CN114231597A - Circulating tumor DNA detection method based on DNAzyme circulating activation - Google Patents
Circulating tumor DNA detection method based on DNAzyme circulating activation Download PDFInfo
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Abstract
The invention discloses a circulating tumor DNA detection method based on DNAzyme circulating activation, which comprises the following steps: the probe A with ferrocene modified at the tail end is modified on the surface of a gold electrode, a probe B-C hybrid is connected to the probe A, the probe C can be replaced under the drive of target nucleic acid, so that methylene blue signal molecules modified at the tail end of the probe B are close to the surface of the gold electrode, the probe A is catalyzed by activating DNAzyme to crack and release ferrocene signal molecules, free probe sequences can further activate other DNAzymes, signals are amplified, and quantitative analysis on the target nucleic acid can be realized by researching the intensity change comparison of double signals. The invention can realize the ultra-sensitive quantitative analysis of the circulating tumor DNA concentration by comprehensively analyzing the dual electrochemical signal change triggered by the target circulating tumor DNA, and has the advantages of simple detection method, easy operation, strong specificity on the circulating tumor DNA and good application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a circulating tumor DNA detection method based on DNAzyme circulating activation.
Background
Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker. ctDNA is generally shed from a tumor site into the blood circulation, and tumor traces in the blood can be detected by ctDNA detection. By detecting and analyzing ctDNA, some information with important reference significance can be provided for the research of tumors. Compared with the tissue DNA biomarker needing surgery or biopsy, the ctDNA is convenient to obtain and low in cost; the use of ctDNA analysis enables continuous monitoring, provides results faster than tissue analysis, and potentially provides comprehensive data of all tumor site genetic changes in a patient. In addition, ctDNA can be used to predict response to a particular therapy, determine treatment mechanisms, drug resistance, and the like. For example, the first ctDNA test in clinical use is the EGFR mutation assay used to predict the response of non-small cell lung cancer patients to EGFR tyrosine kinase inhibitors. To introduce ct DNA detection into routine clinical applications, medical personnel need to receive training of testing skills such as PCR and sequencing at present, and in addition, the existing detection methods or kits for various ctDNA are complex and can meet clinical application requirements at reasonable cost by simplification and standardization.
Disclosure of Invention
The invention aims to solve the technical problem of providing a circulating tumor DNA detection method based on DNAzyme circulating activation aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a circulating tumor DNA detection method based on DNAzyme circulating activation, which comprises the following steps:
providing a DNA probe A which contains a neck ring structure and the tail end of which is modified with a first signal molecule, wherein the DNA probe A is modified on the surface of a gold electrode;
providing a probe B-C hybrid obtained by hybridizing a DNA probe B and a DNA probe C which are not completely complementarily paired, connecting the probe B-C hybrid to the DNA probe A through a base complementary pairing antigen, and fixing the probe B-C hybrid on the surface of the gold electrode, wherein the tail end of the DNA probe B is modified with a second signal molecule;
detecting target ctDNA (circulating tumor DNA) by using an electrochemical detection method and a gold electrode modified with a DNA probe A and a probe B-C hybrid and combining a DNAzyme activating strategy: when the target ctDNA exists, the target ctDNA can replace the DNA probe C in the probe B-C hybrid and form a DNAzyme structure with the DNA probe A, DNA probe B, so that a second signal molecule modified at the tail end of the DNA probe B is close to the surface of the gold electrode, and the signal intensity of the second signal molecule is increased; and activating the DNAzyme to catalyze the cracking of the DNA probe A, releasing a first signal molecule, so that the signal intensity of the first signal molecule is reduced, and a free probe sequence formed after the DNA probe A is cracked further activates other DNAzymes, thereby amplifying signals; and finally, analyzing the change of the signal intensity of the first signal molecule and the second signal molecule by an electrochemical detection method to realize the quantitative detection of the target ctDNA.
Preferably, the first signal molecule is ferrocene.
Preferably, the second signal molecule is methylene blue.
Preferably, the DNAzyme is activated by adding a coenzyme factor to the reaction system.
Preferably, wherein the coenzyme factor is Mg2+。
Preferably, the method specifically comprises the following steps:
1) pretreating a gold electrode;
2) modification of DNA Probe A: preparing an electrode fixing solution, preparing a DNA probe A solution by using the electrode fixing solution, inserting a pretreated gold electrode into the DNA probe A solution for modification, cleaning the gold electrode, and soaking in mercaptohexanol for reaction;
3) quantitative analysis of target ctDNA: respectively preparing a DNA probe B solution and a DNA probe C solution, then mixing, and heating and then cooling the obtained mixed solution to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction, then cleaning the gold electrode, soaking the gold electrode into a target ctDNA solution to be detected for reaction, and then adding Mg into the target ctDNA solution2+Continuing the reaction;
and (3) carrying out electrochemical analysis on the gold electrodes before and after the reaction in the step 3), and calculating to obtain the concentration of the target ctDNA through a standard curve which is prepared in advance and represents the strength change relation between the concentration of the target ctDNA and the electrochemical signal of the gold electrode.
Preferably, the method specifically comprises the following steps:
1) pretreating a gold electrode;
2) modification of DNA Probe A: firstly preparing an electrode fixing solution with the pH of 7.4, wherein the fixing solution comprises: preparing a DNA probe A solution with the concentration of 0.8 mu M by using the electrode fixing solution, inserting a pretreated gold electrode into the DNA probe A solution for modification for 8 hours, cleaning the gold electrode, and soaking in mercaptohexanol for reaction for 0.5 hour;
3) quantitative analysis of target ctDNA: respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour, then washing the gold electrode by using double distilled water, soaking the gold electrode into a target ctDNA solution to be detected for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA solution2+Continuing the reaction for 1 hour;
and (3) carrying out electrochemical analysis on the gold electrode reacted in the step 3), and calculating to obtain the concentration of the target ctDNA through a standard curve which is prepared in advance and represents the strength change relation between the concentration of the target ctDNA and the electrochemical signal of the gold electrode.
Preferably, the step 1) is specifically: soaking the gold electrode in the goby solution for 5 minutes, then washing the gold electrode clean by using double distilled water, sequentially polishing the gold electrode by adopting 3000-5000 meshes of abrasive paper, and sequentially polishing the gold electrode by continuously adopting aluminum paste with the diameters of 1.0, 0.3 and 0.05 mu m until the mirror surface is smooth; continuously using double distilled water to wash the electrode, placing the gold electrode in alcohol for 5 minutes of ultrasonic treatment, and then performing ultrasonic treatment in ultrapure water for 5 minutes; and then placing the gold electrode into 0.5M sulfuric acid electrolyte, carrying out cyclic voltammetry scanning from 0 to 1.6V, finally cleaning by using double distilled water and drying by using nitrogen for later use.
Preferably, among them, the standard curve is prepared by the following method:
3-1) pretreating a gold electrode;
3-2) preparing a series of target ctDNA standard solutions with the concentrations of 0,0.1fM,1fM,10fM,100fM,1pM,10pM,100pM,1nM,10nM and 100 nM;
respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour;
3-3) the following operations are respectively carried out for each concentration of target ctDNA standard solution:
washing the gold electrode obtained in 3-2) with double distilled water, soaking the gold electrode into a target ctDNA standard solution for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA standard solution2+Continuing the reaction for 1 hour;
carrying out electrochemical analysis on the reacted gold electrode to obtain the electrochemical signal intensity I of the reacted ferroceneFCAnd intensity of electrochemical Signal I of methylene blueMBCalculating the electrochemical signal intensity difference delta I ═ IMB-IFC；
And performing curve fitting by taking the logarithmic concentration of the target ctDNA standard solution as an abscissa and the electrochemical signal intensity difference delta I corresponding to the group of target ctDNA standard solutions as an ordinate to obtain the standard curve.
Preferably, the sequence of the target ctDNA is:
5’-GTTGGAGCTAGTGGCGTAG-3’；
the sequence of the DNA probe A is as follows:
SH-TTTTCTACGCCACATATTCCAACCTAT/rA/GGAAGAGATGACGTTGGAGCTAGTGGCGTAG-Fc；
the sequence of the DNA probe B is as follows:
TCATCTCTTCTCCGAGCCGGTCGAAATAGGTTGGAGCTAGTGG-MB；
the sequence of the DNA probe C is as follows: CATCGCCACTAGCTCCAACCTATTT are provided.
The present invention also provides a nucleic acid sensor comprising the DNA probe A, DNA probe B and the DNA probe C as described above, which detects ctDNA using the method as described above.
The invention has the beneficial effects that: according to the invention, a circularly activated DNAzyme reaction system is constructed through interaction among four segments of DNA, ultra-sensitive quantitative analysis of ctDNA concentration can be realized through comprehensive analysis of dual electrochemical signal change triggered by target ctDNA, the detection limit can reach 25aM, and the detection method is simple, easy to operate, strong in ctDNA specificity and good in application prospect.
Drawings
FIG. 1 is a schematic diagram of a DNAzyme cycle activation-based circulating tumor DNA detection method of the present invention;
FIG. 2 is a square wave voltammogram demonstrating the feasibility of the present invention;
FIG. 3 is an AC impedance plot and cyclic voltammogram demonstrating the feasibility of the invention;
FIG. 4 shows the quantitative results and the selective verification results of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
The present embodiment relates to a circulating tumor DNA detection method based on DNAzyme circulating activation, the principle of the method is shown in FIG. 1, and the method comprises:
providing a DNA probe A which contains a neck ring structure and the tail end of which is modified with a ferrocene signal molecule, wherein the DNA probe A is modified on the surface of a gold electrode through a gold-sulfhydryl bond;
providing a probe B-C hybrid obtained by hybridizing a DNA probe B and a DNA probe C which are not completely complementarily paired, connecting the probe B-C hybrid to the annular part of the DNA probe A through a base complementary pairing principle so as to fix the probe B-C hybrid on the surface of a gold electrode, and modifying the tail end of the DNA probe B with a methylene blue signal molecule;
detecting the target ctDNA by adopting an electrochemical detection method and utilizing a gold electrode modified with a DNA probe A and a probe B-C hybrid and combining a DNAzyme activation strategy: when the target ctDNA exists, the target ctDNA can replace the DNA probe C in the probe B-C hybrid and form a DNAzyme (deoxyribozyme) structure with the DNA probe A, DNA probe B, so that a methylene blue signal molecule modified at the tail end of the DNA probe B is close to the surface of the gold electrode, and the signal intensity of the methylene blue signal molecule is increased; and through activating DNAzyme to catalyze the cracking of the DNA probe A, releasing ferrocene number molecules, so that the signal intensity of the ferrocene signal molecules is reduced, and free probe sequences formed after the DNA probe A is cracked further activate other DNAzymes, thereby amplifying signals; and finally, analyzing the change of the signal intensity of the ferrocene signal molecules and the methylene blue signal molecules by an electrochemical detection method to realize the quantitative detection of the target ctDNA.
Wherein Mg is added into the reaction system2+To activate the DNAzyme.
The nucleic acid sequences involved in this example are shown in table 1 below:
TABLE 1 DNA and RNA sequences
In this embodiment, the circulating tumor DNA detection method based on DNAzyme circulating activation specifically includes the steps of:
1) pretreatment of a gold electrode:
soaking the gold electrode in a goby solution (the ratio of concentrated sulfuric acid to hydrogen peroxide is 3: 1) for 5 minutes, then washing the gold electrode clean by using double distilled water, sequentially polishing the gold electrode by adopting 3000-5000 meshes of abrasive paper, and sequentially polishing the gold electrode by adopting aluminum paste with the diameters of 1.0, 0.3 and 0.05 mu m until the mirror surface is smooth; continuously using double distilled water to wash the electrode, placing the gold electrode in alcohol for 5 minutes of ultrasonic treatment, and then performing ultrasonic treatment in ultrapure water for 5 minutes; and then placing the gold electrode into 0.5M sulfuric acid electrolyte, carrying out cyclic voltammetry scanning (30 circles) from 0 to 1.6V, finally cleaning by using double distilled water and drying by using nitrogen for later use.
2) Modification of DNA Probe A: firstly, preparing an electrode fixing solution with the pH of 7.4, wherein the fixing solution comprises: preparing a DNA probe A solution with the concentration of 0.8 mu M by using the electrode fixing solution, then inserting the pretreated gold electrode into 0.3mL of the DNA probe A solution for modifying for 8 hours, then washing the gold electrode, and then soaking in 0.3mL of mercaptohexanol for reaction for 0.5 hour;
3) quantitative analysis of target ctDNA: respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and then cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour, then washing the gold electrode by using double distilled water, soaking the gold electrode into a target ctDNA solution to be detected for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA solution2+Continuing the reaction for 1 hour;
and (3) carrying out electrochemical analysis on the gold electrode reacted in the step 3), and calculating to obtain the concentration of the target ctDNA through a standard curve which is prepared in advance and represents the strength change relation between the concentration of the target ctDNA and the electrochemical signal of the gold electrode.
The preparation method of the standard curve comprises the following steps:
3-1) pretreating a gold electrode, which is the same as the step 1);
3-2) preparing a series of target ctDNA standard solutions with the concentrations of 0,0.1fM,1fM,10fM,100fM,1pM,10pM,100pM,1nM,10nM and 100 nM;
respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour;
3-3) the following operations are respectively carried out for each concentration of target ctDNA standard solution:
washing the gold electrode obtained in 3-2) with double distilled water, soaking the gold electrode into a target ctDNA standard solution for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA standard solution2+Continuing the reaction for 1 hour;
the electrochemical detection adopts CHI660D electrochemical workstation to measure electrochemical signals. The electrochemical system comprises a platinum counter electrode, a saturated calomel reference electrode and a gold working electrode. Cyclic voltammetry parameters: 0.55 to-0.2V scan range, 50mV/s scan rate; alternating current impedance parameters: amplitude 5mV, frequency range 0.1Hz to 100000 Hz; square wave volt-ampere parameters: amplitude 25mV, step 4mV, frequency 50Hz, sweep range 0.6 to-0.6V. The electrolyte of the cyclic voltammetry and the alternating-current impedance method is 5mM potassium ferricyanide; the electrolyte for square wave voltammetry was 20mM Tris-HCl (pH 7.45).
The principle, feasibility and the like of the method are experimentally verified as follows:
referring to FIG. 2, the square voltammogram under several conditions includes a bare electrode (bare), an electrode modified with a DNA Probe A (Probe A), an electrode modified with a Probe A and a hybrid of probes B-C, and Mg2+After the action (Probe A/B/C + Mg2+) Electrode modified with probe A and probe B/C hybrid, and Mg2+And ctDNA (Probe A/B/C + Mg)2++ctDNA)。
As can be seen from fig. 2, no significant current peak was observed in the square wave voltammogram of the bare electrode; after the probe A is modified, an obvious current peak appears at 0.3V, and belongs to a ferrocene molecule modified at the tail end of the probe A; after the electrode is further reacted with probe B/C, in Mg2+In the presence of the probe, the obtained square wave voltammogram is basically consistent with the square wave voltammogram, because the methylene blue at the tail end of the probe B is far away from the electrode, a remarkable current peak cannot be generated; on the other hand, when the target ctDNA is present, probe C can be displaced into the solution and form a DNAzyme structure with DNA probe A, DNA, probe B sequence can be further reacted with probe A, the distance between methylene blue and gold electrode is reduced, and the DNAzyme structure is formed in Mg2+With the assistance of the probe A, the probe A can be cut off, and a sequence modified with ferrocene is released into a solution, so that the obvious reduction of the ferrocene peak value and the generation of a new peak (methylene blue) at-0.3V are observed in a square wave voltammogram.
Fig. 3(a) is an ac impedance diagram: the electrode modified with the hybrid of the probe A and the probe B-C and Mg2+After the action, the electrode modified with the hybrid of the probe A and the probe B-C is further mixed with Mg2+After action and ctDNA action. FIG. 3(B) is the corresponding cyclic voltammogram.
And (3) characterizing various properties of the surface of the electrode after each part reacts through electrochemical alternating current impedance and a cyclic voltammogram. As shown in fig. 3A, the ac impedance of the bare electrode is represented by a linear region, which proves that the gold electrode has good conductivity; after the probe A is modified, a semicircular area appears, and the diameter of the semicircular area is in positive correlation with the impedance value, so that the effective fixation of the probe A is verified; after the probes B-C are further modified, the diameter of the semicircular area is further increased, and the hybridization of the fixed probes B-C can further block the electron transfer of the electric signal molecules (potassium ferricyanide) with the same charge in the electrolyte, so that the impedance value is increased; after ctDNA is added, although the probe C is replaced, the interaction of the probe B and the probe A can improve the density of DNA molecules near the electrode, so that the repulsion effect on potassium ferricyanide is increased, and the impedance value is further increased; if Mg is added to the system2+Can assist DNAzyme to circularly activate and cut the probe A, and after a large number of DNA sequences with negative electricity leave an electrode interface, the repulsion action on potassium ferricyanide is greatly reduced, so that a smaller impedance value (a semicircular area) is presented. The cyclic voltammogram of fig. 3B provides the same trend, with lower peak current at higher DNA density on the electrode surface, and the trend of peak current in the cyclic voltammogram is more consistent with the expectation.
Electrochemical analysis is performed on the reacted gold electrode, and fig. 4(a) shows a square wave voltammetry curve (0,0.1fM,1fM,10fM,100fM,1pM,10pM,100pM,1nM,10nM,100nM) obtained by detecting ctDNA with different concentrations by using the method of the present invention, and square wave voltammetry peak changes of ferrocene and methylene blue after the reaction are obtained. Intensity of electrochemical signal IFC IMBIs calculated as Δ I ═ IMB-IFC；
And (3) performing curve fitting by taking the logarithmic concentration of the target ctDNA standard solution as an abscissa and the electrochemical signal intensity difference Δ I corresponding to the set of target ctDNA standard solutions as an ordinate to obtain a standard curve, as shown in fig. 4 (B).
Fig. 4(C) is a comparison of square wave voltammograms after a reaction triggered by mismatched ctDNA molecules and target ctDNA molecules; fig. 4(D) is a comparison of the difference in electrochemical signal intensity after the reaction triggered by the mismatched ctDNA molecule and the target ctDNA molecule.
In this example, the electrochemical double signal intensity change caused by ctDNA with different concentrations was tested, as shown in fig. 4A, the electrochemical signal intensity of ferrocene gradually decreased and the electrochemical signal intensity of methylene blue gradually increased with the increase of ctDNA concentration. FIG. 4B is a graph of the corresponding electrochemical signal variation versus intensity, which is linearly related to the logarithmic ctDNA concentration and has a wide linear range (10)-16To 10-7M). Furthermore, the selectivity of the detection method can be verified by comparing the strength of electrochemical signals. By introducing 7 mismatched ctDNA sequences to carry out experiments and comparing corresponding square wave voltammetry curves (figure 4C), it can be seen that the mismatched sequences can not cause the reduction of ferrocene peak basically, in addition, methylene blue signals are not generated, and the comparison of related electrochemical signal difference values can be more intuitively shown in figure 4D, which shows that the method of the invention has good specificity to ctDNA.
Example 2
This example provides a nucleic acid sensor that includes DNA probe A, DNA probe B and DNA probe C in example 1, and that detects ctDNA using the method of example 1.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A circulating tumor DNA detection method based on DNAzyme circulating activation is characterized by comprising the following steps:
providing a DNA probe A which contains a neck ring structure and the tail end of which is modified with a first signal molecule, wherein the DNA probe A is modified on the surface of a gold electrode;
providing a probe B-C hybrid obtained by hybridizing a DNA probe B and a DNA probe C which are not completely complementarily paired, connecting the probe B-C hybrid to the DNA probe A through a base complementary pairing antigen, and fixing the probe B-C hybrid on the surface of the gold electrode, wherein the tail end of the DNA probe B is modified with a second signal molecule;
detecting the target ctDNA by adopting an electrochemical detection method and utilizing a gold electrode modified with a DNA probe A and a probe B-C hybrid and combining a DNAzyme activation strategy: when the target ctDNA exists, the target ctDNA can replace the DNA probe C in the probe B-C hybrid and form a DNAzyme structure with the DNA probe A, DNA probe B, so that a second signal molecule modified at the tail end of the DNA probe B is close to the surface of the gold electrode, and the signal intensity of the second signal molecule is increased; and activating the DNAzyme to catalyze the cracking of the DNA probe A, releasing a first signal molecule, so that the signal intensity of the first signal molecule is reduced, and a free probe sequence formed after the DNA probe A is cracked further activates other DNAzymes, thereby amplifying signals; and finally, analyzing the change of the signal intensity of the first signal molecule and the second signal molecule by an electrochemical detection method to realize the quantitative detection of the target ctDNA.
2. The method for detecting circulating tumor DNA based on DNAzyme circulation activation of claim 1, wherein the first signal molecule is ferrocene and the second signal molecule is methylene blue.
3. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 2, wherein DNAzyme is activated by adding coenzyme factor to the reaction system.
4. The method for detecting circulating tumor DNA based on DNAzyme circulation activation of claim 3, wherein the coenzyme factor is Mg2+。
5. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 4, wherein the method comprises the following steps:
1) pretreating a gold electrode;
2) modification of DNA Probe A: preparing an electrode fixing solution, preparing a DNA probe A solution by using the electrode fixing solution, inserting a pretreated gold electrode into the DNA probe A solution for modification, cleaning the gold electrode, and soaking in mercaptohexanol for reaction;
3) quantitative analysis of target ctDNA: respectively preparing a DNA probe B solution and a DNA probe C solution, then mixing, and heating and then cooling the obtained mixed solution to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction, then cleaning the gold electrode, soaking the gold electrode into a target ctDNA solution to be detected for reaction, and then adding Mg into the target ctDNA solution2+Continuing the reaction;
and (3) carrying out electrochemical analysis on the gold electrodes before and after the reaction in the step 3), and calculating to obtain the concentration of the target ctDNA through a standard curve which is prepared in advance and represents the strength change relation between the concentration of the target ctDNA and the electrochemical signal of the gold electrode.
6. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 5, wherein the method comprises the following steps:
1) pretreating a gold electrode;
2) modification of DNA Probe A: firstly, preparing an electrode fixing solution with the pH of 7.4, wherein the fixing solution comprises: preparing a DNA probe A solution with the concentration of 0.8 mu M by using the electrode fixing solution, inserting a pretreated gold electrode into the DNA probe A solution for modification for 8 hours, cleaning the gold electrode, and soaking in mercaptohexanol for reaction for 0.5 hour;
3) quantitative analysis of target ctDNA: respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour, then washing the gold electrode by using double distilled water, soaking the gold electrode into a target ctDNA solution to be detected for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA solution2+Continuing the reaction for 1 hour;
and (3) carrying out electrochemical analysis on the gold electrode reacted in the step 3), and calculating to obtain the concentration of the target ctDNA through a standard curve which is prepared in advance and represents the strength change relation between the concentration of the target ctDNA and the electrochemical signal of the gold electrode.
7. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 5 or 6, wherein the step 1) is specifically: soaking the gold electrode in the goby solution for 5 minutes, then washing the gold electrode clean by using double distilled water, sequentially polishing the gold electrode by adopting 3000-5000 meshes of abrasive paper, and sequentially polishing the gold electrode by continuously adopting aluminum paste with the diameters of 1.0, 0.3 and 0.05 mu m until the mirror surface is smooth; continuously using double distilled water to wash the electrode, placing the gold electrode in alcohol for 5 minutes of ultrasonic treatment, and then performing ultrasonic treatment in ultrapure water for 5 minutes; and then placing the gold electrode into 0.5M sulfuric acid electrolyte, carrying out cyclic voltammetry scanning from 0 to 1.6V, finally cleaning by using double distilled water and drying by using nitrogen for later use.
8. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 5 or 6, wherein the standard curve is prepared by:
3-1) pretreating a gold electrode;
3-2) preparing a series of target ctDNA standard solutions with the concentrations of 0,0.1fM,1fM,10fM,100fM,1pM,10pM,100pM,1nM,10nM and 100 nM;
respectively preparing a DNA probe B solution and a DNA probe C solution with the concentration of 2 mu M, then mixing the solutions in equal volumes, heating the obtained mixed solution to 95 ℃ for 5 minutes, and cooling to room temperature; soaking the gold electrode obtained in the step 2) into the mixed solution for reaction for 1 hour;
3-3) the following operations are respectively carried out for each concentration of target ctDNA standard solution:
washing the gold electrode obtained in 3-2) with double distilled water, soaking the gold electrode into a target ctDNA standard solution for reaction for 1 hour, and then adding 35mM Mg into the target ctDNA standard solution2+Continuing the reaction for 1 hour;
carrying out electrochemical analysis on the reacted gold electrode to obtain the electricity of the reacted ferroceneChemical Signal Strength IFCAnd intensity of electrochemical Signal I of methylene blueMBCalculating the electrochemical signal intensity difference delta I ═ IMB-IFC；
And performing curve fitting by taking the logarithmic concentration of the target ctDNA standard solution as an abscissa and the electrochemical signal intensity difference delta I corresponding to the group of target ctDNA standard solutions as an ordinate to obtain the standard curve.
9. The method for detecting circulating tumor DNA based on DNAzyme circulation activation according to claim 1, wherein the sequence of the target ctDNA is:
5’-GTTGGAGCTAGTGGCGTAG-3’；
the sequence of the DNA probe A is as follows:
SH-TTTTCTACGCCACATATTCCAACCTAT/rA/GGAAGAGATGACGTTGGAGCTAGTGGCGTAG-Fc；
the sequence of the DNA probe B is as follows:
TCATCTCTTCTCCGAGCCGGTCGAAATAGGTTGGAGCTAGTGG-MB；
the sequence of the DNA probe C is as follows: CATCGCCACTAGCTCCAACCTATTT are provided.
10. A nucleic acid sensor comprising the DNA probe A, DNA probe B and the DNA probe C according to any one of claims 1 to 9, which detects ctDNA by the method according to any one of claims 1 to 9.
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