CN110484601B - Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof - Google Patents
Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof Download PDFInfo
- Publication number
- CN110484601B CN110484601B CN201910819285.4A CN201910819285A CN110484601B CN 110484601 B CN110484601 B CN 110484601B CN 201910819285 A CN201910819285 A CN 201910819285A CN 110484601 B CN110484601 B CN 110484601B
- Authority
- CN
- China
- Prior art keywords
- electrode
- temperature
- alkaline phosphatase
- gene
- bstnbi
- 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.)
- Active
Links
Images
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
- C12Q1/682—Signal amplification
-
- 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
Abstract
The invention discloses a preparation method of a temperature-controllable electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase, which comprises a gold disc thermode, capture probes, streptavidin-labeled alkaline phosphatase and nicking endonuclease Nt.BstNBI, wherein the capture probes are respectively labeled with biotin and sulfydryl at two ends which are complementary with a target p53 sequence. When p53 exists, hybridizing with the capture probe, then inducing Nt.BstNBI to cut, releasing a target substance p53 to perform next hybridization enzyme cutting circulation, and applying current to an electrode to change the enzyme cutting temperature so as to improve the activity of enzyme, so that the enzyme cutting process is quicker and more thorough; when the electrode is detected before and after enzyme digestion, the ALP catalytic activity is improved by changing the temperature of the electrode, the difference value of the peak current before and after enzyme digestion is amplified, the reduction value of the oxidation peak current and the concentration of the target p53 are in a linear relation, and the high-sensitivity detection of the p53 gene is realized.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a temperature-controllable electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase, and a preparation method and application thereof.
Background
The p53 gene was discovered in the 70's of the 20 th century, is a gene encoding a protein that inhibits tumor development and growth, is a central regulator of cellular responses to stress, and is one of the most common mutant genes in human cancers. Therefore, establishing a rapid and sensitive detection means for p53 gene is of great value for early diagnosis of cancer.
The current biosensor technology has been developed to a stage where a signal generated from a target of low concentration can be amplified through a series of amplification steps, and the nicking endonuclease assisted amplification technology (NESA) has also come into play. NESA can modulate amplification of a single target DNA to cyclic generation of multiple triggers, thereby enabling ultra-low concentration detection of targets. The basic design of NESA is to hybridize the target DNA with the probe to generate a nicking endonuclease inhibition site, which is specifically recognized by the enzyme to make the probe to crack and release the target DNA, and then start the next hybridization, cracking and dissociation cycle to realize the amplification of the signal.
BstNBI is one of the nicking endonucleases, which has a significant selectivity for a specific DNA sequence 5'-GAGTC-3' in double-stranded DNA (dsDNA), which is just part of the complementary sequence of the p53 gene, so it is often used as a helper enzyme for detecting p 53. Similarly, bstNBI is a protein whose DNA cleavage activity is also affected by temperature, and whose activity directly affects the detection of p53 gene. Therefore, the combination of Nt.BstNBI and thermode technology can improve the detection sensitivity and reduce the detection limit.
Disclosure of Invention
The invention aims to provide a temperature-controllable electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase and a preparation method thereof. The sensor disclosed by the invention has the advantages of simple structure, simple preparation process, short detection time, high sensitivity and good selectivity, and provides a rapid and low-cost detection method for the tumor suppressor gene p53 in the field of medical diagnosis.
In order to realize the purpose of the invention, the target p53 is specifically combined with a capture probe modified on a gold disc thermode, and p53 is released by utilizing the recognition and shearing action of nicking endonuclease Nt. The method comprises the following steps:
(1) Designing a capture probe DNA chain CP, wherein the DNA chain CP is complementarily paired with the target p53 gene to form double-stranded DNA containing the recognition sequence 5'-GAGTC-3' of Nt. BstNBI is induced to cut the captured DNA at the fourth base in the 3' end direction after the sequence, and the target substance p53 is released to perform the next hybridization enzyme digestion cycle; sulfhydrylation is carried out on the 5' end of the CP chain, so that the CP is modified to the surface of the gold wire thermode through a gold-sulfur bond; biotin is modified at the 3' end of the CP chain, and is combined with alkaline phosphatase through streptavidin; wherein the DNA sequence of the capture probe CP is as follows: 5' -SH- (CH) 2 ) 6 –GAGTC TTCCA GTGTG ATGA-biotin-3', the DNA sequence of the target p53 gene is: 5 'TCATC ACACT GGAAG ACTC-3';
(2) Polishing the gold disc thermode into a mirror surface, ultrasonically cleaning the mirror surface by using secondary distilled water, and drying to obtain the processed gold disc thermode;
(3) Dropwise adding a buffer solution containing a thiolated capture probe with a 3' end labeled with biotin onto the gold-plate thermoelectric electrode treated in the step (2), then sealing residual sites on the surface of the electrode with mercaptohexanol, and further sealing active sites on the surface of the electrode with bovine serum albumin to obtain a capture probe modified gold-plate thermoelectric electrode;
(4) And (4) dropwise adding streptavidin-labeled alkaline phosphatase diluted by 5 mu L of buffer solution onto the surface of the electrode obtained in the step (3), and obtaining the alkaline phosphatase modified gold-plate thermode after the reaction is finished.
(5) And (4) soaking the electrode obtained in the step (4) in a buffer solution containing target p53 genes and nicking endonuclease Nt.BstNBI with different concentrations to perform a hybrid enzyme digestion cycle, and obtaining the electrochemical p53 gene sensor after the reaction is finished.
In the step (2), polishing is carried out by polishing aluminum oxide powder on chamois leather; the ultrasonic cleaning time is 40-60 s, and the drying is nitrogen blow-drying.
In the step (3), the buffer solution is a mixed solution containing 10mM Tris-HCl and 1mM EDTA, and the pH value is 7.2-7.6.
In the step (3), the concentration of the capture probe is 1-2 MuM; the modification temperature is 20-25 ℃; the modification time is 1.5-2.5 h;
in the step (3), the concentration of mercaptohexanol is 0.1-4 mM, and the immersion time of the electrode is 1h; the bovine serum albumin concentration is 0.1-2%, and the electrode immersion time is 1h;
in the step (4), the buffer solution is a mixture containing 10 to 20mM PBS and 100 to 200mM NaCl, and the pH value is 7.2 to 7.6.
In the step (4), the concentration of the streptavidin-labeled alkaline phosphatase is 80-120 mug/mL, the reaction temperature is 20-25 ℃, and the reaction time is 0.2-1 h.
In the step (5), the concentration of the nicking endonuclease Nt.BstNBI is 200-500 units/mL, the circular reaction temperature of the hybrid enzyme digestion is 0-40 ℃, and the reaction time is 0.25-2 h.
In the step (5), the temperature of the gold disc thermal electrode is regulated and controlled by externally added direct current to control the temperature of the hybrid enzyme digestion cycle reaction process.
The invention also provides an application of the sensor in detecting the p53 gene, which comprises the following detection steps:
(1) Soaking a gold-plate thermal electrode modified by a thiolated capture probe with alkaline phosphatase modified at the 3' end in a detection solution containing ferrocene methanol and sodium ascorbyl phosphate, forming a three-electrode system with a silver-silver chloride reference electrode and a platinum wire counter electrode, and performing CV detection to obtain the oxidation peak current I of the ferrocene methanol 0(FcM) ；
(2) Detecting the finally obtained electrochemical p53 gene sensor in a detection solution containing 0.1 mM ferrocene methanol and 0.5 mM sodium ascorbyl phosphate by CV, and comparing the oxidation peak current of the obtained ferrocene methanol with the I obtained in the step (1) 0(FcM) And (5) taking the difference and obtaining an absolute value: i Delta I FcM |=|I FcM -I 0(FcM) L, |; at | Δ I FcM I, making a linear regression equation on the logarithm of the p53 concentration to obtain a working curve;
in the step (1), the pH of the ferrocene methanol and sodium ascorbyl phosphate detection solution is 8.0, and the mixed solution contains 0.1M diethanolamine, 0.1 mM ferrocene methanol and 0.5 mM sodium ascorbyl phosphate; the detection temperature is controlled by regulating the temperature of the gold plate hot electrode through the external direct current.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts the sulfhydrylation DNA which is complementary with the target p53 as the capture probe, thereby improving the selectivity of the DNA electrochemical biosensor and the practicability of the DNA electrochemical biosensor in complex clinical samples;
(2) The invention applies the target cyclic signal amplification technology of nicking endonuclease Nt.BstNBI to the detection of p53 gene, realizes the high-sensitivity detection (the detection limit reaches 95 aM) of the target p53, and has the advantages of simple and convenient operation, low cost, quick detection and the like.
(3) The biosensor is constructed on the surface of the gold disc thermode, so that the nicking endonuclease Nt.BstNBI and the alkaline phosphatase are in the optimal catalytic condition only by changing the surface temperature of the electrode without integrally heating the solution, the solution convection is enhanced, the mass transfer rate is improved, the activities of the nicking endonuclease Nt.BstNBI and the alkaline phosphatase are enhanced, the enzyme digestion reaction and the electrochemical-chemical reaction process are promoted, the electrode response signal is increased, and the detection sensitivity is improved.
Drawings
FIG. 1 is a schematic diagram showing the preparation process of the electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase with controllable temperature according to the present invention;
FIG. 2 is a graph showing the effect of temperature on the stability of a gold wire thermode modified with capture probes;
FIG. 3 is a graph showing the effect of electrode temperature on alkaline phosphatase activity;
fig. 4 is a graph of the effect of electrode temperature on the activity of nt.bstnbi;
FIG. 5 is an electrochemical response of the electrochemical p53 gene sensor of the present invention to different concentrations of p53 gene at different electrode temperatures;
FIG. 6 is an electrochemical response of the electrochemical p53 gene sensor of the present invention to various DNAs.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail with reference to examples, and it will be understood by those skilled in the art that the following examples are only preferred examples of the present invention in order to better understand the present invention, and thus should not be construed as limiting the scope of the present invention.
Example 1
A method for preparing a temperature-controlled electrochemical p53 gene sensor based on signal amplification of nicking endonuclease nt, bstnbi and alkaline phosphatase, as shown in fig. 1, comprising the following steps:
(1) Designing a capture probe DNA chain CP, wherein the capture probe DNA chain CP is complementarily paired with a target p53 gene to form double-stranded DNA containing a recognition sequence 5'-GAGTC-3' of Nt. CP (CP)Sulfhydrylation is carried out on the 5' end of the chain, so that the CP is modified to the surface of the gold wire thermode through a gold-sulfur bond; biotin is modified at the 3' end of the CP chain, and the CP chain is combined with alkaline phosphatase through streptavidin; wherein the DNA sequence of the capture probe CP is as follows: 5' -SH- (CH) 2 ) 6 -GAGTC TTCCA gtgtgtg ATGA-biotin-3', the DNA sequence of the target p53 gene is: 5 'TCATC ACACT GGAAG ACTC-3';
(2) Gold disk thermode on chamois using 0.05 mm Al 2 O 3 Polishing into a mirror surface, and ultrasonically cleaning for 40-60 s by using secondary distilled water; then circulating volt-ampere in 0.5M sulfuric acid solution, cleaning with secondary water after the sweep rate is 0.1V/s is stabilized, and drying with nitrogen;
(3) Treating a capture probe DNA chain CP with the concentration of 1mM in a buffer solution containing 10mM Tris-HCl and 1mM EDTA with the pH value of 7.4 for 2h to open a disulfide bond; then 5 mL of the solution is dripped on the gold plate thermoelectric electrode processed in the step (2), and the modification time is 2h at 25 ℃; then, blocking the residual sites on the surface of the electrode by using mercaptohexanol with the concentration of 2 mM for 1h; further sealing the active sites on the surface of the electrode by bovine serum albumin with the concentration of 1 percent for 1h; finally, 5 mu L of alkaline phosphatase marked with streptavidin is dripped on the surface of the electrode, and the gold plate thermal electrode modified by the alkaline phosphatase is obtained after the reaction is finished;
(4) BstNBI was mixed with p53 at a concentration of 200unit/ml of nicking endonuclease, 50mM Tris-HCl,100mM NaCl, and 10mM MgCl, pH 7.4 2 And (3) soaking the electrode obtained in the step (3) in a buffer solution of 100 mu g/ml BSA (bovine serum albumin) for a hybrid enzyme digestion cycle, reacting at 40 ℃ for 0.5h, wherein the reaction temperature is controlled by regulating the temperature of a gold disc thermal electrode by adding direct current, and obtaining the electrochemical p53 gene sensor after the reaction is finished.
Example 2
Temperature influence on the stability of the gold plate thermode modified by the capture probe CP is examined, and as shown in FIG. 2, the specific steps are as follows:
(1) Forming a three-electrode system by the alkaline phosphatase immobilized capture probe modified gold plate thermode obtained in the step (3) in the example 1, a silver-silver chloride reference electrode and a platinum wire counter electrode, and performing CV detection in 0.1M DEA detection solution containing 0.1 mM ferrocenemethanol and 0.5 mM sodium ascorbyl phosphate to obtain an initial CV curve of the ferrocenemethanol;
(2) The gold plate thermal electrode modified by the capture probe immobilized with alkaline phosphatase, which is subjected to initial detection, is soaked in a Tris-HCl buffer solution containing 10mM and having the temperature of 30,35,40,45 and 50 ℃ and the pH value of 7.4 for 1h, and after soaking, detection is carried out by CV in a DEA detection solution containing 0.1 mM of ferrocenyl methanol and 0.5 mM of sodium ascorbyl phosphate, so as to obtain the final peak current of the obtained ferrocenyl methanol. As shown in fig. 2, comparing CV curves before and after soaking, it can be seen that when the soaking temperature exceeds 40 ℃, the stability of the gold plate thermode modified by the capture probe is reduced, which indicates that the temperature range which can be studied by the present invention is below 40 ℃.
Example 3
Forming a three-electrode system by using the alkaline phosphatase immobilized capture probe modified gold disc hot electrode obtained in the step (3) in the example 1, a silver-silver chloride reference electrode and a platinum wire counter electrode, and performing CV detection in a 0.1M DEA detection solution containing 0.1 mM ferrocenyl methanol and 0.5 mM sodium ascorbyl phosphate, wherein the electrode temperatures are changed to 0 ℃, 25 ℃ and 40 ℃, so as to respectively obtain an initial CV curve of the ferrocenyl methanol; as shown in FIG. 3, it is demonstrated that an increase in electrode temperature increases the activity of alkaline phosphatase and the electrochemical signal is greater.
Example 4
Experiments are sequentially and respectively carried out by changing the reaction temperature in the step (4) of the example 1 from 0 to 40 ℃, and the example 1 is carried out under other reaction conditions; the alkaline phosphatase-immobilized capture probe-modified gold disk thermode obtained in step (3) of example 1 was examined in a 0.1M DEA assay solution containing 0.1 mM ferrocenemethanol and 0.5 mM sodium ascorbyl phosphate to obtain the oxidation peak current I of ferrocenemethanol 0(FcM) (ii) a The electrochemical biosensor finally obtained in step (4) of example 1 was assayed by CV in 0.1M DEA assay solution containing 0.1 mM ferrocenemethanol and 0.5 mM sodium ascorbyl phosphate, and the resulting oxidation peak current and the resulting I were measured 0(FcM) And (5) taking the difference and obtaining an absolute value: i Delta I FcM |=|I FcM -I 0(FcM) L, |; at | Δ I FcM | vs. temperature as shown in FIG. 4, it can be seen that | Δ I increases with temperature FcM BstNBI activity is increased by increasing temperature, which promotes faster enzyme digestion.
Example 5
Is the electrochemical response characteristic of the electrochemical p53 gene sensor of the invention to p53 with different concentrations.
The electrochemical p53 gene sensor of the invention is adopted, and two ends of the electrochemical p53 gene sensor respectively modify sulfydryl and single-stranded DNA (sequence: 5' -SH- (CH)) 2 ) 6 -GAGTC TTCCA gtgtgtg ATGA-biotin-3') as capture probe, with p53 gene (sequence: 5'-TCATC ACACT GGAAG ACTC-3') as target DNA. All procedures were as in example 1 above, wherein the electrochemical response characteristics of the electrochemical p53 gene sensor of the present invention were examined by varying the concentration of the target DNA (0, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM).
The detection process comprises the following steps: the alkaline phosphatase-immobilized capture probe-modified gold disk thermode obtained in step (3) of example 1 was examined in a 0.1M DEA assay solution containing 0.1 mM ferrocenemethanol and 0.5 mM sodium ascorbyl phosphate to obtain the oxidation peak current I of ferrocenemethanol 0(FcM) (ii) a The electrochemical p53 gene sensor finally obtained in step (4) of example 1 was detected by CV in 0.1M DEA detection solution containing 0.1 mM ferrocenyl methanol and 0.5 mM sodium phosphate ascorbate, and the oxidation peak current of the obtained ferrocenyl methanol and the obtained I 0(FcM) Taking the difference and obtaining the absolute value: i Delta I FcM |=|I FcM -I 0(FcM) L, |; at | Δ I FcM And (4) performing a linear regression equation on the logarithm of the p53 concentration to obtain a working curve.
The detection result is shown in FIG. 5. When the hybrid enzyme digestion cycle and the detection process are both carried out at 25 |. DELTA.I FcM The concentration of | and p53 is linear between 1 pM and 10nM, and the detection limit is 1 pM; when the hybridization cycle and the detection are carried out at 40 ℃, | Δ I FcM The concentration of | and p53 is linear in the range of 100 aM-10 nMIn relation, the detection limit is 95aM, and is reduced by 4 orders of magnitude compared with that at 25 ℃, which shows that the activity of Nt.BstNBI and alkaline phosphatase is enhanced by increasing the temperature, and the high-sensitivity detection of the p53 gene is realized.
Example 6
Is the electrochemical response of the electrochemical p53 gene sensor of the invention to different DNAs.
The method of the invention is adopted, and the two ends respectively modify the single-stranded DNA (sequence: 5' -SH- (CH)) 2 ) 6 -GAGTC TTCCA GTGTGTG ATGA-biotin-3') as capture probe, and p53 gene (sequence: 5 'TCATC ACACT GGAAG ACTC-3'), single base mismatch DNA (sequence: 5 'TCATC ACACTGGAAG AATC-3'), double-base mismatch DNA (sequence: 5 'TCATC ACACT GGATC ACTC-3') and control DNA (sequence: 5 'GACGT CAGAC TTCCT GCGA-3'). All procedures were as in example 1 above, wherein all DNA sequence concentrations were 10nM, and the electrochemical response of the electrochemical p53 gene sensor of the invention to different DNAs was examined. The detection result is shown in FIG. 6, and it can be seen from the figure that the electrochemical p53 gene sensor of the present invention has electrochemical responses with different intensities for different sequence DNAs, and thus has a good choice for detecting p53 gene.
SEQUENCE LISTING
<110> Fuzhou university
<120> temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof
<160>5
<170>PatentIn version 3.5
<210>1
<211>19
<212>DNA
<213>Artificial Sequence
<400>1
gagtcttcca gtgtgatga 19
<210>2
<211>19
<212>DNA
<213>Artificial Sequence
<400>2
tcatcacact ggaagactc 19
<210>3
<211> 19
<212>DNA
<213>Artificial Sequence
<400>3
tcatcacact ggaagaatc 19
<210>4
<211>19
<212>DNA
<213>Artificial Sequence
<400>4
tcatcacact ggatcactc 19
<210>5
<211>19
<212>DNA
<213>Artificial Sequence
<400>5
gacgtcagac ttcctgcga 19
Claims (7)
1. A temperature-controllable electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt, bstNBI and alkaline phosphatase, wherein the preparation method of the electrochemical p53 gene sensor comprises the following steps:
(1) Designing a capture probe DNA chain CP, wherein the capture probe DNA chain CP is complementarily paired with a target p53 gene; sulfhydrylation is carried out on the 5 'end of a capture probe DNA chain CP chain, and biotin is modified on the 3' end; the DNA sequence of the capture probe DNA strand CP is: 5' -SH- (CH) 2 ) 6 -GAGTC TTCCA GTGTGTG ATGA-biotin-3', the sequence of the target p53 gene is: 5 'TCATC ACACT GGAAG ACTC-3';
(2) Firstly, polishing the gold disc thermal electrode, then ultrasonically cleaning the gold disc thermal electrode by secondary distilled water, and drying to obtain the gold disc thermal electrode with a clean and smooth surface;
(3) Dropwise adding a buffer solution containing the capture probe DNA chain CP obtained in the step (1) on the gold-plate thermode processed in the step (2) for modification, then sealing residual sites on the surface of the electrode by using mercaptohexanol, and further sealing active sites on the surface of the electrode by using bovine serum albumin to obtain the capture probe modified gold-plate thermode;
(4) Dripping 5 mu L of alkaline phosphatase marked with streptavidin on the surface of the electrode obtained in the step (3), and obtaining an alkaline phosphatase modified gold disc hot electrode after the reaction is finished;
(5) Soaking the electrode obtained in the step (4) in a buffer solution containing target p53 genes and nicking endonuclease Nt.BstNBI with different concentrations to perform hybrid enzyme digestion circulation, and obtaining the electrochemical p53 gene sensor after the reaction is finished;
in the step (3), the concentration of the capture probe DNA chain CP is 1-2 MuM; the modification temperature is 20-25 ℃; the modification time is 1.5 to 2.5 hours; in the step (4), the concentration of alkaline phosphatase marked with streptavidin is 80-120 mug/mL, the reaction temperature is 20-25 ℃, and the reaction time is 0.2-1 h; in the step (5), the concentration of the nicking endonuclease Nt.BstNBI is 200-500 unit/mL, the temperature of the hybrid enzyme digestion cycle reaction is 0-40 ℃, and the reaction time is 0.25-2 h.
2. The temperature-controlled electrochemical p53 gene sensor based on signal amplification of nicking endonuclease nt.bstnbi and alkaline phosphatase as claimed in claim 1, wherein in the step (2), the polishing is performed by polishing with alumina powder on a chamois leather; the ultrasonic cleaning time is 20-60 s, and the drying is nitrogen blow-drying.
3. The temperature-controlled electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase as claimed in claim 1, wherein in the step (3), the buffer solution is a mixture solution containing 10mM Tris-HCl and 1mM EDTA, and the pH value is 7.2-7.6.
4. The temperature-controlled electrochemical p53 gene sensor based on signal amplification of nicking endonuclease Nt.BstNBI and alkaline phosphatase as claimed in claim 1, wherein in the step (3), mercaptohexanol concentration is 0.1-4 mM, and electrode immersion time is 1h; bovine serum albumin concentration is 0.1-2%, and the electrode immersion time is 1h.
5. The temperature-controlled electrochemical p53 gene sensor based on signal amplification by nicking endonuclease Nt. BstNBI and alkaline phosphatase in accordance with claim 1, wherein the buffer solution in step (5) is a mixture of 10-20 mM PBS and 100-200 mM NaCl at a pH of 7.2-7.6.
6. The temperature-controlled electrochemical p53 gene sensor based on signal amplification of endonuclease nt.bstnbi and alkaline phosphatase as claimed in claim 1, wherein in the step (5), the temperature of gold disk thermode is controlled by applying dc current to control the temperature of the hybridization cycle.
7. Use of an electrochemical p53 gene sensor according to any one of claims 1 to 6 in a method for detecting a target p53 gene, wherein the detection step is as follows:
(1) Soaking an alkaline phosphatase modified gold plate thermal electrode in a detection solution containing ferrocene methanol and sodium ascorbyl phosphate, forming a three-electrode system with a silver-silver chloride reference electrode and a platinum wire counter electrode, and performing CV detection to obtain an oxidation peak current I of the ferrocene methanol 0(FcM) (ii) a Wherein, the detection solution of ferrocene methanol and sodium ascorbyl phosphate is a mixed solution with pH of 8.0 and containing 0.1M diethanolamine, 0.1 mM ferrocene methanol and 0.5 mM sodium ascorbyl phosphate;
(2) Detecting electrochemical p53 gene sensor in detection solution containing 0.1 mM ferrocene methanol and 0.5 mM sodium ascorbyl phosphate by CV, and measuring the oxidation peak current I of obtained ferrocene methanol FcM With the I obtained in step (1) 0(FcM) Taking absolute value, | Delta I FcM |=|I FcM -I 0(FcM) L, |; at | Δ I FcM I, making a linear regression equation on the logarithm of the p53 concentration to obtain a working curve;
the use is not in the field of disease diagnosis and treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910819285.4A CN110484601B (en) | 2019-08-31 | 2019-08-31 | Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910819285.4A CN110484601B (en) | 2019-08-31 | 2019-08-31 | Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110484601A CN110484601A (en) | 2019-11-22 |
| CN110484601B true CN110484601B (en) | 2022-12-09 |
Family
ID=68555914
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910819285.4A Active CN110484601B (en) | 2019-08-31 | 2019-08-31 | Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110484601B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986213B (en) * | 2021-03-12 | 2022-03-22 | 福州大学 | Raman spectrum sensor for detecting oral cancer DNA |
| CN113552191B (en) * | 2021-07-28 | 2023-11-21 | 江苏师范大学 | Construction method of proportional electrochemical sensor for detecting methylated DNA based on multilayer DNA amplification loop |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596662A (en) * | 2016-12-08 | 2017-04-26 | 福州大学 | Temperature-controllable electrochemical DNA biosensor and preparation method thereof |
| CN106596675A (en) * | 2016-12-19 | 2017-04-26 | 福州大学 | Temperature-controllable enzymatic catalysis-based type electrochemical hydrogen peroxide sensor |
| CN107144619A (en) * | 2017-06-21 | 2017-09-08 | 福州大学 | The electrochemical DNA biosensor of a kind of temperature-controllable based on enzymatic and preparation method thereof |
| WO2018010681A1 (en) * | 2016-07-14 | 2018-01-18 | 青岛大学 | Electrochemical biosensor based on aptamer/nano-silver probes and exo i enzyme |
-
2019
- 2019-08-31 CN CN201910819285.4A patent/CN110484601B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018010681A1 (en) * | 2016-07-14 | 2018-01-18 | 青岛大学 | Electrochemical biosensor based on aptamer/nano-silver probes and exo i enzyme |
| CN106596662A (en) * | 2016-12-08 | 2017-04-26 | 福州大学 | Temperature-controllable electrochemical DNA biosensor and preparation method thereof |
| CN106596675A (en) * | 2016-12-19 | 2017-04-26 | 福州大学 | Temperature-controllable enzymatic catalysis-based type electrochemical hydrogen peroxide sensor |
| CN107144619A (en) * | 2017-06-21 | 2017-09-08 | 福州大学 | The electrochemical DNA biosensor of a kind of temperature-controllable based on enzymatic and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 基于信号放大技术的电致化学发光传感器的应用研究;杨林林;《中国优秀硕士学位论文全文数据库-工程科技I辑》;20180615;3.2实验部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110484601A (en) | 2019-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10605761B2 (en) | Electrochemical biosensor based on aptamer/nano silver probe and EXO I enzyme | |
| Qian et al. | Ultrasensitive electrochemical detection of DNA based on Zn2+ assistant DNA recycling followed with hybridization chain reaction dual amplification | |
| Tao et al. | A new mode for highly sensitive and specific detection of DNA based on exonuclease III-assisted target recycling amplification and mismatched catalytic hairpin assembly | |
| WO2016062101A1 (en) | Modified electrode for detecting ndm-1 and preparation method therefor and use thereof | |
| Wang et al. | Multiple signal amplification electrogenerated chemiluminescence biosensors for sensitive protein kinase activity analysis and inhibition | |
| CN110501411B (en) | Electrochemical biosensor for enzyme-free detection of ampicillin and preparation method and application thereof | |
| CN109738503B (en) | Positive feedback amplification electrochemical sensor based on exonuclease III | |
| Ding et al. | Electrochemical biosensor for p53 gene based on HRP-mimicking DNAzyme-catalyzed deposition of polyaniline coupled with hybridization chain reaction | |
| CN110484601B (en) | Temperature-controllable electrochemical p53 gene sensor and preparation method and application thereof | |
| CN112649479A (en) | Multi-clamp-string cascade assembly construction universal electrochemical biosensor ultrasensitive detection target based on assistance of tetrahedral tripod | |
| Wang et al. | Ultraspecific electrochemical DNA biosensor by coupling spontaneous cascade DNA branch migration and dual-signaling sensing strategy | |
| Song et al. | A novel assay strategy based on isothermal amplification and cascade signal amplified electrochemical DNA sensor for sensitive detection of Helicobacter pylori | |
| Xie et al. | A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires | |
| Hou et al. | Sensitive electrochemical assay for T4 polynucleotide kinase activity based on dual-signaling amplification coupled with exonuclease reaction | |
| CN110106232A (en) | Based on target catalysis without the unmarked double tail hybrid organisms sensors of enzyme and preparation method | |
| Wang et al. | Catalytic hairpin assembly-programmed formation of clickable nucleic acids for electrochemical detection of liver cancer related short gene | |
| Xiao et al. | Amplified electrochemiluminescence of luminol based on hybridization chain reaction and in situ generate co-reactant for highly sensitive immunoassay | |
| CN106596662A (en) | Temperature-controllable electrochemical DNA biosensor and preparation method thereof | |
| CN109507254B (en) | Electrochemical biosensor for detecting kanamycin and application thereof | |
| Mao et al. | DNAzyme-driven DNA walker biosensor for amplified electrochemical detection of T4 polynucleotide kinase activity and inhibition | |
| CN106290521B (en) | Preparation method of electrochemical sensor for ADRB1-1165G & gtC gene polymorphism detection | |
| Cao et al. | An ultrasensitive biosensor for virulence ompA gene of Cronobacter sakazakii based on boron doped carbon quantum dots-AuNPs nanozyme and exonuclease III-assisted target-recycling strategy | |
| CN109856211B (en) | Preparation method and application of electrochemical biosensor for simultaneously detecting Exo I and TdT | |
| CN109613096B (en) | DNA-related enzyme electrochemical biosensor constructed based on DNA-copper nanoclusters and application thereof | |
| Wang et al. | Coupling of background reduction with rolling circle amplification for highly sensitive protein detection via terminal protection of small molecule-linked DNA |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |