阅读说明：本技术 一种基于四面体dna纳米探针的电化学分析方法及应用 (Electrochemical analysis method based on tetrahedral DNA (deoxyribonucleic acid) nanoprobe and application ) 是由 郑峻松 陈曦 朱全敬 李艳 黄辉 汪莉娜 朱垂雨 方立超 邓均 刘华敏 李承红 于 2021-08-13 设计创作，主要内容包括：本发明属于DNA甲基化分析技术领域,公开了一种基于四面体DNA纳米探针的电化学分析方法及应用,包括：四面体探针结构序列、不同的DNA甲基化靶序列、信号放大序列及比对序列。在四面体探针固定时间3h、靶序列杂交时间1h、HpaII内切酶酶切时间2h的条件下体现出最优性能,于1amol/L至1pmol/L的范围内表现出较好的检测能力,检出限达到0.93amol/L,灵敏度高。本发明的电化学生物传感技术在DNA甲基化检测方面具有独特的优点：能在较短时间内完成对甲基化DNA靶序列的特异性分析,操作方便、价格低廉、灵敏度高、特异性好、稳定性强,能有效地用于血清等复杂体系中的DNA甲基化检测。(The invention belongs to the technical field of DNA methylation analysis, and discloses an electrochemical analysis method based on a tetrahedral DNA nanoprobe and application thereof, wherein the electrochemical analysis method comprises the following steps: a tetrahedral probe structure sequence, different DNA methylation target sequences, a signal amplification sequence and an alignment sequence. The method has the advantages that the optimal performance is realized under the conditions that the tetrahedral probe fixing time is 3h, the target sequence hybridization time is 1h and the digestion time of the HpaII endonuclease is 2h, the good detection capability is realized in the range of 1amol/L to 1pmol/L, the detection limit reaches 0.93amol/L, and the sensitivity is high. The electrochemical biosensing technology of the invention has unique advantages in the aspect of DNA methylation detection: can complete the specificity analysis of the methylated DNA target sequence in a short time, has convenient operation, low price, high sensitivity, good specificity and strong stability, and can be effectively used for DNA methylation detection in complex systems such as serum and the like.)

1. A tetrahedral DNA nanoprobe, wherein the tetrahedral DNA nanoprobe comprises: a tetrahedral probe structure sequence, different DNA methylation target sequences, a signal amplification sequence and an alignment sequence;

the tetrahedral thiolation structural sequence is SEQ ID NO: 1; the tetrahedral thiolation structural sequence is SEQ ID NO: 2; the tetrahedral thiolation structural sequence is SEQ ID NO: 3; the structural sequence of the tetrahedral probe is SEQ ID NO: 4;

the unmethylated target sequence is SEQ ID NO: 5; the methylation target sequence is SEQ ID NO: 6; the single base mismatched methylation target sequence is SEQ ID NO: 7; the target sequence of the multi-base mismatch methylation is SEQ ID NO: 8;

the hybrid chain reaction sequence 1 is SEQ ID NO: 9; the hybrid chain reaction sequence 2 is SEQ ID NO: 10; general signal probe amplification method alignment target sequence T1 is SEQ ID NO: 11; the sequence of the signal probe by the common signal probe amplification method is SEQ ID NO: 12.

2. an electrochemical analysis method using the tetrahedral DNA nanoprobe of claim 1, wherein the electrochemical analysis method comprises:

taking out all DNA sequence freeze-dried powder from-20 ℃, and adding TE buffer solution with corresponding volume to dissolve the DNA sequence freeze-dried powder to 100 umol/L;

secondly, diluting the S1-S4 sequence to 50umol/L by using TE buffer solution, sucking 1uL of the diluted sequence respectively, adding the diluted sequence into the same EP tube, adding 41uLTM buffer solution and 5uLTCEP solution, wherein the total amount is 50uL, and the final concentration is 1 umol/L; performing vortex mixing, performing instantaneous centrifugation, setting a PCR instrument at 95 ℃ for 2min and 4 ℃ for 1min, synthesizing a tetrahedral DNA nanoprobe by adopting a one-step method, performing vortex mixing on an EP tube, performing instantaneous centrifugation, and storing at 4 ℃ for later use;

thirdly, preparing 10% polyacrylamide gel and 9 samples, respectively sucking 10uL of each group of samples into a 200uLEP tube, respectively adding 2uL of loading buffer solution, lightly blowing, uniformly mixing, respectively adding 10uL into 2-10 lanes, and finally adding 10uLDNA markers into 1 lane;

fourthly, carrying out electrophoresis at the voltage of 150V for 5min, adjusting to 80V, continuing electrophoresis for 2h, and cutting off the power supply until the bromophenol blue in the loading buffer solution approaches the bottom of the gel; pouring 70mL of ultrapure water into a large-size culture dish, adding 30uL of nucleic acid dye, and uniformly mixing; taking out the gel, transferring the gel to a culture dish, shaking and dyeing for 30min, taking out the gel, and putting the gel into a digital gel image analysis system for analysis;

fifthly, peeling off the complete mica sheet thin layer by using a pair of tweezers, covering the surface of the freshly peeled mica with 50uL of 1% APTES solution, acting for 30min to passivate the mica, then thoroughly washing the mica with a large amount of ultrapure water, and drying the mica by using nitrogen; and then dropwise adding 15uL tetrahedral DNA nanoprobe, acting for 20min to enable the nano-probe to be naturally adsorbed on the surface of the mica sheet, gently leaching with ultrapure water, then placing into a drying box, and carrying out atomic force microscope detection after water is evaporated.

3. The electrochemical analysis method of claim 2, wherein the processing of the working electrode of the electrochemical analysis method comprises: soaking chamois leather with ultrapure water, making bare gold electrode perpendicular to chamois leather surface, and coating Al with 0.5um particle size₂O₃Polishing in homogenate for 2min, washing with ultrapure water, and adding 0.05um Al₂O₃Polishing and grinding the homogenate to a mirror surface, and thoroughly washing with ultrapure water; then respectively ultrasonically cleaning for 5min by using ultrapure water, absolute ethyl alcohol and ultrapure water in sequence; thoroughly washing with ultrapure water, blow-drying with nitrogen, immersing the electrode in freshly prepared piranha solution for activation for more than 15min, taking out, thoroughly washing with ultrapure water, and blow-drying with nitrogen for later use;

a three-electrode detection system is set up on an electrochemical workstation, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and a polished gold electrode is used as a working electrode; performing electrochemical cleaning in 0.5mol/L sulfuric acid solution by cyclic voltammetry until a standard and repeated curve appears;

after the electrochemical cleaning, the electrodes were rinsed thoroughly with ultra pure water, blown dry with nitrogen and then immersed in [ Fe (CN) ]₆]^3+/4+Performing cyclic voltammetry characterization in the solution until a standard and repeated curve appears and the potential difference of an oxidation/reduction peak is less than 0.1V, which represents that the bare gold electrode is cleaned completely and is named AuE for subsequent experiments; if the potential difference is greater than 0.1V, the capacitor is discarded.

4. The electrochemical analysis method according to claim 2, wherein the electrochemical analysis method comprises the following steps of electrodepositing nanogold: immersing the bare gold electrode into the nano-gold working solution to carry out electrochemical deposition of nano-gold by a potential dissolution analysis method;

after the electrodeposition is finished, the electrode is named AuNPs/AuE, the electrode is rinsed thoroughly with ultrapure water, nitrogen is blown dry, and the characterization of a scanning electron microscope and an electrochemical cyclic voltammetry is carried out on the electrode.

5. The electrochemical analysis method of claim 2, wherein the electrochemical analysis method is a method of operating an electrochemical analyzerModification of polar tetrahedral DNA nanoprobes: inversely inserting the prepared AuNPs/AuE on a foam board, dripping 4uL tetrahedral DNA nanoprobes (1umol/L) to the surface of an electrode, and covering an electrode plastic sleeve; after incubation at room temperature for 3h, the electrodes were rinsed thoroughly with PBS and ultrapure water, respectively, blown dry with nitrogen, designated TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+After the cyclic voltammetry characterization is carried out in the solution, the solution is washed by ultrapure water and dried by nitrogen.

6. The electrochemical assay of claim 2, wherein detection of a methylated DNA target sequence by the electrochemical assay: diluting a target DNA sequence B0/B1 to 1nmol/L by using hybridization buffer solution, heating and denaturing for 5min at 95 ℃ on a PCR instrument, uniformly mixing by vortex, and dripping 10uL to the surface of each electrode after instantaneous centrifugation; the electrodes were inserted vertically into a foam plate and placed in a water bath cabinet, incubated at 37 ℃ for 1h and then removed, the electrodes were rinsed thoroughly with PBS and ultrapure water, respectively, and blown dry with nitrogen, the electrodes being designated DNA/TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+After cyclic voltammetry characterization is carried out in the solution, the solution is washed by ultrapure water and dried by nitrogen;

taking out 10 Xcut from-20 deg.CThe buffer solution was diluted to the working concentration (1X) with ultrapure water on an ice box, and the stock HpaII restriction enzyme was taken out from-20 ℃ and applied to an ice box with 1 XcutDiluting the buffer solution to 50 Unit/mL; dropping 10uL onto the surface of each vertically placed electrode, incubating at 37 ℃ for 2h in a water bath, taking out, rinsing thoroughly with PBS and ultrapure water, and blowing dry with nitrogen, the electrode being HpaII/DNA/TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

7. The electrochemical analysis method of claim 6, wherein 100 is addedDiluting umol/L H1 and H2 sequences to 1umol/L with TE buffer solution, heating and denaturing at 95 ℃ for 5min on a PCR instrument, and then placing at 4 ℃ for renaturation for more than 15min to enable the H1 and H2 sequences to spontaneously form stem-loop structures; mixing H1 and H2 at a ratio of 1:1, diluting to 0.1umol/L with HCR amplification buffer solution, mixing uniformly by vortex, centrifuging instantaneously, and dripping 15uL to the surface of each electrode; after incubation in a water bath cabinet at 37 ℃ for 2h, the electrode was thoroughly rinsed with PBS and ultrapure water, respectively, and blown dry with nitrogen, as described for HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

8. The electrochemical analysis method of claim 7, wherein the streptavidin-labeled horseradish peroxidase S-HRP solution is removed from-20 ℃, diluted to 0.01mg/mL with the diluent, 5uL is added dropwise to the surface of each electrode, covered with an electrode plastic cover, and after incubation at room temperature for 20min, the electrode is thoroughly rinsed with PBS and ultrapure water to remove unbound S-HRP, and dried with nitrogen gas, and the electrode is labeled as HRP/HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

9. The electrochemical analysis method of claim 2, wherein the three-electrode system is placed in a TMB detection solution for amperometric and cyclic voltammetry, the TMB detection solution being replaced for each detection; setting parameters of a current analysis method: InitE: 0.1V, RunTime: 100s, QuietTime: 2s, Sensitivity1e-0.05A/V, and the rest parameters are set by default; setting parameters of cyclic voltammetry: InitE: 0.7V, HighE: 0.7V, LowE: 0V, Final E: 0V, InitialScanPolary: negative, ScanRate: 0.05V/s, SweepSegments: 2, Sensitivity1e-0.05A/V, with the remaining parameters using default settings.

10. Use of the tetrahedral DNA nanoprobe of claim 1 in the high sensitivity detection of DNA methylation.

Technical Field

The invention belongs to the technical field of DNA methylation biosensing analysis, and particularly relates to an electrochemical analysis method based on a tetrahedral DNA nanoprobe and application thereof.

Background

At present: DNA methylation plays an important role in the gene transcription process, and can cause the occurrence and development of Alzheimer's disease, cardiovascular and cerebrovascular diseases and malignant tumors once abnormality occurs. At present, various DNA methylation analysis methods exist, such as methods based on bisulfite conversion, such as methylation specific polymerase chain reaction, fluorescence quantification, methylation specific high-resolution melting curve, pyrosequencing and the like, and analysis methods based on methylation specific recognition molecules, such as methylation restriction enzymes, methyl binding domain proteins, anti-5-methylcytosine antibodies, zinc finger proteins and the like, and methods such as high performance liquid chromatography, high performance capillary electrophoresis, mass spectrometry, surface enhanced Raman spectroscopy and the like are utilized. Generally speaking, the traditional methods can complete the analysis of DNA methylation, but due to the reasons of complicated operation, poor stability, poor specificity, DNA degradation and the like, and the need of specific large-scale equipment, experimental sites and professional data analysis technicians, the clinical application is limited, and the conditions for developing the DNA methylation analysis of clinical specimens are difficult to satisfy. Therefore, the development of a DNA methylation detection method with convenient operation, high sensitivity and good specificity has great clinical application value.

Through the above analysis, the problems and defects of the prior art are as follows: the traditional DNA methylation analysis method is complex in operation, poor in stability and specificity, can cause degradation of DNA to be detected, needs specific large-scale equipment, an experimental site and professional data analysis technicians, and is relatively limited in clinical application.

The difficulty and significance of solving the above problems and defects: with the development of medicine, the role of DNA methylation in the occurrence and development of diseases is increasingly prominent, and the DNA methylation plays an important role in the 'early discovery, early diagnosis and early treatment' of diseases, and the traditional analysis method is difficult to meet the current clinical detection requirement of DNA methylation. The biosensor analysis method has the advantages of convenient operation, low cost, high sensitivity, easy miniaturization, particular suitability for bedside detection and capability of providing accurate detection results in a short time. The project aims to establish an ultrasensitive DNA methylation electrochemical biosensor analysis method, provide an accurate and sensitive detection means for the diagnosis of cancer and other methylation related diseases, and solve the technical requirement of clinical samples on DNA methylation ultrasensitive detection.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an electrochemical analysis method based on a tetrahedral DNA nanoprobe and application thereof.

The invention is thus achieved, a tetrahedral DNA nanoprobe, comprising: tetrahedral probe structure sequence, different DNA methylation target sequence, signal amplification sequence and alignment sequence:

the tetrahedral thiolation structural sequence is SEQ ID NO: 1; the tetrahedral thiolation structural sequence is SEQ ID NO: 2; the tetrahedral thiolation structural sequence is SEQ ID NO: 3; the structural sequence of the tetrahedral probe is SEQ ID NO: 4;

the unmethylated target sequence is SEQ ID NO: 5; the methylation target sequence is SEQ ID NO: 6; the single base mismatched methylation target sequence is SEQ ID NO: 7; the target sequence of the multi-base mismatch methylation is SEQ ID NO: 8;

the hybrid chain reaction sequence 1 is SEQ ID NO: 9; the hybrid chain reaction sequence 2 is SEQ ID NO: 10; general signal probe amplification method alignment target sequence T1 is SEQ ID NO: 11; the sequence of the signal probe by the common signal probe amplification method is SEQ ID NO: 12.

another object of the present invention is to provide an electrochemical analysis method using the tetrahedral DNA nanoprobe, the electrochemical analysis method comprising:

taking out all DNA sequence freeze-dried powder from-20 ℃, and adding TE buffer solution with corresponding volume to dissolve the DNA sequence freeze-dried powder to 100 umol/L;

secondly, diluting the S1-S4 sequence to 50umol/L by using TE buffer solution, sucking 1uL of the diluted sequence respectively, adding the diluted sequence into the same EP tube, adding 41uLTM buffer solution and 5uL of TCEP solution, wherein the total amount is 50uL, and the final concentration is 1 umol/L; performing vortex mixing, performing instantaneous centrifugation, setting a PCR instrument at 95 ℃ for 2min and 4 ℃ for 1min, synthesizing a tetrahedral DNA nanoprobe by adopting a one-step method, performing vortex mixing on an EP tube, performing instantaneous centrifugation, and storing at 4 ℃ for later use;

thirdly, preparing 10% polyacrylamide gel and 9 samples, respectively sucking 10uL of each group of samples into a 200uL EP tube, respectively adding 2uL of loading buffer solution, gently blowing, uniformly mixing, respectively adding 10uL into 2-10 lanes, and finally adding 10uL of DNA marker into 1 lane;

fourthly, carrying out electrophoresis at the voltage of 150V for 5min, adjusting to 80V, continuing electrophoresis for 2h, and cutting off the power supply until the bromophenol blue in the loading buffer solution approaches the bottom of the gel; pouring 70mL of ultrapure water into a large-size culture dish, adding 30uL of nucleic acid dye, and uniformly mixing; taking out the gel, transferring the gel to a culture dish, shaking and dyeing for 30min, taking out the gel, and putting the gel into a digital gel image analysis system for analysis;

fifthly, peeling off the complete mica sheet thin layer by using a pair of tweezers, covering the surface of the freshly peeled mica with 50uL of 1% APTES solution, acting for 30min to passivate the mica, then thoroughly washing the mica with a large amount of ultrapure water, and drying the mica by using nitrogen; and then dropwise adding 15uL tetrahedral DNA nanoprobe, acting for 20min to enable the nano-probe to be naturally adsorbed on the surface of the mica sheet, gently leaching with ultrapure water, then placing into a drying box, and carrying out atomic force microscope detection after water is evaporated.

Further, the processing of the working electrode of the electrochemical analysis method includes: soaking chamois leather with ultrapure water, making bare gold electrode perpendicular to chamois leather surface, and coating Al with 0.5um particle size₂O₃Polishing in homogenate for 2min, washing with ultrapure water, and adding 0.05um Al₂O₃Polishing and grinding the homogenate to a mirror surface, and thoroughly washing with ultrapure water; then respectively ultrasonically cleaning for 5min by using ultrapure water, absolute ethyl alcohol and ultrapure water in sequence; thoroughly washing with ultrapure water, blow-drying with nitrogen, immersing the electrode in freshly prepared piranha solution for activation for more than 15min, taking out, thoroughly washing with ultrapure water, and blow-drying with nitrogen for later use;

a three-electrode detection system is set up on an electrochemical workstation, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and a polished gold electrode is used as a working electrode. Performing electrochemical cleaning in 0.5mol/L sulfuric acid solution by cyclic voltammetry until a standard and repeated curve appears;

after the electrochemical cleaning, the electrodes were rinsed thoroughly with ultra pure water, blown dry with nitrogen and then immersed in [ Fe (CN) ]₆]^3+/4+Performing cyclic voltammetry characterization in the solution until a standard and repeated curve appears and the potential difference of an oxidation/reduction peak is less than 0.1V, which represents that the bare gold electrode is cleaned completely and is named AuE for subsequent experiments; if the potential difference is greater than 0.1V, the capacitor is discarded.

Further, the electrodeposited nanogold of the electrochemical analysis method: immersing the bare gold electrode into the nano-gold working solution to carry out electrochemical deposition of nano-gold by a potential dissolution analysis method;

after the electrodeposition is finished, the electrode is named AuNPs/AuE, the electrode is rinsed thoroughly with ultrapure water, nitrogen is blown dry, and the characterization of a scanning electron microscope and an electrochemical cyclic voltammetry is carried out on the electrode.

Further, the tetrahedron DNA nanoprobe modification of the working electrode of the electrochemical analysis method is as follows: the prepared AuNPs/AuE was inserted upside down on a foam plate, 4uL of tetrahedral DNA nanoprobes (1umol/L) were added dropwise to the electrode surface, and the electrode plastic cover was covered. After incubation at room temperature for 3h, the electrodes were rinsed thoroughly with PBS and ultrapure water, respectively, blown dry with nitrogen, designated TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+After the cyclic voltammetry characterization is carried out in the solution, the solution is washed by ultrapure water and dried by nitrogen.

Further, the detection of the methylated DNA target sequence by the electrochemical assay method: diluting a target DNA sequence B0/B1 to 1nmol/L by using hybridization buffer solution, heating and denaturing for 5min at 95 ℃ on a PCR instrument, uniformly mixing by vortex, and dripping 10uL to the surface of each electrode after instantaneous centrifugation; the electrodes were inserted vertically into a foam plate and placed in a water bath cabinet, incubated at 37 ℃ for 1h and then removed, the electrodes were rinsed thoroughly with PBS and ultrapure water, respectively, and blown dry with nitrogen, the electrodes being designated DNA/TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+After cyclic voltammetry characterization is carried out in the solution, the solution is washed by ultrapure water and dried by nitrogen;

taking out from-20 DEG CThe buffer solution was diluted with ultrapure water on an ice box to the working concentration (1X), and the stock HpaII restriction enzyme was taken out from-20 ℃ and used on an ice boxDiluting the buffer solution to 50 Unit/mL; dropping 10uL onto the surface of each vertically placed electrode, incubating at 37 ℃ for 2h in a water bath, taking out, rinsing thoroughly with PBS and ultrapure water, and blowing dry with nitrogen, the electrode being HpaII/DNA/TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

Further, 100umol/L of H1 and H2 sequences are diluted to 1umol/L by TE buffer solution, heated and denatured for 5min at 95 ℃ on a PCR instrument, and then placed at 4 ℃ for renaturation for more than 15min, so that the H1 and H2 sequences spontaneously form a stem-loop structure; mixing H1 and H2 at a ratio of 1:1,diluting to 0.1umol/L by using HCR amplification buffer solution, uniformly mixing by vortex, instantly centrifuging, and dropwise adding 15uL to the surface of each electrode; after incubation in a water bath cabinet at 37 ℃ for 2h, the electrode was thoroughly rinsed with PBS and ultrapure water, respectively, and blown dry with nitrogen, as described for HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

Further, a streptavidin-labeled horseradish peroxidase S-HRP solution was taken out from-20 ℃, diluted to 0.01mg/mL with the diluent, 5uL was added dropwise to the surface of each electrode, covered with an electrode plastic sheath, incubated at room temperature for 20min, and then thoroughly washed with PBS and ultrapure water to remove unbound S-HRP, and dried with nitrogen gas in the form of HRP/HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+And (4) performing cyclic voltammetry characterization in the solution, then washing with ultrapure water, and drying with nitrogen.

Further, the three-electrode system is put into TMB detection liquid for detection of amperometry and cyclic voltammetry, and the TMB detection liquid is replaced for each detection. Setting parameters of a current analysis method: InitE: 0.1V, RunTime: 100s, QuietTime: 2s, Sensitivity1e-0.05A/V, with the remaining parameters using default settings. Setting parameters of cyclic voltammetry: InitE: 0.7V, HighE: 0.7V, LowE: 0V, Final E: 0V, InitialScanPolary: negative, ScanRate: 0.05V/s, SweepSegments: 2, Sensitivity1e-0.05A/V, with the remaining parameters using default settings.

The invention also aims to provide an application of the tetrahedral DNA nanoprobe in high-sensitivity detection of DNA methylation.

By combining all the technical schemes, the invention has the advantages and positive effects that: the electrochemical biosensing technology of the invention has unique advantages in the aspect of DNA methylation detection: the specificity analysis of the methylated DNA target sequence can be completed in a short time, and the method is convenient to operate, low in price, good in specificity and strong in stability; meanwhile, the detection sensitivity capability is greatly improved due to the comprehensive action of a multiple signal amplification system, the methylation detection of trace DNA can be completed in complex systems such as serum and the like, and a response signal obviously different from a non-methylated DNA sequence is obtained, so that an accurate and sensitive detection result is provided for the diagnosis of cancers and other methylation related diseases. In addition, instruments and equipment involved in the invention are all miniaturized equipment and can be used for carrying out DNA methylation detection work in various clinical laboratories.

Drawings

FIG. 1 is a flow chart of an electrochemical analysis method provided in an embodiment of the present invention.

FIG. 2 is a schematic atomic force microscope image of a tetrahedral DNA nanoprobe provided in an embodiment of the present invention.

FIG. 3 is a schematic representation of polyacrylamide gel electrophoresis characterization of tetrahedral DNA nanoprobes provided by embodiments of the present invention.

FIG. 4 shows the results of the electrode coating with nanogold before and after the application of sulfuric acid solution (A) and [ Fe (CN)₆]^3+/4+CV curve in the solution (B), SEM image of the surface of the electrode covered with the nano-gold and a progressive amplification image thereof, wherein the scales are 300um (C), 300nm (D) and 50nm (E) respectively.

FIG. 5 is a schematic diagram of a CV characterization curve of a step modification process of a biosensor provided in an embodiment of the present invention.

FIG. 6 is a schematic diagram of the optimization experiment results of the tetrahedral DNA nanoprobe immobilization time (A), the target sequence hybridization time (B) and the HpaII enzyme digestion time (C) provided by the embodiment of the present invention.

Fig. 7 is a schematic diagram comparing signal amplification methods provided by embodiments of the present invention.

FIG. 8 is a cyclic voltammetry curve (A) for a methylated target sequence as provided in an example of the invention; amperometric detection curves (B) of methylated and unmethylated target sequences; amperometric detection curves (C) of methylated target sequences at different concentrations; amperometry measurements of different concentrations of methylated target sequence detect the concentration-signal curve and the linear range (D).

FIG. 9 shows amperometric detection signals (A) of a perfectly complementary target sequence (B1), a single-base mismatched target sequence (BC1) and a multiple-base mismatched target sequence (BCx) according to the example of the present invention; schematic representation of amperometric detection signal (B) of the sensor stored at 4 ℃ for 0-30 days.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides an electrochemical analysis method based on a tetrahedral DNA nanoprobe and application thereof, and the invention is described in detail with reference to the accompanying drawings.

As shown in fig. 1, the electrochemical analysis method provided by the present invention comprises the following steps:

s101: designing and synthesizing a specific tetrahedral DNA nano probe, a target sequence and a signal probe;

s102: step-by-step self-assembly and characterization of the gold electrode;

s103: optimizing the detection condition of DNA methylation electrochemical sensing;

s104: DNA methylation electrochemical sensing detection and signal analysis;

those skilled in the art of electrochemical analysis provided by the present invention can also perform other steps, and the electrochemical analysis method provided by the present invention in fig. 1 is only one specific example.

The tetrahedral DNA nanoprobe provided by the embodiment of the invention utilizes PrimerPremier and DNAsis software to design a tetrahedral probe structure sequence, different DNA methylation target sequences, a signal amplification sequence and an alignment sequence:

(1) tetrahedral thiolation structure sequence (S1, 5' thiol modification) SEQ ID NO: 1:

5’-SH-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC-3’

(2) tetrahedral thiolation structure sequence (S2, 5' thiol modification) SEQ ID NO: 2:

5’-SH-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC-3’

(3) tetrahedral thiolation structure sequence (S3, 5' thiol modification) SEQ ID NO: 3:

5’-SH-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT-3’

(4) tetrahedral probe structure sequence (S4) SEQ ID NO: 4:

5’-ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTATTTAGGTCGCAACACCCGGCGAGCACTGCGACCT-3’

(5) unmethylated target sequence (B0) SEQ ID NO: 5:

5’-AGTCTAGGATTCTGAGTGGGTTAAAGGTCGCAGTGCTCGCCGGGTGT-3’

(6) methylated target sequence (B1) SEQ ID NO: 6:

5’-AGTCTAGGATTCTGAGTGGGTTAAAGGTCGCAGTGCTCGCCGGGTGT-3’

(7) single base mismatched methylated target sequence (BC1) SEQ ID NO: 7:

5’-AGTCTAGGATTCTGAGTGGGTTAAAGGTCGCAGTGCTCGCCAGGTGT-3’

(8) multi-base mismatched methylation target sequence (BCx) SEQ ID NO: 8:

5’-AGTCTAGGATTCTGAGTGGGTTAAACGTAGCAGGGCTTGCCAGGTGT-3’

(9) hybrid strand reaction sequence 1(H1, 5' modified biotin) SEQ ID NO: 9:

5’-biotin-TTAACCCACTCAGAATCCTAGACTCAAAGTAGTCTAGGATTCTGAGTG-3’

(10) hybrid chain reaction sequence 2(H2, 5' modified biotin) SEQ ID NO: 10:

5’-biotin-AGTCTAGGATTCTGAGTGGGTTAACACTCAGAATCCTAGACTACTTTG-3’

(11) general signal probe amplification method alignment of target sequence (T1) SEQ ID NO: 11:

5’-TAGTGACTAGGTCGCAGTGCTCGCCGGGTGT-3’

(12) general signal probe amplification method signal probe sequence (R1, 3' modified biotin) SEQ ID NO: 12:

5’-AGTCACTA-biotin-3’

the technical solution of the present invention is further described below with reference to the accompanying drawings.

The electrochemical analysis method provided by the invention specifically comprises the following steps:

(1) all DNA sequence lyophilized powder was removed from-20 ℃ and dissolved to 100umol/L by adding the corresponding volume of TE buffer (10mmol/LTris, 1mmol/LEDTA, pH 8.0) as required by the reagent manufacturer.

(2) Diluting the S1-S4 sequence to 50umol/L with TE buffer, pipetting 1uL respectively, adding into the same EP tube, adding 41uLTM buffer (20mmol/L Tris, 50mmol/L MgCl)₂pH 8.0) and 5uL of TCEP solution (30mmol/L), the total amount being 50uL and the final concentration of DNA being 1 umol/L. Vortex mixing, instantaneous centrifuging, setting PCR instrument according to parameters (95 deg.C for 2min, 4 deg.C for 1min), and synthesizing tetrahedral DNA nanoprobe by one-step method. The EP tubes were then vortexed and centrifuged instantaneously and stored at 4 ℃ until use.

(3) 10% polyacrylamide gel and 9 samples (the specific components are shown in table 1) are prepared, and the tetrahedral DNA nanoprobe is manufactured in the same process. Respectively sucking 10uL of each group of samples into a 200uL EP tube, respectively adding 2uL of loading buffer solution, gently blowing and uniformly mixing, respectively adding 10uL of the loading buffer solution into 2-10 lanes, and finally adding 10uL of DNA marker into 1 lane. Electrophoresis is carried out for 5min under the voltage of 150V, then electrophoresis is continued for 2h after the voltage is adjusted to 80V, and the power supply is cut off until bromophenol blue in the loading buffer solution approaches the bottom of the gel. 70mL of ultrapure water was poured into a large-size petri dish, and 30uL of nucleic acid dye was added and mixed well. Taking out the gel, transferring the gel to a culture dish, shaking and dyeing for 30min, taking out the gel, and putting the gel into a digital gel image analysis system for analysis.

TABLE 1 composition of the samples of each group

(4) And (4) stripping the complete mica sheet thin layer by using a pair of tweezers to avoid the residue of the scraps. The freshly peeled mica surface was covered with 50uL of 1% APTES solution, passivated by exposure to 30min, thoroughly rinsed with large amounts of ultra-pure water and blown dry with nitrogen. And then dropwise adding 15uL tetrahedral DNA nanoprobe, acting for 20min to enable the nano-probe to be naturally adsorbed on the surface of the mica sheet, gently leaching with ultrapure water, then placing into a drying box, and carrying out atomic force microscope detection after water is evaporated.

Treatment of the working electrode in an embodiment of the invention: by means of ultrasoundsSoaking chamois leather in pure water, and making bare gold electrode be perpendicular to chamois leather surface, and in the condition of 0.5um grain size Al₂O₃Polishing in homogenate for 2min, washing with ultrapure water, and adding 0.05um Al₂O₃Polishing and grinding the homogenate to a mirror surface, and thoroughly washing with ultrapure water. Then, the ultrasonic cleaning was carried out for 5min in the order of ultrapure water, absolute ethanol and ultrapure water. Thoroughly rinsing with ultrapure water, blowing to dry with nitrogen, immersing the electrode in freshly prepared piranha solution (volume ratio of 98% concentrated sulfuric acid to 30% hydrogen peroxide is 3:1) for activation for more than 15min, taking out, thoroughly rinsing with ultrapure water, and blowing to dry with nitrogen for later use. A three-electrode detection system is set up on an electrochemical workstation, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and a polished gold electrode is used as a working electrode. Electrochemical washes were performed with cyclic voltammetry in 0.5mol/L sulfuric acid solution until a standard, repeated curve appeared (parameter settings: Inite: -0.3V, HighE: 1.6V, LowE: -0.3V, FinalE: 0V, InitialScanPolary: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, remaining parameters using default settings). After the electrochemical cleaning, the electrodes were rinsed thoroughly with ultra pure water, blown dry with nitrogen and then immersed in [ Fe (CN) ]₆]^3+/4+Solution (1mmol/L K)₃Fe(CN)₆、1mmol/L K₄Fe(CN)₆And 0.1mol/L KCl) until a standard, repeated curve occurs and the potential difference of the oxidation/reduction peak is less than 0.1V (parameter set: InitE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinalE: 0V, InitialScanPolary: positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, the rest parameters are set by default), which represents that the bare gold electrode is cleaned completely, and is named AuE and can be used for subsequent experiments; if the potential difference is greater than 0.1V, the capacitor is discarded.

In the examples of the present invention, nanogold was electrodeposited: immersing bare gold electrode in nano gold working solution (10mmol/L HAuCl)₄0.5mol/L sulfuric acid) of gold nanoparticles (parameter set: InitE: -0.2V, depositionitiime: 60s, QuiteTime: 2s, default settings for the remaining parameters). After the electrodeposition is finished, theIt was named AuNPs/AuE, and the electrodes were rinsed thoroughly with ultra pure water, blown dry with nitrogen, and characterized by scanning electron microscopy and electrochemical cyclic voltammetry (parameter settings: IniteE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinaE: 0V, InitialScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity 1-1 e-0.05A/V, and the remaining parameters were set as default).

In the examples of the present invention, the tetrahedral DNA nanoprobe modification of the working electrode: the prepared AuNPs/AuE is inversely inserted on a foam board, 4uL and 1umol/L tetrahedral DNA nanoprobes are dripped on the surface of the electrode, and the plastic cover of the electrode is covered. After incubation at room temperature for 3h, the electrodes were rinsed thoroughly with PBS (0.01mol/L, pH 7.4) and ultrapure water, dried with nitrogen, designated TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+The solution was subjected to cyclic voltammetry characterization (parameters: IniteE: -0.3V, HighE: 0.7V, LowE: -0.3V, Finale: 0V, InitialScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, and the remaining parameters were set as default), rinsed with ultrapure water, and dried with nitrogen for the next step.

Detection of methylated DNA target sequences in the examples of the invention: 100umol/L of the target DNA sequence (B0/B1) was hybridized with hybridization buffer (20mmol/L MgCl₂1mol/L NaCl dissolved in PBS) to 1nmol/L, heating and denaturing at 95 ℃ for 5min on a PCR instrument, mixing uniformly by vortex, centrifuging instantaneously, then respectively dripping 10uL to the surface of each electrode, vertically inserting the electrode into a foam plate and putting the foam plate into a water bath, incubating at 37 ℃ for 1h, taking out, respectively rinsing the electrode thoroughly with PBS and ultrapure water and drying with nitrogen, wherein the electrode is marked as DNA/TSP/AuNPs/AuE, and the application is as follows [ Fe (CN))₆]^3+/4+The solution was subjected to cyclic voltammetry characterization (parameters: InitE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinalE: 0V, initialsScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, and the remaining parameters were set as default), followed by rinsing with ultrapure water and blowing with nitrogen.

Taking out proper amount from-20 DEG CBuffer (containing 50mmol/L potassium acetate, 20mmol/L Tris-acetic acid, 10mmol/L magnesium acetate, 100ug/mL BSA, pH 7.9), diluted with ultrapure water on an ice box to the working concentration (1X), and HpaII restriction enzyme stock (10000Unit/mL) was removed from-20 deg.C, and put on an ice box with ultrapure waterThe buffer was diluted to 50 Unit/mL. Dropping 10uL onto the surface of each vertically placed electrode, incubating at 37 ℃ for 2h in a water bath, taking out, rinsing thoroughly with PBS and ultrapure water, and blowing dry with nitrogen, the electrode being HpaII/DNA/TSP/AuNPs/AuE, in [ Fe (CN)₆]^3+/4+The solution was subjected to cyclic voltammetry characterization (parameters: InitE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinalE: 0V, initialsScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, and the remaining parameters were set as default), followed by rinsing with ultrapure water and blowing with nitrogen.

Diluting the H1 and H2 sequences of 100umol/L to 1umol/L by TE buffer solution, heating and denaturing at 95 ℃ for 5min on a PCR instrument, and then placing at 4 ℃ for renaturation for more than 15min to ensure that the H1 and H2 sequences spontaneously form stem-loop structures. H1 and H2 were mixed at a ratio of 1:1, and the mixture was amplified with HCR amplification buffer (1mol/L NaCl, 50mmol/L Na)₂HPO₄pH 7.5) to 0.1umol/L, vortexing, mixing, centrifuging instantaneously, and adding 15uL dropwise to the surface of each electrode. After incubation in a water bath cabinet at 37 ℃ for 2h, the electrode was thoroughly rinsed with PBS and ultrapure water, respectively, and blown dry with nitrogen, as described for HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+The solution is subjected to cyclic voltammetry characterization (the parameter settings are IniteE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinalE: 0V, InitialScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, and the other parameters are set by default), then is washed with ultrapure water, and is subjected to the next operation after being dried by nitrogen.

The streptavidin-labeled horseradish peroxidase (S-HRP) solution was removed from-20 ℃, diluted to 0.01mg/mL with a diluent (1% BSA, 0.5% casein), 5uL was added dropwise to the surface of each electrode, and the electrode was covered with a coverThe electrode was thoroughly rinsed with PBS and ultrapure water after incubation for 20min at room temperature to remove unbound S-HRP, and dried with nitrogen gas, and was labeled as HRP/HCR/HpaII/DNA/TSP/AuNPs/AuE in [ Fe (CN)₆]^3+/4+The solution was subjected to cyclic voltammetry characterization (parameters: InitE: -0.3V, HighE: 0.7V, LowE: -0.3V, FinalE: 0V, initialsScanPolarity: Positive, ScanRate: 0.05V/s, SweepSegments: 10, Sensitivity1e-0.05A/V, and the remaining parameters were set as default), followed by rinsing with ultrapure water and blowing with nitrogen.

The three-electrode system was placed in TMB detection solution (Ready-to-use solution, pre-H₂O₂) The detection of amperometry and cyclic voltammetry is carried out, and the TMB detection solution is replaced for each detection. Setting parameters of a current analysis method: InitE: 0.1V, RunTime: 100s, QuietTime: 2s, Sensitivity1e-0.05A/V, with the remaining parameters using default settings. Setting parameters of cyclic voltammetry: InitE: 0.7V, HighE: 0.7V, LowE: 0V, Final E: 0V, InitialScanPolary: negative, ScanRate: 0.05V/s, SweepSegments: 2, Sensitivity1e-0.05A/V, with the remaining parameters using default settings.

The technical effects of the present invention will be described in detail with reference to experiments.

(1) Optimization of experimental conditions: the time for fixing the tetrahedral DNA nanoprobe on the surface of the electrode is respectively set to 0, 1, 2, 3 and 4h, the time for hybridizing the target DNA is respectively set to 0, 0.5, 1, 1.5, 2 and 2.5h, the time for carrying out enzyme digestion on HpaII is respectively set to 0, 0.5, 1, 1.5, 2 and 2.5h, and the final signal difference of the amperometry is detected under the condition that other conditions are not changed, so that the optimal reaction condition is found.

(2) Detection of concentration gradient of target DNA: target DNA solutions with different concentrations are respectively prepared, the concentration gradient is gradually increased to 100nmol/L from 1amol/L by taking 10 times as the gradient, and the concentration gradient is detected in 12 orders of magnitude, and the final signal difference of the amperometry is recorded.

(3) Alignment of Signal amplification method 100umol/L T1, R1 sequences were diluted to 1umol/L with TE buffer, respectively, diluted to 0.1umol/L with hybridization buffer, again vortexed, mixed, and centrifuged instantaneously for experiments in "no amplification" and "AuNPs amplification" groups in Table 2. The electrodes were divided into 5 groups for comparative experiments, and the rest of the experimental operations were performed as before, except for the differences in the items described in the table below.

Table 2 comparative experimental condition settings

(4) Repeatability, specificity and stability experiments: 1pmol/L of target DNA solution is prepared, 10uL of the target DNA solution is respectively dripped to the surfaces of 15 electrodes (3 electrodes in each group) in 5 groups treated in the same batch, and repeated experimental detection is carried out according to the experimental operation and parameters.

A solution of 1pmol/L of a perfectly complementary target DNA (B1) and a mismatched target DNA (BC1/BCx) was prepared, and the base mismatch discrimination ability of the sensor was examined by repeating the above experiment 3 times.

Diluting a target DNA sequence of 100umol/L to 1, 10, 20, 50 and 100fmol/L by using serum, respectively dripping 10uL to different electrode surfaces for hybridization after vortex mixing and instantaneous centrifugation, repeating the operation for 3 times according to the experiment, and simulating a real detection sample to verify the detection capability of the sensor.

Preparing 4 electrodes according to the experimental operation, flushing the electrodes with PBS and ultrapure water after the first amperometric detection is finished, drying the electrodes with nitrogen, covering an electrode plastic sleeve, and storing the electrodes in a refrigerator at 4 ℃. Then every 10 days, the electrode is detected by amperometry, washed again and stored in a refrigerator at 4 ℃ until the third detection (day 30) is finished.

(5) Atomic force microscopy characterization of tetrahedral DNA nanoprobes: as shown in FIG. 2, the atomic force microscope image shows that the mica plate substrate is relatively flat, and after natural sedimentation and fixation, the tetrahedral DNA nanoprobe is in a peak shape under the mirror, and the average height of the tetrahedral DNA nanoprobe is about 4.73 nm.

(6) Polyacrylamide gel electrophoresis characterization of tetrahedral DNA nanoprobes: as shown in FIG. 3, the stained gel was developed under an ultraviolet lamp, and the samples were well separated without tailing. The loaded samples in each lane show obvious electrophoresis rate difference in the gel according to different components, wherein the electrophoresis rate of the tetrahedral DNA nanoprobe (S1+ S2+ S3+ S4) positioned in the 10 th lane is the slowest.

(7) And (3) processing the gold electrode: the gold electrode is subjected to polishing, ultrasonic cleaning and piranha solution activation, and then is subjected to electrochemical cleaning in a sulfuric acid solution until a stable and repeated curve appears. As shown by the black solid line A in FIG. 4, a large cathodic reduction peak at +0.93V and multiple anodic oxidation peaks at + 1.15- +1.6V can be seen. With [ Fe (CN) ]₆]^3+/4+The solution is characterized by cyclic voltammetry, and the obtained stable curve is as shown by a black solid line in B of figure 3, and an anodic oxidation peak appears at +0.18V, a cathodic reduction peak appears at +0.26V, the two peaks have symmetrical peak types, and the potential difference is about 0.08V.

After the electrode is electroplated with gold, the gold is plated in a sulfuric acid solution and [ Fe (CN)₆]^3+/4+The solutions both showed a phenomenon of increase in peak current, as shown by the dotted lines of a and B of fig. 4. The gold-plated electrode was examined by scanning electron microscopy, and C, D, E in FIG. 4 shows that a large number of particles with an average particle size of about 40. + -.5 nm were collected on the surface of the electrode.

(8) Modification of the biosensor: the prepared sensor is covered by a tetrahedral DNA nanoprobe, hybridized with a target sequence, and then treated by HpaII, HCR and S-HRP, wherein after each step is finished, the sensor is coated with Fe (CN)₆]^3+/4+When cyclic voltammetry characterization is performed in solution, as shown in fig. 5, AuE and AuNPs/AuE present larger peak currents, and as subsequent operations are performed, the peak currents of the electrodes gradually decrease, indicating that the modification of each step of the electrochemical biosensor is successful.

(9) Optimization of experimental conditions: the detection result of the optimized experimental amperometric method of the tetrahedral DNA nanoprobe with the fixing time from 0 to 4h is shown in A of figure 6, the detection signal of the amperometric method is gradually increased along with the increase of the fixing time, and the signal of the amperometric method tends to be flat after exceeding 3 h; as shown in fig. 6B, the detection signal rapidly increases with the increase of the hybridization time, and the rise of the amperometric signal after 1h tends to be gentle; as shown in fig. 6C, the restriction enzyme HpaII optimized from 0 to 2.5h (n ═ 3) showed that the amperometric detection signal of the unmethylated target sequence B0 gradually decreased with increasing cleavage time, and the decrease of the amperometric signal tended to be gradual over 2h, while the amperometric detection signal of the methylated target sequence B1 did not change much.

(10) Contrast signal amplification of signal amplification method: the current analysis method detection result of the comparative experiment is shown in fig. 7, the current shows a rising trend along with the addition of the signal amplification system, and the HCR lifting effect is more obvious than that of AuNPs. The current of the AuNPs + HCR group was increased by about 3.9 times compared to the experimental group without signal amplification.

(11) Detection of target DNA: FIG. 8A is a cyclic voltammetry characterization curve of the sensor in TMB substrate, and in the blank control (black solid line), two pairs of redox peaks are visible, and as the concentration of the target sequence increases, the current of the reduction peak at +0.24V also increases, resulting in asymmetric change.

FIG. 8B is the amperometric detection curve of 1nmol/L of methylated target sequence (B1) and unmethylated target sequence (B0) respectively detected by the sensor, and it can be seen that the signal of the unmethylated target sequence is almost close to the blank signal value, while the signal of the methylated target sequence is stronger.

FIG. 8C is a current analysis detection curve of the sensor for detecting methylated target sequences at different concentrations, which is a blank control and a target sequence of 1amol/L to 100nmol/L (increasing by 10 times of gradient) from top to bottom, wherein the current is positively correlated with the concentration of the target sequence, and the average of 3 repeated experiments in each group is plotted as shown in FIG. 8D. The D-inset of fig. 8 shows that the log of the current detected by the sensor and the target sequence concentration exhibits a good linear relationship in the concentration range of 1 to 1pmol/L, and the linear equation can be expressed as Y-4130.28 LogC +206.09 and the correlation coefficient R-0.9909.

The sensors used 1pmol/L of methylated target sequence in TMB solution to detect 5 groups by amperometry, and the average detection signals obtained for each group are shown in Table 3, with the total average current ≈ 1780 + -86 nA and RSD ≈ 4.846%.

TABLE 3 results of repeated experiments

(12) Specificity and stability of the sensor: FIG. 9A shows the amperometric detection signals of 1pmol/L of the perfectly complementary methylated target sequence (B1), the single base mismatched methylated target sequence (BC1) and the multi base mismatched methylated target sequence (BCx), showing that the currents of the mismatched sequences are significantly reduced.

FIG. 9B shows the amperometric detection signal after 1pmol/L of methylated target sequence was stored at 4 ℃ after the first amperometric detection, taken out again after 10 days, and repeatedly stored-taken out for 3 times. It can be seen that the detection signal gradually decreased with time, and the detection signal was about 91.97% of the original signal at day 30.

(13) Serum recovery experiments: the addition of the methylated target sequence to the serum of healthy persons followed by detection of amperometric signals resulted in a recovery of between 96.02 and 105.43% and an RSD of between 1.22 and 6.74% as shown in Table 4.

TABLE 4 serum recovery test results

In conclusion, the tetrahedral DNA nanoprobe used by the invention has the advantages of effective synthesis and high yield, and successfully forms a three-dimensional structure according to the assumption. The electrochemical sensing detection result proves that each step of modification of the sensor is successful and effective, and various biomolecules are modified on the surface of the electrode. The sensor shows the optimal performance under the conditions that the tetrahedral probe is fixed for 3h, the target sequence hybridization time is 1h and the digestion time of the HpaII endonuclease is 2h, and shows better detection capability in the range of 1amol/L to 1pmol/L, the detection limit reaches 0.93amol/L and the sensitivity is high. The detection experiment of the base mismatch methylation target sequence and the human serum labeled recovery experiment result prove that the sensor has good specificity and anti-interference capability, and the storage stability of 30 days is also verified. The result proves that the DNA methylation electrochemical biosensor can complete the detection of trace methylated DNA target sequences and can provide an accurate and sensitive method for early diagnosis and treatment tracking of DNA methylation related diseases.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> China people liberation army, military and medical university

<120> electrochemical analysis method based on tetrahedral DNA nanoprobe and application

<141> 2021-08-13

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatac 55

<210> 2

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcaactgcct ggtgataaaa cgacactacg tgggaatcta ctatggcggc tcttc 55

<210> 3

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttcagactta ggaatgtgct tcccacgtag tgtcgtttgt attggaccct cgcat 55

<210> 4

<211> 89

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acattcctaa gtctgaaaca ttacagcttg ctacacgaga agagccgcca tagtatttag 60

gtcgcaacac ccggcgagca ctgcgacct 89

<210> 5

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agtctaggat tctgagtggg ttaaaggtcg cagtgctcgc cgggtgt 47

<210> 6

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agtctaggat tctgagtggg ttaaaggtcg cagtgctcgc cgggtgt 47

<210> 7

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agtctaggat tctgagtggg ttaaaggtcg cagtgctcgc caggtgt 47

<210> 8

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtctaggat tctgagtggg ttaaacgtag cagggcttgc caggtgt 47

<210> 9

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttaacccact cagaatccta gactcaaagt agtctaggat tctgagtg 48

<210> 10

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agtctaggat tctgagtggg ttaacactca gaatcctaga ctactttg 48

<210> 11

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tagtgactag gtcgcagtgc tcgccgggtg t 31

<210> 12

<211> 8

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agtcacta 8