Electrochemical detection method for circulating tumor DNA in human body and application of electrochemical detection method

Abstract

The invention discloses an electrochemical detection method for circulating tumor DNA in a human body and application thereof, and the electrochemical detection method comprises the following steps: dissolving a to-be-detected sample in a buffer solution to obtain a sample buffer solution; incubating an electrochemical sensor electrode in the sample buffer solution to obtain an electrochemical sensor electrode incubated with a sample to be detected; a working electrode of the electrochemical sensor electrode is a gold electrode or an electrode with a gold coating; placing the electrochemical sensor electrode incubated by the sample to be detected in electrolyte, and performing electrochemical signal detection by using differential pulse voltammetry or cyclic square wave voltammetry; and analyzing the electrochemical signal. The detection method disclosed by the invention can be used for accurately detecting small-component ctDNA to achieve the effect of early screening of a generic cancer species, has a semi-quantitative characteristic, can be used for quantitatively analyzing a biological marker methylated ctDNA of a tumor, and can be used for reflecting the load change of the tumor by monitoring the concentration of the methylated ctDNA to realize the monitoring of the dynamic change of the tumor; and reference is provided for treatment effect and treatment scheme adjustment.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensor detection, and relates to an electrochemical detection method and application of circulating tumor DNA in the human body. Background Art

[0002] Tumor cells can release small fragments of DNA into the blood circulation system, and these DNA fragments are circulating tumor DNA (ctDNA), which belongs to the category of cfDNA. CfDNA (cell-free DNA) is the general term for cell-free DNA in plasma. CtDNA plays a very important clinical role in cancer diagnosis and treatment. CtDNA detection can help judge the possible expected survival period and recurrence risk of cancer patients, and can also help and guide doctors to select clinical treatment plans. Moreover, it can be used to guide the selection of targeted therapies for diseases such as lung cancer, and can also be used for early screening of cancer.

[0003] Patent CN118817796A discloses a method for detecting circulating tumor DNA. Specifically, a human free DNA sample is adsorbed on a gold working electrode, and a square wave voltammetry method is used by an electrochemical workstation to measure ctDNA in the sample, thereby establishing a method for detecting ctDNA in free DNA using square wave voltammetry.

[0004] The basic principle of square wave voltammetry is that the potential is scanned in a stepped form and a square wave voltage is superimposed, and analysis is carried out by measuring the current difference within the square wave period. However, in square wave voltammetry, the higher the sensitivity, the lower the detection limit, and the lower the detection limit, the greater the influence of the background current. The high background current will mask the signal of low-concentration analytes. The detection characteristics of square wave voltammetry make it unable to have the characteristics of high sensitivity. Therefore, the detection ability of square wave voltammetry for low-concentration analytes is weak, and it cannot meet the actual needs of precise detection of minute amounts of ctDNA in medicine, resulting in certain difficulties in widely applying electrochemical sensor-related detection methods to the medical industry.

[0005] In practical applications, the concentration of ctDNA in blood is extremely low, and extremely high sensitivity is required for the detection method. The detection characteristics between the signal-to-noise ratio and sensitivity of square wave voltammetry limit its optimization, which restricts its application in the electrochemical detection of ctDNA in the human body.

[0006] Based on the above situation, the present invention aims to provide an electrochemical-based ctDNA detection method to make up for the defects of square wave voltammetry, achieve precise detection of minute concentrations of ctDNA, further expand its application scope, and can be further widely applied in the medical field to provide a new cancer detection method for relevant detection scenarios. Summary of the Invention

[0007] To overcome the deficiencies of the prior art, the object of the present invention is to provide a method for detecting circulating tumor DNA in the human body. This detection method can accurately detect trace amounts of ctDNA, achieve the effect of early screening for various cancers, and has semi-quantitative characteristics. It can quantitatively analyze the methylated ctDNA, a biomarker of tumors, and reflect the changes in tumor burden by monitoring the concentration of methylated ctDNA, so as to monitor the dynamic changes of tumors and provide a reference for treatment effects and adjustment of treatment plans.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] An electrochemical detection method for circulating tumor DNA in the human body, characterized in that differential pulse voltammetry or cyclic square wave voltammetry in electrochemical analysis methods is used to detect circulating tumor DNA.

[0010] Furthermore, the specific steps of using differential pulse voltammetry or cyclic square wave voltammetry in the electrochemical analysis method to detect circulating tumor DNA are as follows:

[0011] S1. Dissolve the sample to be tested in a buffer solution to obtain a sample buffer solution;

[0012] S2. Incubate the electrochemical sensor electrode in the sample buffer solution to obtain an electrochemical sensor electrode incubated with the sample to be tested;

[0013] S3. Connect the electrochemical sensor electrode incubated with the sample to be tested to an electrochemical workstation, then place the electrochemical sensor electrode incubated with the sample to be tested in an electrolyte solution, and use differential pulse voltammetry or cyclic square wave voltammetry to perform electrochemical signal detection and analyze the electrochemical signals.

[0014] Furthermore, the pH value of the buffer solution is 7.0 - 7.5.

[0015] Furthermore, the buffer solution is one or a mixture of more than one of PBS buffer solution, SSC buffer solution, and TE buffer solution.

[0016] Furthermore, the incubation duration of the electrochemical sensor electrode is 8 - 16 h at a temperature of 0 - 8 °C and 0.1 - 2 h at a temperature of 20 - 30 °C.

[0017] Furthermore, the electrochemical sensor electrode includes a working electrode, specifically a gold electrode or an electrode with a gold coating.

[0018] Furthermore, the electrochemical sensor electrode also includes a reference electrode and an auxiliary electrode; the reference electrode is silver silver hydride, and the auxiliary electrode is any one of carbon, platinum, or platinum black.

[0019] Further, before the gold electrode is incubated in the sample buffer, it also includes pre-treatment steps of polishing, cleaning, and drying the electrode.

[0020] Further, the electrolyte contains potassium ferricyanide.

[0021] Further, the concentration of potassium ferricyanide is 5 - 15 mmol / L.

[0022] Further, the test parameters of differential pulse voltammetry (DPV) are: initial potential -0.3 - 0.1 V, termination potential 0.4 - 0.6 V, potential increment 1 - 5 mV, pulse height 10 - 100 mV, pulse period 20 - 200 ms, pulse width 10 - 100 ms, sampling width 5 - 50 ms, silent time 3 - 20 s.

[0023] Further, the characteristic peak in the characteristic curve obtained by differential pulse voltammetry is between 0.05 V and 0.2 V.

[0024] Further, the test parameters of cyclic square wave voltammetry (CSWV) are: initial potential -0.5 - 0.3 V, termination potential 0.4 - 0.6 V, pulse potential increment 1 - 10 mV, pulse height 10 - 100 mV, frequency 1 - 100 Hz, number of scanning cycles 3 - 10 cycles, sampling width 5 - 50 ms, silent time 5 - 20 s.

[0025] Further, the characteristic peak in the characteristic curve obtained by cyclic square wave voltammetry is between 0.05 V and 0.25 V.

[0026] The present invention uses differential pulse voltammetry (DPV) or cyclic square wave voltammetry (CSWV) in electrochemistry methods to predict the amount of ctDNA according to the difference in electrical signals before and after the electrochemical sensor electrode incubates ctDNA.

[0027] As attached Figure 1 shown, taking differential pulse voltammetry as an example, the principle is as follows: when no substance is adsorbed, the current value of the bare gold electrode is the highest; when nucleic acid of a normal human sample is adsorbed, since it still maintains a globular and highly helical state on the gold electrode surface, the effect of hindering electron transfer is limited, and the current slightly decreases; when nucleic acid of a cancer patient sample is adsorbed, ctDNA presents a dispersed state on the gold electrode, and the hindering effect is stronger than that of the normal sample, and the current significantly decreases.

[0028] The detection method of the present invention can be used for qualitative detection of ctDNA, preliminary screening of tumors, and providing reference for diagnosis. And the detection method of the present invention can detect low-concentration circulating tumor DNA and has high sensitivity.

[0029] As Figure 2As shown, differential pulse voltammetry was used to detect wild-type ctDNA at a concentration of 0.32 - 5.00 ng / μL, and a voltammetric characteristic curve was obtained. In Figure 2 a - e, it can be seen that the results of differential pulse voltammetry show that the current value decreases significantly relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of wild-type ctDNA at different concentrations, and also has an obvious response to 0.32 ng / μL wild-type ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention has high sensitivity to wild-type ctDNA, up to 10 -9 mol / L.

[0030] As Figure 3 shown, differential pulse voltammetry was used to detect mutant ctDNA at a concentration of 0.32 - 5.00 ng / μL, and a voltammetric characteristic curve was obtained. In Figure 3 a - e, it can be seen that the results of differential pulse voltammetry show that the current value decreases significantly relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of mutant ctDNA at different concentrations, and also has an obvious response to 0.32 ng / μL mutant ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention has high sensitivity to wild-type ctDNA, up to 10 -9 mol / L.

[0031] As Figure 4 shown, differential pulse voltammetry was used to detect methylated ctDNA at a concentration of 0.32 - 5.00 ng / μL, and a voltammetric characteristic curve was obtained. In Figure 4 a - e, it can be seen that the results of differential pulse voltammetry show that the current value decreases significantly relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of methylated ctDNA at different concentrations, and also has an obvious response to 0.32 ng / μL methylated ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided in Example 3 of the present invention has high sensitivity to wild-type methylated ctDNA, up to 10 -9 mol / L.

[0032] As Figure 5 shown, cyclic square wave voltammetry was used to detect wild-type ctDNA at a concentration of 0.32 - 5.00 ng / μL, and a voltammetric characteristic curve was obtained. In Figure 5As can be seen from a to e, the results of cyclic square wave voltammetry show that the current value of wild-type ctDNA has a significant decrease relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of mutant ctDNA at different concentrations, and also has an obvious response to wild-type ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention has high sensitivity to wild-type ctDNA, up to 10 -9 mol / L.

[0033] As Figure 6 shown, the cyclic square wave voltammetry was used to detect mutant ctDNA with a concentration of 0.32 - 5.00 ng / μL, and the obtained voltammetric characteristic curve. As Figure 6 can be seen from a to e, the results of cyclic square wave voltammetry show that the current value of mutant ctDNA has a significant decrease relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of mutant ctDNA at different concentrations, and also has an obvious response to mutant ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention has high sensitivity to mutant ctDNA, up to 10 -9 mol / L.

[0034] As Figure 7 shown, the cyclic square wave voltammetry was used to detect methylated ctDNA with a concentration of 0.32 - 5.00 ng / μL, and the obtained voltammetric characteristic curve. As Figure 7 can be seen from a to e, the results of cyclic square wave voltammetry show that the current value of methylated ctDNA has a significant decrease relative to the cell-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of methylated ctDNA at different concentrations, and also has an obvious response to methylated ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention has high sensitivity to methylated ctDNA, up to 10 -9 mol / L.

[0035] The detection method of the present invention has the characteristic of semi-quantification and can be used for quantitative analysis of methylated ctDNA. The value of ctDNA methylation detection in tumor diagnosis and treatment lies in its ability to reflect the change of tumor burden, monitor the treatment response, and provide reference for treatment effect and adjustment of treatment plan.

[0036] As shown in the appendix Figure 8 shown, there is a certain linear relationship between the fitting curves of the response differences of cyclic square wave voltammetry for detecting methylated ctDNA with a concentration of 0.32 - 5.00 ng / μL, and the R of the fitting curve of methylated ctDNA 2It is equal to 0.9916. This indicates that the method for detecting circulating tumor DNA in the human body provided by the present invention is a semi - quantitative method, which has high accuracy and reliability in low - concentration methylated ctDNA and can accurately predict the concentration of methylated ctDNA.

[0037] The method provided by the present invention can be applied to the early screening of pan - cancer types and the monitoring of tumor treatment.

[0038] The beneficial effects of the present invention are as follows:

[0039] The present invention adsorbs a human free DNA sample on a gold working electrode and uses an electrochemical workstation to measure ctDNA in the sample by differential pulse voltammetry or cyclic square wave voltammetry, thereby establishing a method for detecting ctDNA in free DNA by square wave voltammetry, differential pulse voltammetry or cyclic square wave voltammetry.

[0040] The detection method of the present invention can accurately detect trace amounts of ctDNA, achieving the effect of early screening of pan - cancer types, and has the characteristic of semi - quantification. It can quantitatively analyze the tumor biomarker methylated ctDNA, reflect the change of tumor burden by monitoring the concentration of methylated ctDNA, realize the monitoring of tumor dynamic changes, and provide a reference for treatment effect and adjustment of treatment plan. Description of the Drawings

[0041] The present invention is further described with reference to the drawings, but the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the following drawings.

[0042] Figure 1 It is the schematic diagram of detecting ctDNA by the method for detecting circulating tumor DNA in the human body provided by the present invention;

[0043] Figure 2 It is the voltammetry characteristic curve diagram of analyzing wild - type ctDNA with different concentrations by DPV electrochemistry in Example 1;

[0044] Figure 3 It is the voltammetry characteristic curve diagram of analyzing mutant wild - type ctDNA with different concentrations by DPV electrochemistry in Example 2;

[0045] Figure 4 It is the voltammetry characteristic curve diagram of analyzing methylated ctDNA with different concentrations by DPV electrochemistry in Example 3;

[0046] Figure 5 It is the voltammetry characteristic curve diagram of analyzing wild - type ctDNA with different concentrations by CSWV electrochemistry in Example 4;

[0047] Figure 6Example 5 shows the voltammetry characteristic curves of mutant ctDNA with different concentrations analyzed by CSWV electrochemistry;

[0048] Figure 7 Example 6 shows the voltammetry characteristic curves of methylated ctDNA with different concentrations analyzed by CSWV electrochemistry;

[0049] Figure 8 Example 6 shows the fitting curve of the CSWV response difference detected by cyclic square wave voltammetry for methylated ctDNA with a concentration range of 0.32 - 5.00 ng / μL. Detailed implementation manners

[0050] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with the following specific embodiments and with reference to the accompanying drawings.

[0051] In the embodiments of the present invention, an electrochemical sensor is used to convert chemical signals into electrical signals, and the electrical signals are detected by differential pulse voltammetry or cyclic square wave voltammetry. Since ctDNA has a higher affinity for the gold surface, qualitative detection of ctDNA can be achieved by comparing the electrochemical signals with those of free DNA from healthy individuals. According to a large number of experiments, the electrical signals of free DNA from healthy individuals (also known as healthy person DNA) and free DNA from tumor patients (also known as tumor patient DNA) are each in a relatively stable range. This method can be used for preliminary screening of tumors and provide a reference for diagnosis.

[0052] In this embodiment, the test samples used are from the plasma of tumor patients, and the control samples used in the embodiments of the present invention are from the plasma of healthy individuals. The plasma of tumor patients is taken from the remaining plasma samples clinically tested at the Sun Yat - sen University Cancer Center from February 2024 to September 2024. The plasma of healthy individuals is taken from the remaining plasma samples clinically tested in the Department of Oncology. Most patients with malignant tumors have been diagnosed by pathological examination. Healthy individuals have undergone a comprehensive physical examination, including at least chest X - ray, abdominal ultrasound, and serum tumor biomarkers, and no obvious tumor evidence has been found.

[0053] The electrochemical sensor used in the embodiments of the present invention consists of a PBC buffer solution, an electrode, and an electrolyte solution. The electrode includes a working electrode, a reference electrode, and a counter electrode. The working electrode is a gold electrode, the reference electrode is a silver / silver chloride electrode, and the auxiliary electrode is carbon. The working electrode of the electrode is a gold electrode or an electrode with a gold coating. The electrolyte solution is a mixture of 0.1 mmol / L potassium ferricyanide and 0.1 mol / L potassium chloride solution prepared with PBS solution.

[0054] Instruments used: The self - developed electrochemical workstation of Guangzhou Yuxin Hanji Technology Co., Ltd., model MECART PTS - Pr10; software version: MECStat_upgrade(240711).

[0055] Example 1

[0056] The method for detecting circulating tumor DNA in the human body provided in this example includes the following steps:

[0057] Sample reagent: 15 μL, 50 ng / μL 5EGFR WT reagent, where EGFR WT is wild-type ctDNA;

[0058] Control sample reagent: 15 μL, 50 ng / μL plasma-free DNA, which is provided by the plasma of healthy people;

[0059] Testing method and parameters: Differential pulse voltammetry, and the testing parameters are: initial potential -0.1 V, termination potential 0.6 V, potential increment 5 mV, pulse height 50 mV, pulse period 100 ms, pulse width 50 ms, sampling width 25 ms, silent time 3 s;

[0060] The specific detection steps are as follows:

[0061] (1) Prepare the sample buffer: Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a 5.00 ng / μL sample buffer to be measured. Pipette 75 μL of the 5.00 ng / μL sample buffer to be measured and add 75 μL of PBS for semi-dilution. In the same way, obtain sample buffers to be measured with concentrations of 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0062] (2) Pretreat the working electrode of the sample to be measured: Dropwise coat the sample buffers to be measured with concentrations of 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL on the gold electrode, incubate for 16 h in a closed environment at 4°C, and then rinse with deionized water for 2 - 3 s to obtain an electrochemical sensor electrode incubated with the sample to be measured with concentrations of 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0063] (3) Repeat step (2) to prepare an electrochemical sensor electrode incubated with the control sample. Then connect the electrochemical sensor electrode incubated with the control sample to an electrochemical workstation, place the electrochemical sensor electrode incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain a differential pulse voltammetry characteristic curve;

[0064] (4) Connect the pair of electrochemical sensor electrodes incubated with the sample to be measured obtained in step (2) to an electrochemical workstation, place the electrochemical sensor electrode incubated with the sample to be measured in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain a differential pulse voltammetry characteristic curve;

[0065] (5) Read the differential pulse voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0066] The characteristic curves obtained by detecting wild-type ctDNA with a concentration of 0.32 - 5.00 ng / μL using the differential pulse voltammetry of Example 1 are as Figure 2 shown. The results of differential pulse voltammetry show that the current value of wild-type ctDNA decreases significantly compared with the plasma-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of wild-type ctDNA with different concentrations, and also has an obvious response to 0.32 ng / μL wild-type ctDNA. It shows that the detection method of circulating tumor DNA in the human body provided in this Example 1 has high sensitivity to wild-type ctDNA, up to 10 -9 mol / L.

[0067] Example 2

[0068] The detection method of circulating tumor DNA in the human body provided in this example includes the following steps:

[0069] Sample reagent: 15 μL, 50 ng / μL EGFR T790M reagent, EGFR T790M is mutant ctDNA;

[0070] Control sample reagent: 15 μL, 50 ng / μL plasma-free DNA, plasma-free DNA is provided by healthy human plasma;

[0071] Test method and parameters: Differential pulse voltammetry, test parameters are: initial potential -0.1 V, termination potential 0.6 V, potential increment 5 mV, pulse height 50 mV, pulse period 100 ms, pulse width 50 ms, sampling width 25 ms, silent time 3 s;

[0072] Test method and parameters: Differential pulse voltammetry, initial potential -0.3 V, termination potential 0.6 V, potential increment 1 mV, pulse height 100 mV, pulse period 20 ms, pulse width 10 ms, sampling width 50 ms, silent time 20 s;

[0073] The specific detection steps are as follows:

[0074] (1) Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a 5.00 ng / μL sample buffer to be measured. Pipette 75 μL of the 5.00 ng / μL sample buffer to be measured and then add 75 μL of PBS for semi-dilution. In the same way, obtain sample buffers to be measured with concentrations of 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0075] (2) Pretreatment of the electrode of the sample to be measured: Drop 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the buffer solution of the sample to be measured on the gold electrode respectively, incubate for 16 h in a closed environment at 4 °C, and then rinse with deionized water for 2 - 3 s to obtain the electrochemical sensor electrodes incubated with 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the sample to be measured;

[0076] (3) Repeat step (2) to prepare the electrochemical sensor electrodes incubated with the control sample, then connect the electrochemical sensor electrodes incubated with the control sample to the electrochemical workstation, and then place the electrochemical sensor electrodes incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain the differential pulse voltammetry characteristic curve;

[0077] (4) Connect the pair of electrochemical sensor electrodes incubated with the sample to be measured obtained in step (2) to the electrochemical workstation, and then place the electrochemical sensor electrodes incubated with the sample to be measured in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain the differential pulse voltammetry characteristic curve;

[0078] (5) Read the differential pulse voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0079] The characteristic curves obtained by using the differential pulse voltammetry of Example 2 to detect mutant ctDNA with a concentration of 0.32 - 5.00 ng / μL are as Figure 3 shown. The results of differential pulse voltammetry show that the current value of mutant ctDNA decreases significantly compared with the plasma-free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of mutant ctDNA with different concentrations, and also has an obvious response to 0.32 ng / μL of mutant ctDNA. It shows that the detection method of circulating tumor DNA in the human body provided in this Example 2 has high sensitivity to mutant ctDNA, up to 10 -9 mol / L.

[0080] Example 3

[0081] The detection method of circulating tumor DNA in the human body provided in this example includes the following steps:

[0082] Sample reagent: 15 μL, 50 ng / μ of EGFR LSEPT9 reagent; EGFR SEPT9 is methylated ctDNA;

[0083] Control sample reagent: 15 μL, 50 ng / μL of plasma-free DNA, and the plasma-free DNA is provided by healthy human plasma;

[0084] Testing method and parameters: Differential pulse voltammetry, initial potential 0.1 V, final potential 0.4 V, potential increment 5 mV, pulse height 10 mV, pulse period 200 ms, pulse width 10 ms, sampling width 5 ms, quiet time 3 s;

[0085] The specific detection steps are as follows:

[0086] (1) Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a 5.00 ng / μL sample buffer solution to be measured. Pipette 75 μL of the 5.00 ng / μL sample buffer solution to be measured and add 75 μL of PBS for semi-dilution. In the same way, obtain sample buffer solutions to be measured with concentrations of 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0087] (2) Pretreatment of the sample electrode to be measured: Dropwise coat 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the sample buffer solution to be measured on the gold electrode, incubate for 16 h in a closed environment at 4°C, and then rinse with deionized water for 2 - 3 s to obtain an electrochemical sensor electrode incubated with 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the sample to be measured;

[0088] (3) Repeat step (2) to prepare an electrochemical sensor electrode incubated with the control sample. Then connect the electrochemical sensor electrode incubated with the control sample to an electrochemical workstation, place the electrochemical sensor electrode incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain a differential pulse voltammetry characteristic curve;

[0089] (4) Connect the electrochemical sensor electrode pair incubated with the sample to be measured obtained in step (2) to an electrochemical workstation, place the electrochemical sensor electrode incubated with the sample to be measured in the electrolyte solution, and perform electrochemical measurement using differential pulse voltammetry to obtain a differential pulse voltammetry characteristic curve;

[0090] (5) Read the differential pulse voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0091] The characteristic curves obtained by using the differential pulse voltammetry in Example 3 to detect methylated ctDNA with a concentration range of 0.32 - 5.00 ng / μL are as Figure 4As shown, the differential pulse voltammetry results show that the current value of methylated ctDNA has a significant decrease compared to the plasma-free DNA provided by the plasma of healthy individuals. The gold electrode has a significant response to the adsorption of methylated ctDNA at different concentrations, and also has a significant response to 0.32 ng / μL of methylated ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided in Example 3 has high sensitivity to wild methylated ctDNA, reaching -9 mol / L.

[0092] Example 4

[0093] The method for detecting circulating tumor DNA in the human body provided in this example includes the following steps:

[0094] Sample reagent: 15 μL of 50 ng / μL 5EGFR WT reagent, where EGFR WT is wild-type ctDNA;

[0095] Control sample reagent: 15 μL of 50 ng / μL plasma-free DNA, provided by the plasma of healthy individuals;

[0096] Testing method and parameters: Using cyclic square wave voltammetry, initial potential -0.3 V, termination potential 0.4 V, pulse potential increment 5 mV, pulse height 20 mV, frequency 20 Hz, number of scanning cycles 3, sampling width 10 ms, silent time 5 s;

[0097] The specific detection steps are as follows:

[0098] (1) Prepare the sample buffer: Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a 5.00 ng / μL sample buffer to be tested. Pipette 75 μL of the 5.00 ng / μL sample buffer to be tested and add 75 μL of PBS for semi-dilution. In the same way, obtain sample buffers to be tested with concentrations of 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0099] (2) Electrode pretreatment: Dropwise apply 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the sample buffer to be tested on the gold electrode, incubate for 16 h in a closed environment at 4°C, and then rinse with deionized water for 2 - 3 s to obtain an electrochemical sensor electrode incubated with 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL of the sample to be tested;

[0100] (3) Repeat step (2) to prepare an electrochemical sensor electrode incubated with the control sample, then connect the electrochemical sensor electrode incubated with the control sample to the electrochemical workstation, place the electrochemical sensor electrode incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain a cyclic square wave voltammetry characteristic curve;

[0101] (4) Connect the electrochemical sensor electrode pair incubated with the test sample obtained in step (2) to an electrochemical workstation, then place the electrochemical sensor electrode incubated with the test sample in an electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain a cyclic square wave voltammetry characteristic curve;

[0102] (5) Read the cyclic square wave voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0103] The characteristic curves obtained by using the cyclic square wave voltammetry of Example 4 to detect wild-type ctDNA with a concentration of 0.32 - 5.00 ng / μL are as Figure 5 shown. The results of cyclic square wave voltammetry show that the current value of wild-type ctDNA decreases significantly relative to the plasma-free DNA provided by healthy human plasma. The gold electrode pair has an obvious response to the adsorption of mutant ctDNA with different concentrations, and also has an obvious response to wild-type ctDNA. It shows that the detection method of circulating tumor DNA in the human body provided in this Example 4 has high sensitivity to wild-type ctDNA, up to 10 -9 mol / L.

[0104] Example 5

[0105] The detection method of circulating tumor DNA in the human body provided in this example includes the following steps:

[0106] Sample reagent: 15 μL of 50 ng / μL EGFR T790M reagent, and EGFR T790M is mutant ctDNA;

[0107] Control sample reagent: 15 μL of 50 ng / μL plasma-free DNA, and the plasma-free DNA is provided by healthy human plasma;

[0108] Test method and parameters: Using cyclic square wave voltammetry, initial potential -0.3 V, final potential 0.4 V, pulse potential increment 5 mV, pulse height 20 mV, frequency 20 Hz, number of scanning cycles 3, sampling width 10 ms, silent time 5 s;

[0109] The specific detection steps are as follows:

[0110] (1) Prepare a sample buffer solution: Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a 5.00 ng / μL test sample buffer solution. Pipette 75 μL of the 00 ng / μL test sample buffer solution and add 75 μL of PBS for half dilution. In the same way, obtain test sample buffer solutions of 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0111] (2) Electrode pretreatment: Dropwise coat 5.00, 2.50, 1.25, 0.63, 0.32 ng / μL of the sample buffer to be tested on the gold electrode, incubate for 16 h in a closed environment at 4 °C, and then rinse with deionized water for 2 - 3 s to obtain the electrochemical sensor electrodes incubated with 5.00, 2.50, 1.25, 0.63, 0.32 ng / μL of the sample to be tested;

[0112] (3) Repeat step (2) to prepare the electrochemical sensor electrodes incubated with the control sample, then connect the electrochemical sensor electrodes incubated with the control sample to the electrochemical workstation, and then place the electrochemical sensor electrodes incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain the cyclic square wave voltammetry characteristic curve;

[0113] (4) Connect the electrochemical sensor electrodes incubated with the sample to be tested obtained in step (2) to the electrochemical workstation, and then place the electrochemical sensor electrodes incubated with the sample to be tested in the electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain the cyclic square wave voltammetry characteristic curve;

[0114] (5) Read the cyclic square wave voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0115] The characteristic curves obtained by using the cyclic square wave voltammetry in Example 5 to detect mutant ctDNA with a concentration of 0.32 - 5.00 ng / μL are as Figure 6 shown. The results of cyclic square wave voltammetry show that the current value of mutant ctDNA decreases significantly compared with the plasma free DNA provided by healthy human plasma. The gold electrode has an obvious response to the adsorption of mutant ctDNA with different concentrations, and also has an obvious response to mutant ctDNA. It shows that the detection method of circulating tumor DNA in the human body provided in this Example 5 has high sensitivity to mutant ctDNA, up to 10 -9 mol / L.

[0116] Example 6

[0117] The detection method of circulating tumor DNA in the human body provided in this example includes the following steps:

[0118] Sample reagent: 15 μL, 50 ng / μL of EGFR SEPT9 reagent; EGFR SEPT9 is methylated ctDNA;

[0119] Control sample reagent: 15 μL, 50 ng / μL of plasma free DNA, and the plasma free DNA is provided by healthy human plasma;

[0120] Testing method and parameters: Cyclic square wave voltammetry was adopted, with an initial potential of -0.3 V, a termination potential of 0.4 V, a pulse potential increment of 5 mV, a pulse height of 20 mV, a frequency of 20 Hz, 3 scanning cycles, a sampling width of 10 ms, and a silent time of 5 s;

[0121] The specific detection steps are as follows:

[0122] (1) Prepare the sample buffer: Dissolve the sample reagent in 135 μL of 1×PBS buffer solution with a pH value of 7.2 to obtain a sample buffer solution to be measured at 5.00 ng / μL. Pipette 75 μL of the sample buffer solution to be measured at 00 ng / μL and add 75 μL of PBS for semi-dilution. In the same way, obtain sample buffer solutions to be measured at 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0123] (2) Electrode pretreatment: Dropwise coat the sample buffer solutions to be measured at 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL on the gold electrode, incubate for 16 h in a closed environment at 4°C, and then rinse with deionized water for 2 - 3 s to obtain an electrochemical sensor electrode incubated with the sample buffer solutions to be measured at 5.00, 2.50, 1.25, 0.63, and 0.32 ng / μL;

[0124] (3) Repeat step (2) to prepare an electrochemical sensor electrode incubated with the control sample. Then connect the electrochemical sensor electrode incubated with the control sample to the electrochemical workstation, place the electrochemical sensor electrode incubated with the control sample in the electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain a cyclic square wave voltammetry characteristic curve;

[0125] (4) Connect the electrochemical sensor electrode pair incubated with the sample to be measured obtained in step (2) to the electrochemical workstation, place the electrochemical sensor electrode incubated with the sample to be measured in the electrolyte solution, and perform electrochemical measurement using cyclic square wave voltammetry to obtain a cyclic square wave voltammetry characteristic curve;

[0126] (5) Read the cyclic square wave voltammetry characteristic curves in the range of 0.2 V to 0.3 V in steps (3) and (4) respectively, and obtain the results after processing.

[0127] The characteristic curves obtained by using the cyclic square wave voltammetry of Example 6 to detect methylated ctDNA with a concentration of 0.32 - 5.00 ng / μL are as Figure 7As shown, the results of cyclic square wave voltammetry show that the current value of methylated ctDNA decreases significantly relative to the cell-free DNA provided by the plasma of healthy individuals. The gold electrode has an obvious response to the adsorption of methylated ctDNA at different concentrations, and there is also an obvious response to 0.32 ng / μL of methylated ctDNA. This indicates that the method for detecting circulating tumor DNA in the human body provided in Example 6 has high sensitivity to methylated ctDNA, up to -9 mol / L.

[0128] In addition, there is a certain linear relationship in the fitting curve of the response difference for detecting methylated ctDNA with a concentration of 0.32 - 5.00 ng / μL by cyclic square wave voltammetry. As Figure 8 shown, in the fitting curve of methylated ctDNA, R 2 is equal to 0.9916. This indicates that the method for detecting circulating tumor DNA in the human body provided in Example 6 is a semi-quantitative method, and has high accuracy and reliability in low-concentration methylated ctDNA, and can accurately predict the concentration of methylated ctDNA.

[0129] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0130] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. The technical details not described in detail in the present invention can all be realized by any existing technology in the art. In particular, all the technical features not described in detail in the present invention can be realized by any existing technology.

Claims

1. A method for electrochemical detection of circulating tumor DNA in the human body, characterized in that: Differential pulse voltammetry or cyclic square wave voltammetry in electrochemical analysis method was used to detect circulating tumor DNA.

2. The electrochemical detection method of circulating tumor DNA in human body according to claim 1, characterized in that: The differential pulse voltammetry or cyclic square wave voltammetry in the electrochemical analysis method for detecting circulating tumor DNA specifically comprises the following steps: S1, dissolving the sample to be tested in a buffer solution to obtain a sample buffer solution; S2, incubating the electrochemical sensor electrode in the sample buffer to obtain the electrochemical sensor electrode incubated with the sample to be tested; S3, connecting the electrochemical sensor electrode incubated with the sample to be tested to an electrochemical workstation, and then placing the electrochemical sensor electrode incubated with the sample to be tested in an electrolyte, and using differential pulse voltammetry or cyclic square wave voltammetry to detect and analyze electrochemical signals.

3. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The pH value of the buffer solution is 7.0-7.

5.

4. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The buffer is one of PBS buffer, SSC buffer, TE buffer or a mixture of more than one of them.

5. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The electrochemical sensor electrode is incubated for 8 to 16 hours at a temperature of 0 to 8° C., and for 0.1 to 2 hours at a temperature of 20 to 30° C.

6. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The electrochemical sensor electrode comprises a working electrode, which is a gold electrode or an electrode having a gold coating.

7. The electrochemical detection method of circulating tumor DNA in the human body according to claim 6, characterized in that: The electrochemical sensor electrode also includes a reference electrode and an auxiliary electrode; the reference electrode is silver hydride, and the auxiliary electrode is any one of carbon, platinum or platinum black.

8. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The electrolyte contains potassium ferrocyanide.

9. The electrochemical detection method of circulating tumor DNA in the human body according to claim 8, characterized in that: The concentration of the potassium ferrocyanide is 5-15 mmol / L.

10. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The test parameters of the differential pulse voltammetry are: initial potential -0.3-0.1V, termination potential 0.4-0.6V, potential increment 1-5mV, pulse height 10-100mV, pulse period 20-200ms, pulse width 10-100ms, sampling width 5-50ms, and silent time 3-20s.

11. The electrochemical detection method of circulating tumor DNA in the human body according to claim 10, characterized in that: The characteristic peak in the characteristic curve obtained by the differential pulse voltammetry is between 0.05V and 0.2V.

12. The electrochemical detection method of circulating tumor DNA in the human body according to claim 2, characterized in that: The test parameters of the cyclic square wave voltammetry method are: initial potential -0.5 to 0.3 V, termination potential 0.4 to 0.6 V, pulse potential increment 1 to 10 mV, pulse height 10 to 100 mV, frequency 1 to 100 Hz, scanning number 3 to 10 circles, sampling width 5 to 50 ms, and silent time 5 to 20 s.

13. The electrochemical detection method of circulating tumor DNA in the human body according to claim 12, characterized in that: The characteristic peak in the characteristic curve obtained by the cyclic square wave voltammetry is between 0.05V and 0.25V.

14. Use of the electrochemical detection method of circulating tumor DNA in the human body as described in claims 1-13 in early cancer screening and monitoring tumor treatment.

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