CN116953055A - DNA electrochemical sensor and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a DNA electrochemical sensor, a preparation method and application thereof, and belongs to the technical field of biological sensing. The invention modifies the sulfhydryl modified hairpin DNA probe marked by Methylene Blue (MB) to a gold electrode through self-assembly, and prepares a DNA electrochemical sensor capable of detecting tumor related TP53 gene and single nucleotide polymorphism. The DNA electrochemical sensor has the advantages of simple preparation process, low cost, high sensitivity, good reproducibility, good storage stability and the like, provides a powerful tool for the sensitive and specific detection of TP53 gene mutation sites, and can be used for early diagnosis and early warning of cancers.
Description
Technical Field
The invention belongs to the technical field of biological sensing, and particularly relates to a DNA electrochemical sensor, a preparation method and application thereof in early screening and early warning of tumors/cancers.
Background
Despite the continued advancement of healthcare, cancer remains a major life threatening disease involving multiple changes in gene status and expression, leading to death of over 1.5 tens of thousands of people per day, with early discovery, diagnosis and treatment critical to cancer treatment. Currently, methods for early detection of cancer mainly include blood detection chips, gene detection, nano detection, positron Emission Tomography (PET), computed Tomography (CT), and the like. These methods can provide very accurate results, but still suffer from low sensitivity, high cost, and potential physical or chemical hazards, which are very limited in early cancer detection. Furthermore, techniques of enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) are widely focused due to their high sensitivity and low invasiveness, but they require a long detection time and high operation costs. There is therefore an urgent need to develop a reliable, efficient, sensitive, rapid, inexpensive method for early screening and early warning of cancer that is critical to medical diagnosis.
Tumor suppressor gene TP53 is one of the most common mutant genes in human cancers, involved in cell cycle control and apoptosis regulation. It consists of 11 exons, encoding a 393 amino acid p53 protein, with more than 50% of human tumor and cancer cell lines having alterations in the TP53 gene or p53 protein function. Although most tumor suppressor genes are inactivated by deletion or truncation mutations, the TP53 gene in human tumors typically has a mutation in exons 4-8 encoding the protein DNA binding domain. Among the mutations in this region, the mutation at positions 175, 248, 249, 273, 282 is most frequent and is commonly found in almost all types of cancer. The close relation between TP53 gene and cancer creates conditions for early screening and early warning of cancer.
The DNA electrochemical sensor is characterized in that DNA molecules are used as identification elements and fixed on a substrate electrode, and electrochemical signals are converted into electric signals by the substrate electrode so as to detect the electric signals. DNA electrochemical sensors combine the advantages of DNA and electrochemistry and, in general, include the following: 1) The specificity is good, and the DNA molecule double chains have very high specific recognition through specific base pairing. 2) The stability is good, the thermal stability of the isolated DNA is better than that of most protein (enzyme) molecules, and the prepared sensor can be stored for a long time. 3) The cost is low, the preparation is simple, and the preparation can be synthesized in a large scale. 4) The method has the advantages of high sensitivity, quick electrochemical response and simple operation. However, the electrochemical method directly detects DNA to generate a response signal which is low, and the weak response signal is easy to be interfered by noise, so that baseline deviation occurs, and the sensitivity of the instrument is reduced.
Disclosure of Invention
In view of the above, the present invention aims to provide a DNA electrochemical sensor, a method for preparing the same, and an application thereof in early detection and early warning of tumor/cancer, wherein the DNA electrochemical sensor capable of detecting tumor suppressor gene TP53 and single nucleotide polymorphism thereof is prepared according to the following principle that the DNA electrochemical sensor detects tumor suppressor gene TP53 and single nucleotide polymorphism thereof: after the target TP53 sequence acts with a hairpin DNA capture probe modified by Methylene Blue (MB) immobilized on an electrochemical DNA sensor, the probe structure is rearranged through hydrogen bond action between DNA double chains, so that a marked electrochemical indicator methylene blue is far away from the surface of an electrode, and the TP53 gene can be effectively identified through recording the electric signal change of the methylene blue before and after the action, thereby realizing the effective detection of the target sequence by the DNA electrochemical sensor under nanomolar concentration; according to the difference of differential pulse volt-ampere signals of the complete complementary sequence and the sequence with single nucleotide polymorphism, single base mutation of TP53 genes can be selectively identified, and early detection and early warning of tumors/cancers related to tumor suppressor genes TP53 can be realized.
The invention aims at realizing the following steps:
the invention provides a preparation method of a DNA electrochemical sensor for detecting tumor suppressor gene TP53 and single nucleotide polymorphism thereof, which mainly comprises the following steps:
(1) Reacting the single-stranded DNA probe solution marked by the electrochemical indicator with a reducing reagent solution for 1-2 hours at room temperature according to the mol ratio of 1:1-1000 to obtain a sulfhydryl modified single-stranded DNA probe, wherein the reducing reagent comprises tris (2-carboxyethyl) phosphine, DTT, sulfhydryl ethanol and reduced glutathione;
(2) Dropwise adding the diluted solution of the sulfhydryl modified single-stranded DNA probe obtained in the step (1) onto the surface of an activated gold electrode, and reacting for 12-16 hours at the temperature of 1-10 ℃ to obtain the single-stranded DNA probe modified gold electrode;
(3) Immersing the gold electrode modified by the single-stranded DNA probe prepared in the step (2) into an aqueous solution of a blocking agent, and reacting for 0.5-5 hours at room temperature to obtain the DNA electrochemical sensor, wherein the blocking agent comprises mercapto hexanol and mercapto ethanol.
Further, the electrochemical indicators described in step (1) include, but are not limited to, methylene blue, ferrocene.
Further, the single-stranded DNA probe marked by the electrochemical indicator in the step (1) is a single-stranded DNA probe of a hairpin structure of straight-chain alkane containing disulfide bonds between 3 '-end modified methylene blue or ferrocene and 5' -end modified chain, and the concentration is 0.1-1 mM.
Further, the structure of the single-stranded DNA probe labeled with the electrochemical indicator in the step (1) is as follows: 5'-C6-S-S-C6-GGC ATG AAC CGG AGG CCC ATC TCA TGC C-MB-3'.
Further, the concentration of the reducing reagent solution in the step (1) is 10 to 100mM.
Further, the concentration of the diluted solution of the thiol-modified single-stranded DNA probe in the step (2) is 0.1 to 10. Mu.M, and the dropwise addition volume is 2 to 10. Mu.L.
Further, the treatment process of the activated gold electrode surface in the step (2) is as follows: cleaning in a piranha solution for 5-20 minutes, then sequentially ultrasonically cleaning in absolute ethyl alcohol and deionized water, sequentially polishing the surface of a gold electrode by using aluminum oxide powder with the particle diameters of 1 mu m,0.3 mu m and 0.05 mu m respectively, and then sequentially ultrasonically cleaning in absolute ethyl alcohol and deionized water.
Further, the concentration of the aqueous solution of the blocking agent in the step (3) is 1 to 10mM.
In another aspect, the present invention provides a DNA electrochemical sensor prepared by the above preparation method.
In a further aspect, the invention provides the use of the DNA electrochemical sensor in the detection of tumor suppressor gene TP53 and single nucleotide polymorphisms thereof.
Further, the single nucleotide polymorphisms of the tumor suppressor gene TP53 include, but are not limited to, mutations at positions 175, 248, 249, 273, 282 of the TP53 gene.
The invention also provides a detection method for detecting the single nucleotide polymorphism of the tumor suppressor gene TP53 by using the DNA electrochemical sensor, which mainly comprises the following steps:
(a) Placing a DNA electrochemical sensor, a reference electrode and an auxiliary electrode in a detection buffer system, performing differential pulse voltammetry scanning and recording to obtain peak current I a ；
(b) Taking out the DNA electrochemical sensor, dripping TP53 gene sequence water solution which is completely complementary with DNA in the DNA electrochemical sensor on the surface of the sensor, carrying out hybridization reaction for 30-40 minutes, and leaching with a buffer solution to obtain the DNA electrochemical sensor after the hybridization reaction;
(c) Re-immersing the hybridized DNA electrochemical sensor in the detection buffer system in the step (a), performing differential pulse voltammetry scanning and recording a response signal I b ；
(d) Replacing the completely complementary TP53 gene sequence with the TP53 gene aqueous solution with single nucleotide polymorphism, repeating the steps (b) and (c) to obtain a response signal I c ；
(e) By comparing the percent current drop ((I) a -I b )/I a ) And ((I) a -I c )/I a ) The single nucleotide polymorphism of the tumor suppressor gene TP53 was determined.
Further, the buffer system in step (a) is Na 2 HPO 4 And NaH 2 PO 4 Mixed aqueous solution of equimolar concentration of Na 2 HPO 4 And NaH 2 PO 4 The molar concentration of the catalyst is 5-20 mmol/L, and the pH is 6-8.
Compared with the prior art, the invention has the following beneficial effects:
1. the DNA electrochemical sensor prepared by the invention has reliable and accurate detection result, the DNA sensor has better stability than a protein (enzyme) sensor, and the isolated DNA has better thermal stability than most protein (enzyme) molecules, and the prepared sensor can be stored for a long time.
2. The DNA electrochemical sensor prepared by the invention has good specificity, the DNA molecule double chains have very high specific recognition through specific base pairing, and the mutation of single base can cause obvious change of electric signals.
3. The DNA electrochemical sensor prepared by the invention has low price, simple operation and high reaction speed.
4. A common problem in mutation analysis is that the difference in dissolution temperature between a perfectly matched hybridization sequence and a hybridization sequence containing a Single Nucleotide Polymorphism (SNP) may be only 1-2 ℃, resulting in relatively low selectivity for SNP, and if mutation occurs in the loop region of the hairpin structure, the DNA electrochemical sensor of the invention will increase this difference, thereby improving the discrimination between the complete sequence and the mutated sequence, and potentially enabling early screening and early warning of cancer by detecting the single nucleotide polymorphism of the TP53 gene at an early stage.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 shows the reduction process of thiol groups of DNA capture probes in example 1 of the present invention;
FIG. 2 is a schematic diagram of the preparation of a DNA electrochemical sensor according to example 2 of the present invention;
FIG. 3 is a cyclic voltammogram of the gold electrode (a) and the prepared DNA electrochemical biosensor (b) after activation in the preparation process of the DNA electrochemical sensor of example 2 of the present invention;
FIG. 4 is a cyclic voltammogram (a) and a plot (b) of peak current versus square root of scan rate for a DNA electrochemical sensor prepared in example 2 of the present invention at different scan rates;
FIG. 5 is a plot of the log of cDNA concentration versus percent peak current drop of the DNA electrochemical sensor in example 3 of the present invention;
FIG. 6 is a bar graph showing the current change rate of the peak of the SBM sequence in which the DNA electrochemical sensor detects the mutation at 248 locus of the cDNA sequence and PT53 gene according to example 4 of the present invention;
FIG. 7 is a graph showing the peak change of 10 consecutive scans of the DNA electrochemical biosensor in example 5 of the present invention.
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
The following are the main instruments and equipment used in the examples of the present invention, and other experimental conditions not specifically noted were performed according to conventional or instrument manufacturer recommended conditions.
The instrument used for electrochemical detection is a portable electrochemical workstation PalmSens4, three-electrode system; DNA sequences (including TP53 gene fragments) were purchased from Biotechnology (Shanghai) Inc. The DNA sequences in the examples are shown in Table 1.
TABLE 1 oligonucleotide base sequences
EXAMPLE 1 reduction of DNA Probe to thiol-modified Single-stranded DNA Probe
mu.L of a 0.1mM DNA probe (sDNA) solution was mixed with 1. Mu.L of 10mM tris (2-carboxyethyl) phosphine (TCEP), and reacted at room temperature for 1 hour to obtain a thiol-modified single-stranded DNA probe.
Example 2 preparation of DNA electrochemical biosensor
The preparation method of the DNA electrochemical biosensor mainly comprises the following steps:
(1) Surface area of 0.02cm 2 Gold electrode in piranha solution (H) 2 O 2 :H 2 SO 4 =1:3, v/v) for 15 minutes, then repeatedly sonicated 3 times in absolute ethanol and deionized water for 3 minutes each; sequentially using aluminum oxide powder with particle diameters of 1 μm,0.3 μm and 0.05 μmPolishing, repeatedly and ultrasonically cleaning in absolute ethyl alcohol and deionized water for 3 times for 3 minutes each time; putting the electrode into 0.5M sulfuric acid solution to circularly scan and activate between 0 and 1.7V, and when the scanning curves are completely coincident, taking out the electrode, flushing with deionized water and drying with nitrogen;
(2) Diluting the methylene blue marked and sulfhydryl modified single-stranded DNA probe solution obtained in the step example 1 to 1 mu M, dripping 5 mu L to the surface of the gold electrode activated in the step (1), reacting for 16 hours at the temperature of 4 ℃, repeatedly flushing with deionized water and 10mM PBS buffer solution (PH 7.0), and drying with nitrogen to obtain the single-stranded DNA probe modified gold electrode.
(3) Immersing the gold electrode modified by the single-stranded DNA probe prepared in the step (2) into a 1mM mercaptohexanol (blocking agent) aqueous solution, and reacting for 1 hour at room temperature to obtain the DNA electrochemical biosensor.
In FIG. 3, cyclic voltammogram a is obtained by placing activated gold electrode in 5mM potassium ferricyanide buffer solution, scanning cyclic voltammogram between-0.1 and 0.6V, the oxidation-reduction peak potential difference of the curve is less than 75mV, which indicates that potassium ferricyanide reaction on the surface of gold electrode belongs to complete reversible reaction, and the surface of gold electrode is free of impurities and is completely activated; in FIG. 3, cyclic voltammogram b is a plot of the prepared DNA electrochemical biosensor in 5mM potassium ferricyanide solution, and the cyclic voltammogram is scanned between-0.1 and 0.6V. The peak current of potassium ferricyanide in the CV curve drops greatly, indicating successful binding of the scna and blocking agent to the gold electrode surface.
FIG. 4 is a graph of seven different scan rates of 50, 100, 200, 300, 400, 500, 600mV/s between-0.45 and 0V in 10mM PBS buffer (pH 7.0), showing that the scan rate is linearly related to the peak current, and the reaction is electrode surface control.
Example 3DNA biosensor for detecting TP53 Gene
TP53 genes (cDNA) with the concentration of 10nM, 100nM and 1 mu M are respectively dripped on the surface of a prepared DNA electrochemical biosensor, hybridization reaction is carried out for 30 minutes at room temperature, deionized water and 10mM PBS buffer solution (PH 7.0) are used for repeatedly flushing, nitrogen is used for drying, 10mM PBS buffer solution is added, and differential pulse voltammetry curve is scanned between-0.45V and 0V. The relationship between the peak current change rate and the logarithm of cDNA concentration is shown in FIG. 5.
Example 4DNA biosensor for detecting single nucleotide polymorphism of TP53 Gene
The prepared DNA electrochemical biosensor is put into 10mM PBS buffer solution, and the differential pulse voltammetry curve is scanned between-0.45 and 0V to obtain peak current I a . Then, 1 mu M concentration cDNA and SBM are respectively dripped on the surface of the DNA electrochemical biosensor, hybridization reaction is carried out for 30 minutes under the condition of room temperature, deionized water and 10mM PBS buffer solution (PH 7.0) are used for repeatedly flushing, after nitrogen is blown dry, 10mM PBS buffer solution is added, and differential pulse voltammetry curve is scanned between-0.45V and 0V to obtain peak current I b And I c . The peak current change rates before and after hybridization reaction were respectively delta cDNA ＝(I a -I b )/I a X 100% and% SBM ＝(I a -I c )/I a 100% and the results are shown in FIG. 6.
Example 5 evaluation of reproducibility and storage stability of DNA electrochemical biosensor
Reproducibility of the DNA electrochemical biosensor of the present invention was studied by differential pulse, and the Relative Standard Deviation (RSD) of the DNA electrochemical biosensor to 1 μm cDNA for 10 consecutive scans was less than 1.5%, indicating that the DNA electrochemical biosensor of the present invention has good reproducibility. In order to evaluate reproducibility between different electrodes of the same batch of the DNA electrochemical biosensor of the present invention, 5 sensors were independently prepared using the same conditions. The relative standard deviation of the peak current change rate of the 5 sensors for the 1 mu M cDNA detection is less than 2.0%, which shows that the electrodes prepared in different batches have good reproducibility. In order to evaluate the storage stability of the DNA electrochemical biosensor of the present invention, the electrode was placed in a PBS solution of pH 7.4, stored at 4 ℃ for 7 days, and compared with a freshly prepared DNA electrochemical biosensor, the change rate of the peak current value of the DNA electrochemical biosensor was reduced by about 6% after storage for 7 days, indicating that the biosensor has good storage stability.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
SEQUENCE LISTING
<110> institute of chemical and physical of Dalian of academy of sciences of China
<120> a DNA electrochemical sensor, and preparation method and application thereof
<130> 20220418
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> DNA
<213> artificial sequence
<400> 1
C6-S-S-C6-ggcatgaacc ggaggcccat ctcatgcc-MB 28
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
gatgggcctc cggttcatgc c 21
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
gatgggcctc cagttcatgc c 21
Claims (10)
1. The preparation method of the DNA electrochemical sensor is characterized by mainly comprising the following steps:
(1) Reacting the single-stranded DNA probe solution marked by the electrochemical indicator with a reducing reagent solution according to the molar ratio of 1:1-1000 to obtain a sulfhydryl modified single-stranded DNA probe, wherein the reducing reagent comprises tris (2-carboxyethyl) phosphine, DTT, sulfhydryl ethanol and reduced glutathione;
(2) Dropwise adding the diluted solution of the sulfhydryl modified single-stranded DNA probe obtained in the step (1) onto the surface of an activated gold electrode, and reacting for 12-16 hours at the temperature of 1-10 ℃ to obtain the single-stranded DNA probe modified gold electrode;
(3) Immersing the gold electrode modified by the single-stranded DNA probe prepared in the step (2) into an aqueous solution of a blocking agent, and reacting for 0.5-5 hours to obtain the DNA electrochemical sensor, wherein the blocking agent comprises mercapto hexanol and mercapto ethanol.
2. The method of claim 1, wherein the electrochemical indicator of step (1) includes, but is not limited to, methylene blue, ferrocene.
3. The method according to claim 1, wherein the single-stranded DNA probe labeled with the electrochemical indicator in the step (1) is a single-stranded DNA probe of a hairpin structure of a straight-chain alkane containing disulfide bonds between 3 '-end modified methylene blue or ferrocene and 5' -end modified chain, and the concentration is 0.1-1 mM; the concentration of the reducing reagent solution is 10-100 mM.
4. The method of claim 3, wherein the structure of the single-stranded DNA probe labeled with the electrochemical indicator in the step (1) is as follows: 5'-C6-S-S-C6-GGC ATG AAC CGG AGG CCC ATC TCA TGC C-MB-3'.
5. The method according to claim 1, wherein the concentration of the diluted solution of the thiol-modified single-stranded DNA probe in the step (2) is 0.1 to 10. Mu.M, and the volume of the solution is 2 to 10. Mu.L.
6. The method according to claim 1, wherein the treatment process of the activated gold electrode surface in step (2) is as follows: cleaning in a piranha solution for 5-20 minutes, then sequentially ultrasonically cleaning in absolute ethyl alcohol and deionized water, sequentially polishing the surface of a gold electrode by using aluminum oxide powder with the particle diameters of 1 mu m,0.3 mu m and 0.05 mu m respectively, and then sequentially ultrasonically cleaning in absolute ethyl alcohol and deionized water.
7. The method according to claim 1, wherein the concentration of the aqueous solution of the blocking agent in the step (3) is 1 to 10mM.
8. A DNA electrochemical sensor produced by the method of any one of claims 1-7.
9. The use of the DNA electrochemical sensor according to claim 8 in the detection of tumor suppressor gene TP53 and single nucleotide polymorphisms thereof.
10. A method for detecting a single nucleotide polymorphism of a tumor suppressor gene TP53 using the DNA electrochemical sensor of claim 8, comprising the steps of:
(a) Placing a DNA electrochemical sensor, a reference electrode and an auxiliary electrode in a detection buffer system, performing differential pulse voltammetry scanning and recording to obtain peak current I a ；
(b) Taking out the DNA electrochemical sensor, dripping TP53 gene sequence water solution which is completely complementary with DNA in the DNA electrochemical sensor on the surface of the sensor, carrying out hybridization reaction for 30-40 minutes, and leaching with a buffer solution to obtain the DNA electrochemical sensor after the hybridization reaction;
(c) Re-immersing the hybridized DNA electrochemical sensor in the detection buffer system in the step (a), performing differential pulse voltammetry scanning and recording a response signal I b ；
(d) Replacing the completely complementary TP53 gene sequence with the TP53 gene aqueous solution with single nucleotide polymorphism, repeating the steps (b) and (c) to obtain a response signal I c ；
(e) General purpose medicinePercentage decrease in current ((I) is compared a -I b )/I a ) And ((I) a -I c )/I a ) The single nucleotide polymorphism of the tumor suppressor gene TP53 was determined.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020146716A1 (en) * | 1997-04-09 | 2002-10-10 | Barton Jacqueline K. | Electrochemical sensor using intercalative, redox-active moieties |
| US20050153285A1 (en) * | 2001-03-13 | 2005-07-14 | Yoshio Umezawa | Electrochemical detection method of complementarity to nucleic acid bases |
| US20110186448A1 (en) * | 2010-01-29 | 2011-08-04 | Mohammad Saeid Hejazi | Detection and discrimination of target dna sequence and single nucleotide polymorphism directly on double-stranded oligonucleotide and pcr product without denaturation of dsdna sample based on pna-dsdna hybrid and utilizing pna probe |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
| CN106706748A (en) * | 2016-12-16 | 2017-05-24 | 北京化工大学 | Aptamer electrochemistry biosensor capable of detecting malachite green and preparation method thereof |
| CN109797200A (en) * | 2019-02-13 | 2019-05-24 | 中国科学院苏州生物医学工程技术研究所 | Ratio-type telomere enzyme active quantitive detection method |
| CN114280127A (en) * | 2022-01-03 | 2022-04-05 | 海南师范大学 | Determination of the gene sequence of Escherichia coli by a nano-gold-graphyne composite modified electrode |
-
2022
- 2022-04-19 CN CN202210412704.4A patent/CN116953055A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020146716A1 (en) * | 1997-04-09 | 2002-10-10 | Barton Jacqueline K. | Electrochemical sensor using intercalative, redox-active moieties |
| US20050153285A1 (en) * | 2001-03-13 | 2005-07-14 | Yoshio Umezawa | Electrochemical detection method of complementarity to nucleic acid bases |
| US20110186448A1 (en) * | 2010-01-29 | 2011-08-04 | Mohammad Saeid Hejazi | Detection and discrimination of target dna sequence and single nucleotide polymorphism directly on double-stranded oligonucleotide and pcr product without denaturation of dsdna sample based on pna-dsdna hybrid and utilizing pna probe |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
| CN106706748A (en) * | 2016-12-16 | 2017-05-24 | 北京化工大学 | Aptamer electrochemistry biosensor capable of detecting malachite green and preparation method thereof |
| CN109797200A (en) * | 2019-02-13 | 2019-05-24 | 中国科学院苏州生物医学工程技术研究所 | Ratio-type telomere enzyme active quantitive detection method |
| CN114280127A (en) * | 2022-01-03 | 2022-04-05 | 海南师范大学 | Determination of the gene sequence of Escherichia coli by a nano-gold-graphyne composite modified electrode |
Non-Patent Citations (1)
| Title |
|---|
| FERNANDO OTERO 等: "Electrochemical biosensor for the detection of a sequence of the TP 53 gene using a methylene blue labelled DNA probe", ELECTROCHIMICA ACTA, vol. 388, 22 May 2021 (2021-05-22), pages 2 - 4 * |
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