CN112322703B - Method for simultaneously detecting two circulating tumor DNAs based on DNA self-assembly structure - Google Patents
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Abstract
The invention discloses a method for simultaneously detecting two circulating tumor DNAs based on a DNA self-assembly structure, belonging to the technical field of biomedicine. According to the invention, a DNA hexahedral nanostructure substrate is constructed, a complete hexahedron is formed in the presence of two ctDNAs, a G-quadruplex-heme composite structure with catalytic activity is formed, aniline polymerization is catalyzed, and polyaniline is formed on the hexahedron, so that the combination of a target sequence and the DNA nanostructure is converted into an electric signal. And due to the base complementary pairing principle, when the target sequence is mutated, no electric signal is generated, so that the specificity of the sensor is enhanced, the mutation of the target sequence can be identified, and meanwhile, the detection sensitivity is improved. Compared with the traditional method for detecting ctDNA, the method has the advantages of short detection time, strong specificity and high sensitivity.
Description
Technical Field
The invention relates to a method for simultaneously detecting two circulating tumor DNAs based on a DNA self-assembly structure, belonging to the technical field of biomedicine.
Background
Ovarian cancer is one of the most lethal cancers in women, and has the characteristics of high mortality rate, difficulty in early detection and the like. Traditional cancer early X-ray detection, ultrasonic detection, cytology detection and the like need to be observed after tumor formation. Circulating tumor DNA (ctDNA) is a nucleic acid molecule that is released into the circulation system by shedding of tumor cell body DNA or after apoptosis, and is a characteristic tumor biomarker. The detection of ctDNA is a means for early detection of cancer, and can be used for diagnosis at a low concentration in early stages of tumor. In the case of ovarian cancer, mutation of the 12 th codon of the Kras gene or mutation of Braf gene V600E occurs, and therefore, detection of these two ctdnas can be used for diagnosis in the early stage of ovarian cancer. The detection method of ctDNA includes Polymerase Chain Reaction (PCR), real-time multiplex PCR, loop-mediated isothermal amplification, etc. However, these methods have high requirements on the expertise of operators, high cost, low accuracy, high detection limit, and require pretreatment of samples, etc., which affect the detection results.
Electrochemical biosensors have attracted much attention because of their advantages such as good selectivity, high stability, high specificity, and high sensitivity. The electrochemical biosensor includes a molecular recognition part that recognizes a target and then converts a bio-signal into an electrical signal, and a signal conversion part. At present, many electrochemical-based ctDNA sensors are developed, such as an enzyme-linked immunosorbent assay, a small molecule modification method, a biological enzyme method and the like, but the problems of high detection limit, weak specificity and the like exist at present.
The self-assembly of DNA is a technology based on the base complementary pairing principle, and a specific configuration is designed according to logic, and a desired structure is formed under a proper condition. The method is widely applied to the fields of drug targeting transportation, small molecule detection and the like. The DNA self-assembly technology is combined with the electrochemical detection based on the gold electrode, so that the functional electrochemical biosensor for detecting the target substance at low concentration can be obtained, and the detection limit of the sensor is reduced. However, at present, no electrochemical biosensor based on DNA self-assembly is available, which is suitable for simultaneously detecting two kinds of circulating tumor DNA.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for simultaneously detecting two circulating tumor DNAs based on a DNA self-assembly structure, which has the advantages of high sensitivity, high specificity, accurate determination and the like.
The invention provides an electrochemical biosensor for simultaneously detecting two circulating tumor DNAs, which comprises an electrode, a DNA hexahedron which is adsorbed on the surface of the electrode and is constructed on the basis of the two circulating tumor DNAs, and an auxiliary probe which is connected to the two circulating tumor DNAs and is used for forming a G-quadruplex-heme composite structure, wherein the G-quadruplex-heme composite structure is used for catalyzing aniline polymerization to form polyaniline and converting a circulating tumor DNA signal into an electric signal.
Further, the electrode is a gold electrode or a glassy carbon electrode with gold nanoparticles modified on the surface.
Further, the two circulating tumor DNAs are respectively Kras gene and Braf gene, and the nucleotide sequences are respectively shown in SEQ ID NO. 1-2.
Further, the DNA hexahedron is obtained by hybridizing Kras gene and Braf gene sequences with a DNA hexahedron substrate, wherein the DNA hexahedron substrate is constructed by four nucleotide sequences; wherein, the four nucleotide sequences respectively contain a polyA sequence which is specifically adsorbed on the surface of the electrode and a sequence which is specifically combined with the Kras gene and the Braf gene sequence.
Further, the four nucleotide sequences are shown as SEQ ID NO.3-6, respectively.
Furthermore, the auxiliary probe is provided with a sequence complementary to the sequences of the Kras gene and the Braf gene.
Furthermore, the nucleotide sequence of the auxiliary probe is shown in SEQ ID NO. 7-8.
The second object of the present invention is to provide a method for simultaneously detecting Kras gene and Braf gene by using the electrochemical biosensor, comprising the steps of:
(1) adding four nucleotide sequences for constructing the DNA hexahedral substrate into a buffer solution, uniformly mixing, performing high-temperature denaturation and renaturation to form the DNA hexahedral substrate, dropwise adding the DNA hexahedral substrate solution onto an electrode, and incubating to obtain the electrode adsorbed with the DNA hexahedral substrate;
(2) dropwise adding a solution containing target Kras gene and Braf gene sequences onto the electrode adsorbed with the DNA hexahedron substrate in the step (1), and incubating to obtain an electrode with a complete DNA hexahedron;
(3) dropwise adding a solution containing the auxiliary probe to the electrode with the complete DNA hexahedron in the step (2), and incubating to obtain an electrode connected with the auxiliary probe;
(4) soaking the electrode prepared in the step (3) in a G-quadruplex forming solution containing heme, and incubating to form an electrode connected with a G-quadruplex-heme complex;
(5) soaking the electrode prepared in the step (4) in an aniline deposition buffer solution, and catalyzing aniline reaction to form polyaniline to be adsorbed on a DNA hexahedron to obtain an electrode adsorbing the polyaniline;
(6) measuring the current value of the electrode obtained in the step (5) by adopting an electrochemical workstation;
(7) drawing a corresponding linear relation curve according to the relation between the measured current value and the target concentration;
(8) and (3) when the concentration of the target sequence in the sample to be detected is detected, measuring the current value of the sample to be detected according to the steps (1) to (6), substituting the current value into the linear relation curve in the step (7), and calculating the concentration of the target sequence in the sample to be detected.
Further, in the step (6), when the electrochemical workstation measures the current value, the Ag/AgCl electrode is used as a reference electrode, and the platinum wire electrode is used as a counter electrode; the electrolyte is acetic acid-sodium acetate solution.
Further, the composition of the G-quadruplex forming liquid is as follows: 8-12 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid and 45-55mmol/L KCl; the aniline deposition buffer composition was: 0.08-0.12mol/L acetic acid-sodium acetate, 80-120mmol/L aniline, and 80-120mmol/L hydrogen peroxide.
The invention has the beneficial effects that:
according to the invention, a DNA hexahedral nanostructure substrate is constructed, a complete hexahedron is formed in the presence of two ctDNAs, a G-quadruplex-heme composite structure with catalytic activity is formed, aniline polymerization is catalyzed, and polyaniline is formed on the hexahedron, so that the combination of a target sequence and the DNA nanostructure is converted into an electric signal. And due to the base complementary pairing principle, when the target sequence is mutated, no electric signal is generated, so that the specificity of the sensor is enhanced, the mutation of the target sequence can be identified, and meanwhile, the detection sensitivity is improved. Compared with the traditional method for detecting ctDNA, the method has the advantages of short detection time, strong specificity and high sensitivity.
In addition, in contrast to the conventional Au-S bond, the use of polyA in combination with gold electrodes in the present invention allows the distance between DNAs to be controlled such that the DNA nanostructures are arranged in a desired spatial density. In this invention, the size of the hexahedral nanostructure of DNA may be changed according to the difference of the target DNA.
Drawings
FIG. 1 is a schematic diagram of a method for simultaneously detecting two ctDNAs based on a DNA self-assembly structure;
FIG. 2 is a standard curve of amperometric detection of target sequence concentration and specificity verification.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The constituent DNA hexahedral nanostructure sequences described in the following examples were purchased from Biotechnology engineering (Shanghai) Inc., and the auxiliary probes were purchased from Biotechnology engineering (Shanghai) Inc.
The composition of the G-quadruplex forming liquid is as follows: 10 mmol/L4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 50mmol/L KCl, pH 8.0;
the aniline deposition buffer composition was: 0.1mol/L acetic acid-sodium acetate, pH 4.3, 100mmol/L aniline, 100mmol/L hydrogen peroxide.
The nucleic acid sequences used in the examples of the present invention are shown in Table 1.
TABLE 1 sequence listing
Example 1: drawing of single target sequence and double target concentration standard curve
Four DNA sequences A1, A2, A3 and A4 forming hexahedral bases of DNA were added to a solution containing 13mmol/L Mg2+The four sequences can be subjected to base complementary pairing according to the expected design to form a DNA hexahedral substrate. Dripping 5 mu L of solution for forming the DNA hexahedral substrate onto the gold electrode, and incubating for 12h at room temperature to enable polyA to be tightly adsorbed on the surface of the gold electrode; washing the gold electrode with 10mmol/L PBS buffer solution, and then blowing ultra-pure nitrogen to dry the surface of the electrode; the gold electrode was immersed in 2 mmol/L6-mercaptohexanol and incubated for 1h to cover the sites that did not bind to DNA. And washing and drying the electrode.
The concentration of the Braf gene sequence was fixed at 1. mu. mol/L, and the concentration of the Kras gene sequence was measured using a standard curve. Mixing the Braf gene sequence of 1 mu mol/L and the Kras gene sequence of different concentrations into 100 mu L PBS, dripping 5 mu L target mixed solution onto the surface of a gold electrode on which a DNA hexahedral nanostructure substrate is incubated, and incubating for 1h at 37 ℃. After the electrode was rinsed and dried, 5. mu.L of auxiliary probe (G1, G2) was added dropwise to the surface of the gold electrode and incubated at 37 ℃ for 1 h. After the electrode is washed and dried, the electrode is soaked in G-tetrad forming liquid containing 200mmol/L of heme, and incubated for 1h at 37 ℃ to form a G-tetrad-heme complex. And washing and drying the electrode, soaking the electrode in an aniline deposition buffer solution, and reacting for 90min at 30 ℃ to enable the aniline to be catalyzed into polyaniline to be adsorbed on the DNA hexahedral nanostructure. The concentration of the Kras gene sequence was then fixed at 1 μmol/L, and standard curve measurements were performed for different concentrations of Braf gene sequences, and the above procedure was repeated. Finally, Kras and Braf gene sequences with the same concentration are added into the detection system, and the operation is repeated to measure the current value. The measurement is carried out by an electrochemical workstation, and the magnitude of the current is measured by differential voltammetry pulsing (DPV). And drawing a corresponding linear relation curve according to the relation between the measured current value and the target concentration.
When the electrochemical workstation for the electrode is adopted to measure the current value, the Ag/AgCl electrode is a reference electrode, the platinum wire electrode is a counter electrode, the electrolyte is 0.1M acetic acid-sodium acetate solution (pH 4.3), the current is measured by using a differential voltammetry pulse method (DPV), and the parameters are as follows: the scanning range is-0.2V-0.2V, the amplitude is 0.05V, the pulse time is 0.05s, and the sampling width is 0.0166 s.
As shown in fig. 2A, the electrical signal intensity increases with increasing Kras sequence concentration, and the linear regression equation for Kras gene is y 0.4515 log c +0.3216, R20.9935, where y represents the electrical signal intensity and C represents the target sequence concentration (. mu.mol/L), the detection limit of this method is 6.36 fmol/L.
As shown in fig. 2B, as the concentration of Braf gene increases, the electric signal also increases gradually, and the linear regression equation is that y is 0.4161, logC +0.2485, R20.9910, where y represents the electrical signal intensity and C represents the target sequence concentration (. mu.mol/L), the detection limit of this method is 4.29 fmol/L.
As shown in FIG. 2C, the electric signal was gradually increased with the increase in the concentration of the Kras gene and the Braf geneIncreasing by a linear regression equation of y 0.315 logC +0.6017, R20.9908, where y represents the electrical signal intensity and C represents the target sequence concentration (. mu.mol/L), the detection limit of this method is 4.77 fmol/L.
Example 2: detection of target sequence concentration in actual sample
In order to further verify the accuracy of the method in determining the concentration of the target sequence in an actual sample, human serum is selected as a sample for detection.
Adding target sequences with different concentrations into a human serum sample, uniformly mixing, taking 5 mu L of the mixture, dropwise adding the mixture to the surface of a gold electrode modified with a DNA hexahedral nanostructure substrate, and incubating for 1h at 37 ℃. After the electrode is washed and dried, 5 mu L of auxiliary probe is dripped on the surface of the gold electrode, and the gold electrode is incubated for 1h at 37 ℃. After the electrode is washed and dried, the electrode is soaked in G-tetrad forming liquid containing 200mmol/L of heme, and incubated for 1h at 37 ℃ to form a G-tetrad-heme complex. And washing and drying the electrode, soaking the electrode in an aniline deposition buffer solution, reacting for 90min at 30 ℃, and catalyzing aniline to generate polyaniline which is adsorbed on the DNA hexahedral nanostructure. And measuring current values by using an electrochemical workstation, and calculating the concentration of the target sequence by substituting the current values into the standard curve.
Specific samples and test results are shown in table 2.
TABLE 2 actual sample testing
| Sample (I) | Concentration of added target | Detected target concentration | Recovery (%) | Relative standard deviation (%) (n ═ 3) |
| 1 | 50nmol/L | 54.2nmol/L | 108.4 | 2.13 |
| 2 | 70pmol/L | 68.5pmol/L | 97.86 | 3.43 |
| 3 | 80fmol/L | 86.5fmol/L | 108.1 | 5.96 |
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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Claims (7)
1. An electrochemical biosensor for simultaneously detecting two circulating tumor DNAs (deoxyribonucleic acids), which is characterized by comprising an electrode, a DNA hexahedron which is adsorbed on the surface of the electrode and is constructed on the basis of the two circulating tumor DNAs, and an auxiliary probe which is connected to the two circulating tumor DNAs and is used for forming a G-quadruplex-heme composite structure, wherein the G-quadruplex-heme composite structure is used for catalyzing aniline polymerization to form polyaniline and converting a circulating tumor DNA signal into an electric signal;
the two circulating tumor DNAs are respectively a Kras gene and a Braf gene, and the nucleotide sequences are respectively shown in SEQ ID NO. 1-2;
the DNA hexahedron is obtained by hybridizing Kras gene and Braf gene sequences with a DNA hexahedron substrate, wherein the DNA hexahedron substrate is constructed by four nucleotide sequences; wherein, the four nucleotide sequences respectively contain a polyA sequence specifically adsorbed on the surface of the electrode and a sequence specifically combined with the Kras gene and Braf gene sequences;
the four nucleotide sequences are respectively shown as SEQ ID NO. 3-6.
2. The electrochemical biosensor as claimed in claim 1, wherein the electrode is a gold electrode or a glassy carbon electrode with surface modified gold nanoparticles.
3. The electrochemical biosensor as claimed in claim 1, wherein the auxiliary probe has a sequence complementary to the sequence of the Kras gene and the Braf gene.
4. The electrochemical biosensor as claimed in claim 3, wherein the nucleotide sequence of the auxiliary probe is shown in SEQ ID No. 7-8.
5. A non-diagnostic method for simultaneously detecting Kras gene and Braf gene using the electrochemical biosensor of any one of claims 1-4, comprising the steps of:
(1) adding four nucleotide sequences for constructing the DNA hexahedral substrate into a buffer solution, uniformly mixing, performing high-temperature denaturation and renaturation to form the DNA hexahedral substrate, dropwise adding the DNA hexahedral substrate solution onto an electrode, and incubating to obtain the electrode adsorbed with the DNA hexahedral substrate;
(2) dropwise adding a solution containing target Kras gene and Braf gene sequences onto the electrode adsorbed with the DNA hexahedron substrate in the step (1), and incubating to obtain an electrode with a complete DNA hexahedron;
(3) dropwise adding a solution containing the auxiliary probe to the electrode with the complete DNA hexahedron in the step (2), and incubating to obtain an electrode connected with the auxiliary probe;
(4) soaking the electrode prepared in the step (3) in a G-quadruplex forming solution containing heme, and incubating to form an electrode connected with a G-quadruplex-heme complex;
(5) soaking the electrode prepared in the step (4) in an aniline deposition buffer solution, and catalyzing aniline reaction to form polyaniline to be adsorbed on a DNA hexahedron to obtain an electrode adsorbing the polyaniline;
(6) measuring the current value of the electrode obtained in the step (5) by adopting an electrochemical workstation;
(7) drawing a corresponding linear relation curve according to the relation between the measured current value and the target concentration;
(8) and (3) when the concentration of the target sequence in the sample to be detected is detected, measuring the current value of the sample to be detected according to the steps (1) to (6), substituting the current value into the linear relation curve in the step (7), and calculating the concentration of the target sequence in the sample to be detected.
6. The non-diagnostic method according to claim 5, wherein in the step (6), when the electrochemical workstation performs the current value measurement, the Ag/AgCl electrode is a reference electrode, and the platinum wire electrode is a counter electrode; the electrolyte is acetic acid-sodium acetate solution.
7. The non-diagnostic method of claim 5, wherein the G-quadruplex forming solution has the composition: 8-12 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid and 45-55mmol/L KCl; the aniline deposition buffer composition was: 0.08-0.12mol/L acetic acid-sodium acetate, 80-120mmol/L aniline, and 80-120mmol/L hydrogen peroxide.
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