CN112098489B - Electrochemical method and system for quantifying DNA methylation degree - Google Patents
Electrochemical method and system for quantifying DNA methylation degree Download PDFInfo
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Abstract
The invention discloses an electrochemical method and system for quantifying DNA methylation degree, which can well reflect the methylation degree of sample DNA by using an electrochemical workstation to test the current adsorption rate of the sample DNA and further determine the tumor risk of the sample based on the result. The detection method of some embodiments of the invention has: 1) High sensitivity, a minimum of 200 μ L of plasma sample is sufficient for detection; 2) The DNA does not need to be pretreated; 3) The detection time is short, and the detection can be completed in about 10 minutes; 4) The detection cost is low, and the detection cost except DNA extraction is lower than 1 yuan; the method is expected to be developed into a detection technology for instant detection.
Description
Technical Field
The invention relates to a method for quantifying the methylation degree of DNA, in particular to an electrochemical method and system for quantifying the methylation degree of cfDNA.
Background
DNA methylation is one of the earliest discovered gene modifications, widely exists in various species, is an important epigenetic modification, is an important component of silent genetics, participates in the processes of animal embryonic development, gene imprinting, X chromosome inactivation and the like, plays an important role in the regulation and control of gene expression, and abnormal methylation can cause the formation of tumors. The detection of the DNA methylation degree has very important significance.
The existing DNA methylation methods include small organic molecule detection methods and biological analysis methods (see reign, wucheng. DNA methylation detection research progress [ J ]. 2012.). The cfDNA methylation detection techniques that are in practical use are mostly based on PCR techniques, including methylation fluorescent quantitative PCR, methylation digital titer PCR, and methylation next-generation sequencing. These techniques have the following disadvantages: 1) DNA pretreatment is required. DNA pretreatment such as sulfite conversion and PCR amplification is required before detection; 2) The cost is expensive. The current one-time expense for the methylation fluorescent quantitative PCR detection is about 500-1000 yuan, the consumable material cost of the methylation digital titer PCR single detection light is $ 50, and the expense for the methylation second-generation sequencing is more expensive; 3) The detection time is longer. Methylation-fluorescent quantitative PCR typically requires 2 hours (excluding DNA pretreatment). Methylation digital titer PCR typically takes 4 to 6 hours (excluding DNA pretreatment). The time required for methylation secondary sequencing is longer, usually greater than 3 days; 4) The sample demand is large. Three assays require 1ml or more plasma samples.
The DNA electrochemical analysis method has many advantages, such as short detection time, simple operation, low cost, easy miniaturization, and can be combined with other technologies, such as photochemistry, to form a visual real-time detection method. There have been a number of reports in the literature that have been applied to the analysis of DNA methylation levels and methyltransferase activity in the past. According to different analysis principles, the method is mainly divided into the following steps: direct electrochemical analysis, indirect electrochemical analysis, electrochemiluminescence analysis, and photoelectrochemical analysis of DNA methylation (see Yan, fan dream, guo wisdom, et al. DNA methylation electrochemical analysis [ J ] chemical progress, 2014,026 (012): 1977-1986.). The existing electrochemical DNA methylation detection technology has the following defects: 1) Modification of the recognition layer, such as probes, receptors, etc., is required; 2) Pretreatment of DNA is required, including sulfite transformation and PCR amplification; 3) Because pretreatment of DNA is required, the overall detection time is longer, and the overall efficiency of detection is reduced.
The liquid biopsy technology is mainly used for diagnosis, curative effect monitoring, drug screening and prognosis evaluation of malignant tumors in malignant tumor clinic. Compared with tissue detection, liquid biopsy has the advantages of non-invasive, repeatable, continuous detection and the like. Compared with the traditional tumor markers, the liquid biopsy technology has higher sensitivity and specificity.
cfDNA is an episomal DNA fragment that is released into the peripheral circulation by human cells, including tumor cells, and methylation modifications also exist, and the degree of methylation can reflect the status of the tumor. The detection of tumor-derived DNA fragments in cfDNA is an important component of current liquid biopsy of malignant tumors.
The development of a label-free, rapid and low-cost DNA methylation degree detection method has very important significance.
Disclosure of Invention
The present invention aims to overcome at least one of the disadvantages of the prior art and to provide a label-free DNA methylation detection method.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a method for electrochemical detection of DNA methylation comprising:
s1) obtaining a DNA sample to be detected;
s2) dissolving a DNA sample to be detected in an aqueous buffer solution to obtain a DNA solution; preferably, the aqueous buffer is a TE buffer, more preferably a TE buffer of pH = 8.0;
s3) immersing a working electrode, a reference electrode and a counter electrode of the electrochemical workstation in electrolyte, and determining a DPV current peak value before adsorption as a baseline current A0, wherein the electrolyte is a buffer solution added with water-soluble ferricyanide and water-soluble ferrocyanide; preferably, the buffer is a PBS buffer;
s4) immersing the working electrode into the DNA solution and fully adsorbing, testing the DPV current peak value after adsorption, and recording as sample current A1;
s5) calculating the DNA methylation degree according to the baseline current A0 and the sample current A1;
wherein, the constant temperature is maintained in the detection process, and the working electrode is a gold electrode.
In some embodiments, the temperature during the assay is maintained at 10 to 30 deg.C, preferably 15 to 25 deg.C, 18 to 22 deg.C, and more preferably about 20 deg.C.
In some examples, the water soluble ferricyanide salt is selected from [ K ] 3 Fe(CN) 6 ]、[Na 3 Fe(CN) 6 ](ii) a The water soluble ferrocyanide salt is selected from [ K 4 Fe(CN) 6 ]Or [ Na ] 4 Fe(CN) 6 ]。
In some examples, the molar ratio of water soluble ferricyanide salt to water soluble ferricyanide salt is (0.95 to 1.05): 1, preferably 1:1.
in some examples, the detection parameters of the electrochemical workstation DPV mode are: tequilibration =0s, ebegin = -0.1V, end =0.5V, E step =0.005V, epulse =0.05V, tpulse =0.05s, scanrate =0.05V/s, currentrange =1uA-1mA.
In some examples, the DNA current adsorption rate is calculated as:
in some examples, when the calculated DNA current adsorption rate is below 10%, the sample is rechecked.
In some examples, the test DNA sample is obtained by magnetic bead adsorption extraction.
In some examples, the DNA sample to be tested comprises a plasma cfDNA sample, a tissue DNA sample.
In a second aspect of the present invention, there is provided:
a tumor screening system, comprising:
a sample DNA methylation degree detection device for implementing the method of the first aspect of the present invention, determining the methylation degree of the sample DNA;
a result judgment device: based on the degree of DNA methylation of the sample, malignant tumors are screened.
In some examples, the degree of methylation of the sample DNA is measured as the current adsorption rate.
In some examples, the tumor includes, but is not limited to, lung cancer, prostate cancer, breast cancer, head and neck squamous cancer, esophageal cancer, lymphoma, gastric cancer, leukemia, pancreatic cancer, liver cancer, intestinal cancer, ovarian cancer, endometrial cancer, gallbladder cancer, cervical cancer, and the like.
The invention has the beneficial effects that:
the detection method of some examples of the invention has: 1) High sensitivity, a minimum of 200 μ L of plasma sample is sufficient for detection; 2) The DNA does not need to be pretreated; 3) The detection time is short, and the detection can be completed in about 10 minutes; 4) The detection cost is low, and the detection cost except DNA extraction is lower than 1 yuan; the method is expected to be developed into a detection technology for instant detection.
Drawings
FIG. 1 is a comparison of the coverage of DNA with different degrees of methylation on a gold surface;
FIG. 2 is a comparison of DNA coverage at different degrees of methylation on a gold surface;
FIG. 3 shows the results of electrochemical detection of DNA with different degrees of methylation;
fig. 4 is the results of electrochemical detection of normal DNA and cancer cfDNA;
FIG. 5 shows the results of electrochemical detection of short-fragment DNA;
FIG. 6 is the effect of different DNA buffers on the results of electrochemical measurements;
FIG. 7 is a graph of the effect of different DNA extraction methods on electrochemical detection results;
FIG. 8 is a graph of the effect of different temperatures on the results of electrochemical measurements;
FIG. 9 is an experimental result of the detection accuracy of the electrochemical detection method;
FIG. 10 is a plot of DNA adsorption rates for different methylation levels;
FIG. 11 shows the results of electrochemical measurements of different tissues;
FIG. 12 is a ROC curve for tissue adsorption rate;
figure 13 is the plasma cfDNA detection results for various tumors;
FIG. 14 is a ROC curve of plasma adsorption rate.
Detailed Description
For convenience of comparison, some operations are as follows:
DNA preparation:
tissue DNA preparation: all tumor tissues and normal tissues are subjected to conventional pathological material drawing, dehydration and paraffin embedding, and are subjected to HE staining after being sliced, and cancer tissue slices are subjected to cancer tissue enrichment after cancer tissues and normal tissue areas are confirmed by two different pathologists together. Tissue DNA extraction was performed using QIAamp DNA tissue extraction kit, according to the kit instructions. Elution was performed with an eluonbuffer (pH 8.0 TE) in the kit and stored in a refrigerator at-80 ℃ for a long period of time.
Plasma cfDNA preparation: cfDNA was extracted by an automatic nucleic acid extractor (magnetic bead adsorption method) in south Korea, and 200ul of plasma samples were added to each case. DNA was eluted with an elumbuffer (pH 8.0 TE) at 100ul per case. cfDNA was frozen at-80 ℃ in a refrigerator after elution.
And (3) DPV detection:
will contain 2.5mM 2 [ K3Fe (CN) 6]And [ K ] 4 Fe(CN) 6 ]The PBS electrolyte was poured into a quartz cell. The working electrode is polished on an automatic polisher by aluminum powder, and is dried in inert gas after being soaked and cleaned by pure water to remove residual aluminum powder. And immersing the polished working electrode, the reference electrode and the counter electrode in electrolyte, and connecting the working electrode, the reference electrode and the counter electrode with an electrode alligator clip of an electrochemical workstation. Opening detection software on a computer connected with the electrochemical workstation, setting the detection mode to be DPV, setting parameters to be tequilibration =0s, ebegin = -0.1V, eend =0.5V, E step =0.005V, epulse =0.05V, tpulse =0.05s, scanrate =0.05V/s, currentrange =1uA-1mA. And detecting that a DPV peak in the positive direction can appear in the software interface when startmeasurement is clicked in the interface. The DPV current peak was recorded as the baseline current. And (3) adsorbing the DNA solution on a working electrode, and comparing the baseline current to observe the current change of the peak value of the DPV curve after adsorption. The calculation formula is as follows:
comparison of binding force of DNA with gold foil at different methylation degrees
Adsorbing 10ng/ul of DNA with different methylation degrees on the surface of the gold foil, and finding out that the coverage areas of the DNA with different methylation degrees on the surface of the gold foil are different through an electron microscope. The coverage of 30% methylated DNA was the highest and 0% methylated DNA was the lowest (FIG. 1).
The DNA coverage of different methylation degrees on the surface of the gold foil is different through electron microscopy. The 30% methylated DNA height is highest and the 0% methylated DNA height is lowest (FIG. 2).
In the experimental process, specific sequences of the DNA are also found to have certain influence on the binding capacity of the gold electrode.
Electrochemical detection:
after DNA having a volume of 5ul and a concentration of 10ng/ul was adsorbed to the polished and cleaned working electrode for 10 minutes, the baseline current (A) was measured and 100% methylated DNA (B) and 0% methylated DNA (C) were adsorbed, respectively, and a large difference in peak current was found. Adsorption was calculated by the formula and found that 100% methylated DNA adsorption was greater than 0% methylated DNA (FIG. 3).
After the working electrode adsorbs DNA, a baseline current (A) is detected, cancer tissue DNA (B) and normal tissue DNA (C) are respectively adsorbed, and the peak current changes greatly. The adsorption rate was calculated by the formula and found to be greater than that of normal DNA (FIG. 4).
In the experimental process, the specific sequence of the DNA also has certain influence on the detection current.
Short fragment DNA assay
The tissue DNA is broken by an ultrasonic breaking instrument until the length of the main fragment is concentrated at 140bp, and the current adsorption rate of the short-fragment tumor tissue DNA and the normal tissue DNA is detected.
To further investigate whether this technique could be used to circulate free DNA, short fragment characteristics of circulating free DNA were simulated by breaking tissue DNA. As a result, it was found that the adsorption rate was increased both for the cancer tissue DNA and the normal tissue DNA after the disruption (FIG. 5).
In conclusion, the detection of the degree of DNA methylation by the electrochemical method is theoretically feasible, but how to obtain a more accurate detection result for the electrochemical detection result by the DNA sequence itself needs to be further researched and solved.
The technical scheme of the invention is further explained by combining experiments.
Effect of buffer composition on DPV assay results
The DNA solutions were tested for TE and 5XSSC, respectively, and the results are shown in FIG. 6.
The same sample is repeatedly tested for 10 times, and the adsorption rate of the TE group is found to be 24.5 +/-5.877%, the adsorption rate of the SSC group is found to be 48.59 +/-23.54%, and the fluctuation amplitude of the same sample of the SSC group is far higher than that of the TE group.
Effect of DNA extraction method on DPV detection results
The effect of extraction of cfDNA by the magnetic bead method (automatic nucleic acid extractor) and the column chromatography method (manual chloroform extraction) on the detection of the results was tested separately, and the same sample was repeated 10 times, with the results shown in fig. 7. The adsorption rate of the column chromatography is 32.01 +/-11.76%, and the adsorption rate of the magnetic bead method is 25.16 +/-7.89%. The results show that the repeatability of the same sample in the column chromatography method is obviously inferior to that in the magnetic bead method. Therefore, the magnetic bead method is preferable for extracting DNA.
Influence of detection temperature on DPV detection result
The same sample DNA was tested for current adsorption at 10 deg.C (B), 15 deg.C (C), 20 deg.C (D), 25 deg.C (E), and 30 deg.C (F), respectively, and the baseline current was A, and the results are shown in FIG. 8.
The experimental results show that ambient temperature has essentially no effect on baseline current, but has an unexpectedly significant effect on the adsorption profile of methylated DNA. The research shows that the adsorption rate of DNA on the gold electrode is the highest and the DNA is more stable at 20 ℃.
Detection accuracy of electrochemical detection method
The research finds that the precision of the technology reaches 1pg/ul (figure 9) by testing the cfDNA adsorption rate of cancer samples and normal samples with different concentrations.
DNA adsorption Rate profiles at different methylation levels
The current adsorption rate of DNA with different methylation levels was tested separately, and the adsorption rate was found to vary with the methylation level. The results are shown in FIG. 10, which shows that the highest adsorption rate is obtained when the methylation level is between 30 and 50%. At greater than 50%, the adsorption rate decreases as the methylation level increases. At less than 30%, the adsorption rate decreases with decreasing methylation levels. At a methylation level of 0%, the adsorption rate reached a minimum level of 10%.
Tissue DNA detection results
The results of taking 15 cases of lung cancer, 15 cases of intestinal cancer, 15 cases of prostate cancer, 15 cases of breast cancer tissue and normal tissue, extracting the DNA from these tissues, and measuring the DNA current adsorption rates of the different tissues by the above-described method are shown in FIG. 11. As can be seen from the figure, there was a very significant difference in the DNA current adsorption rates of the normal tissue and the tumor tissue.
An ROC curve is drawn by using different DNA current adsorption rates (figure 12), and the AUC value is 0.9444, which shows that the detection mode has good sensitivity and high specificity.
Plasma cfDNA detection results
The results of the detection of the DNA current adsorption rates of the plasma of 700 tumor patients and the plasma of 500 normal patients are shown in fig. 13, and it can be seen from the graph that there is a very significant difference between the plasma DNA current adsorption rates of the normal patients and the tumor patients.
An ROC curve is drawn by using different DNA current adsorption rates (figure 14), and the AUC value is 0.8507, which shows that the detection mode has good sensitivity and high specificity.
From the above, it can be seen that the degree of methylation of the sample DNA can be well reflected by testing the current adsorption rate of the sample DNA using the electrochemical workstation, and the tumor risk of the sample can be further determined based on the result.
The detection method of some embodiments of the invention has:
1) High sensitivity, a minimum of 200 μ L of plasma sample is sufficient for detection;
2) The DNA does not need to be pretreated;
3) The detection time is short, and the detection can be completed in about 10 minutes;
4) The detection cost is low, and the detection cost except DNA extraction is lower than 1 yuan; the method is expected to be developed into a detection technology for instant detection.
Claims (9)
1. A method for electrochemical detection of DNA methylation comprising:
s1) obtaining a DNA sample to be detected;
s2) dissolving a DNA sample to be detected in an aqueous buffer solution to obtain a DNA solution; the aqueous buffer is TE buffer pH = 8.0;
s3) immersing a working electrode, a reference electrode and a counter electrode of the electrochemical workstation in electrolyte, and determining a DPV current peak value before adsorption as a baseline current A0, wherein the electrolyte is a buffer solution added with water-soluble ferricyanide and water-soluble ferrocyanide; the buffer solution is a PBS buffer solution;
s4) immersing the working electrode into the DNA solution and fully adsorbing, testing the DPV current peak value after adsorption, and recording as sample current A1;
s5) calculating the DNA methylation degree according to the baseline current A0 and the sample current A1;
wherein, the constant temperature is maintained to 15-25 ℃ in the detection process, and the working electrode is a gold electrode;
the gold electrode is polished by aluminum powder before use;
the detection parameters of the DPV mode of the electrochemical workstation are as follows: t equibration =0s, E begin = -0.1V, E end =0.5V, E step =0.005V, E pulse =0.05V, t pulse =0.05s, scan rate =0.05V/s, current range =1uA-1mA;
the calculation formula of the DNA current adsorption rate is as follows:
2. The electrochemical detection method according to claim 1, characterized in that: the temperature during the detection was maintained at 20 ℃.
3. The electrochemical detection method according to claim 1, characterized in that: the water soluble ferricyanide salt is selected from [ K 3 Fe(CN) 6 ]、[Na 3 Fe(CN) 6 ](ii) a The water-soluble ferrocyanide salt is selected from [ K ] 4 Fe(CN) 6 ]Or [ Na 4 Fe(CN) 6 ]。
4. The electrochemical detection method according to any one of claims 1 to 3, characterized in that: the molar ratio of the water-soluble ferricyanide salt to the water-soluble ferrocyanide salt is (0.95-1.05): 1.
5. the electrochemical detection method according to claim 4, characterized in that: the molar ratio of the water-soluble ferricyanide salt to the water-soluble ferrocyanide salt is 1:1.
6. the electrochemical detection method according to any one of claims 1 to 3, characterized in that: and extracting by using a magnetic bead adsorption method to obtain a DNA sample to be detected.
7. The electrochemical detection method according to any one of claims 1 to 3, characterized in that: the DNA sample to be detected comprises a plasma cfDNA sample and a tissue DNA sample.
8. A tumor screening system, comprising:
sample DNA methylation degree detection device: determining the degree of methylation of the sample DNA using the method of any one of claims 1 to 7;
a result judgment device: based on the degree of DNA methylation of the sample, malignant tumors are screened.
9. The tumor screening system of claim 8, wherein: the tumor is selected from lung cancer, prostatic cancer, breast cancer, head and neck squamous cancer, esophageal cancer, lymphoma, gastric cancer, leukemia, pancreatic cancer, hepatocarcinoma, intestinal cancer, ovarian cancer, endometrial cancer, gallbladder cancer, and cervical cancer.
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