical Detection single-stranded target DNA
CN113293199B (en) Cascade DNA strand displacement reaction-based micro ribonucleic acid detection method and kit
CN107144618A (en) A kind of methods and applications of the protein activation levels of utilization DNA probe part Electrochemical Detection STAT 3
CN108195913B (en) A kind of biosensor and its construction method for Electrochemical Detection HER2
CN105445350A (en) Electrochemical DNA (Deoxyribose Nucleic Acid) biosensor based on peptide nucleic acid and preparation method of electrochemical DNA biosensor
CN109507254A (en) A kind of electrochemica biological sensor and preparation method and application detecting kanamycins
CN109490387A (en) The preparation method of lead ion electrochemical sensor based on aptamer
CN113295740A (en) Thrombin electrochemical sensor based on multi-binding site aptamer
CN106198503B (en) A kind of electrochemiluminescence sandwich biosensor and preparation and application
CN108896639B (en) LncRNA MEG3 dual-detection electrochemical gene sensor, and preparation method and application thereof
CN111122675B (en) Strip-block-strip electrode and VEGF based on strip-block-strip electrode165Sensor, preparation method and detection method thereof
CN109991293B (en) High-flexibility full-solid pH selective electrode and preparation method thereof
CN210376224U (en) Sensor for detecting L-arginine
CN104316705A (en) Preparation method and application of hybridization indicator 5,7-binitro-2-sulfo-acridone
CN105929001B (en) The gold electrode and preparation method and application of specific DNA pseudoknot structure modification
CN104237353B (en) NDM 1 locks nucleic acid probe modified electrode and its preparation method and application
CN107144619A (en) The electrochemical DNA biosensor of a kind of temperature-controllable based on enzymatic and preparation method thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant