Modified electrode for electrochemical nucleic acid detection and preparation method and application thereof

Abstract

The invention discloses a modified electrode for electrochemical nucleic acid detection and a preparation method and application thereof. The modified electrode for electrochemical nucleic acid detection comprises a composite electrode substrate, and a) or b) modified on the surface of a working electrode in the composite electrode substrate: a) a polyaniline membrane and a proton exchange membrane modified on the surface of the polyaniline membrane; b) a proton sensitive membrane; the composite electrode substrate comprises a working electrode, a reference electrode and a counter electrode. The modified electrode provided by the invention realizes real-time detection of nucleic acid by detecting the change of proton concentration in nucleic acid amplification, has no inhibition effect on nucleic acid amplification reaction, and can integrate nucleic acid extraction, amplification and detection; the method has good stability and reproducibility, the detection limit is consistent with that of real-time fluorescent nucleic acid, the detection time is shortened, the cost is low, the equipment size is small, and the method is suitable for household use and real-time fixed-point detection of communities.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical electrode preparation, and specifically relates to a modified electrode for electrochemical nucleic acid detection, a preparation method thereof, and an application in nucleic acid detection. Background Art

[0002] Nucleic acid testing is the gold standard for clinical pathogenic microorganism detection. The most common is fluorescent quantitative PCR (polymerase chain reaction), but this method requires cumbersome processing of nucleic acids and has high requirements for detection equipment or platforms. High-sensitivity RT-PCR instruments are expensive and have high requirements for laboratory cleanliness and operators. In addition, nucleic acid testing is time-consuming. POCT (Point of Care Testing) has the advantage of being fast and is the trend for simple testing. The advantages of electrochemical detection include real-time detection and miniaturization of instruments, but the instability and low sensitivity of electrochemical detection hinder the application of electrochemical detection. Summary of the invention

[0003] In order to overcome the shortcomings of the above-mentioned technology, the purpose of the present invention is to provide a modified electrode for electrochemical nucleic acid detection and its preparation method and application. Using this modified electrode to detect nucleic acid can shorten the detection time, improve the detection sensitivity, and provide a carrier for a new method of nucleic acid detection in a simple scenario.

[0004] In a first aspect, the present invention protects a modified electrode for electrochemical nucleic acid detection, which comprises a composite electrode substrate, and a) or b) modified on the surface of the composite electrode substrate:

[0005] a) Proton sensitive membrane;

[0006] b) polyaniline membrane and proton exchange membrane modified on the surface of polyaniline membrane;

[0007] The composite electrode substrate comprises a working electrode, a reference electrode and a counter electrode.

[0008] The modified electrode for electrochemical nucleic acid detection described above, further, the reference electrode is a silver electrode or a silver / silver chloride electrode, preferably a silver electrode;

[0009] The working electrode is a carbon working electrode;

[0010] The counter electrode is a carbon counter electrode.

[0011] Furthermore, the composite electrode substrate is a screen-printed electrode, and the working electrode, the reference electrode and the counter electrode are printed on the substrate by screen printing.

[0012] Specifically, the preparation of the composite electrode includes the following steps: 1) printing carbon paste on a substrate by screen printing, and forming a working electrode and a counter electrode after a first drying; 2) printing silver paste on an epoxy resin substrate by screen printing, and forming a reference electrode after a second drying; 3) fixing an insulating material on the electrode layer by screen printing to form an insulating layer, and leaving the working areas of the reference electrode, the working electrode and the counter electrode outside, and obtaining the composite electrode after curing.

[0013] More specifically, the substrate is an epoxy resin substrate. The first drying and the second drying are drying at 130°C for 30 minutes. The thickness of the carbon paste used to prepare the working electrode is 1μm to 50μm, specifically 10μm. The thickness of the silver paste used to prepare the reference electrode is 1μm to 50μm, specifically 10μm. Specifically, in terms of mass percentage, the composition of the silver paste is as follows: 40% silver powder, 15% chloroacetic acid resin + polyester resin, 40% divalent ester, 5% isocyanate crosslinker. Among them, the purity of the silver powder is greater than or equal to 99.95%. The thickness of the carbon paste used to prepare the counter electrode is 1μm to 50μm, specifically 10μm. The insulating material is insulating ink. The curing is curing at 100°C for 30 minutes.

[0014] The shapes of the reference electrode and the counter electrode can be arbitrarily adjusted, and can be rectangular or semicircular, and the shape of the working electrode can be arbitrarily adjusted, and can be circular or rectangular. The arrangement order of the reference electrode, the working electrode and the counter electrode can be arranged in a horizontal regular arrangement, or intertwined in the form of concentric circles.

[0015] In the above modified electrode for electrochemical nucleic acid detection, in a), the proton sensitive membrane is composed of a proton sensitive substance and a plasticizer;

[0016] The proton sensitive substance is one or more of trilaurylamine, 4-nonadecanylpyridine, and potassium borate;

[0017] The plasticizer is one or more of polyvinyl chloride, cyclohexanone, and di-n-octyl sebacate;

[0018] The mass ratio of the proton sensitive substance to the plasticizer may be 1:(10000-20000), specifically 1:13333;

[0019] The thickness of the proton sensitive membrane is 50 μm to 200 μm, and specifically may be 100 μm.

[0020] In the above-mentioned modified electrode for electrochemical nucleic acid detection, in b), the polyaniline film modified on the surface of the composite electrode substrate may be a polyaniline film electrodeposited on the surface of the composite electrode substrate.

[0021] In the modified electrode for electrochemical nucleic acid detection, the molecular weight of polyaniline in the polyaniline film may be 186-9300.

[0022] In the modified electrode for electrochemical nucleic acid detection, the proton exchange membrane modified on the surface of the polyaniline membrane can be a proton exchange membrane modified on the surface of the composite electrode substrate by drop coating.

[0023] In the above-mentioned modified electrode for electrochemical nucleic acid detection, in b), the proton exchange membrane may be a Nafion membrane;

[0024] The thickness of the proton exchange membrane may be 5 μm to 500 μm, and specifically may be 100 μm.

[0025] In a second aspect, the present invention protects a method for preparing a modified electrode for electrochemical nucleic acid detection, which is the following method A) or method B):

[0026] The method A) comprises:

[0027] Performing a film-forming treatment on the surface of the working electrode in the composite electrode using a proton sensitive membrane solution to obtain the modified electrode for electrochemical nucleic acid detection;

[0028] The method B) comprises:

[0029] B1) electrochemically depositing a solution containing aniline on the composite electrode substrate to obtain the polyaniline film;

[0030] B2) forming a film on the surface of a working electrode modified with a polyaniline membrane by subjecting a solution of a proton exchange membrane to obtain the modified electrode for electrochemical nucleic acid detection.

[0031] In the above-mentioned preparation method, in method A), the proton sensitive membrane solution is composed of trilaurylamine, potassium borate, polyvinyl chloride, di-n-octyl sebacate and cyclohexanone;

[0032] The concentration of trilaurylamine in the ion-sensitive membrane solution is 3 mM to 6 mM, specifically 4.6 mM;

[0033] The concentration of potassium borate in the ion-sensitive membrane solution is 2 mM to 4 mM, specifically 2.5 mM;

[0034] In terms of g / mL, the concentration of the polyvinyl chloride in the ion-sensitive membrane is 5% to 10%, and specifically 8%;

[0035] The concentration of di-n-octyl sebacate in the ion sensitive membrane is 0.1M-0.6M, specifically 0.4M.

[0036] The proton sensitive membrane is formed by using a drop coating method, that is, a proton sensitive membrane solution is dropped onto the surface of the working electrode in the composite electrode and then dried to form the proton sensitive membrane;

[0037] The amount of the proton sensitive membrane solution is 1 μL to 3 μL, specifically 2 μL;

[0038] The drying may be performed at 37° C. for 15 min.

[0039] In the above preparation method, in method B), the electrochemical deposition comprises the following steps: dropping a solution containing aniline on the composite electrode substrate to cover the working electrode, reference electrode and counter electrode at the same time, and performing the electrochemical deposition using an electrochemical workstation.

[0040] In the above preparation method, in method B), the solution containing aniline may be composed of an aqueous solution of aniline and an inorganic acid; the concentration of the aniline may be 0.01M to 4M, specifically 0.01M; the concentration of the inorganic acid may be 0.1M to 3M, specifically 0.5M. The inorganic acid may specifically be hydrochloric acid.

[0041] In the above preparation method, in method B), the electrochemical deposition can be carried out by cyclic voltammetry scanning method, and the conditions can be as follows: the initial potential is -0.5V to 0V, the end potential is 0.8V to 1.5V, and the scanning rate is 0.01V s -1 ~0.5Vs -1 , the rest time is 1s to 10s, and the number of scans is 5 to 50. As an example, the conditions of the electrochemical deposition are as follows: the initial potential is -0.2V, the end potential is 1.0V, and the scan rate is 0.1V s -1 , the static time is 2s, and the number of scans is 20 circles.

[0042] In the above-mentioned preparation method, in method B), the solution of the proton exchange membrane may be a Nafion solution;

[0043] The proton exchange membrane is formed by using a drop coating method, that is, a proton exchange membrane solution is dropped onto the surface of the working electrode modified with the polyaniline membrane and then dried to form the proton exchange membrane;

[0044] The concentration of the Nafion solution may be 0.5% to 5%, specifically 2%; the solvent may be an organic solvent, such as anhydrous ethanol;

[0045] The drying may be natural air drying, such as natural air drying for 0.5 h.

[0046] In a fourth aspect, the present invention protects the use of the modified electrode for electrochemical nucleic acid detection in detecting proton concentration and / or detecting nucleic acid.

[0047] In a fifth aspect, the present invention protects a method for electrochemical detection of nucleic acids, which is the following method P1 and / or method P2:

[0048] Method P1 comprises the following steps:

[0049] Amplifying the nucleic acid, and simultaneously monitoring the voltage of the working electrode VS the reference electrode in the amplification system using any of the modified electrodes for electrochemical nucleic acid detection described above, and monitoring the amplification reaction in real time according to the change of the voltage over time;

[0050] Method P1 comprises the following steps:

[0051] Amplify nucleic acids of different concentrations respectively, and use any of the modified electrodes for electrochemical nucleic acid detection described above to monitor the voltage of the working electrode VS the reference electrode in the amplification system during the amplification reaction, and record the curve of the change of voltage with amplification time in the nucleic acid amplification reaction of different concentrations;

[0052] According to the curve of voltage variation with amplification time in the nucleic acid amplification reaction of different concentrations, the nucleic acid is quantitatively analyzed. For example, according to the time corresponding to the voltage peak in the curve, the nucleic acid is quantitatively analyzed.

[0053] As an example, the nucleic acid is Staphylococcus aureus genomic DNA. The amplification is performed by heating at 65°C for 40 minutes.

[0054] In the present invention, the modified electrode containing polyaniline and having proton exchange or the modified electrode containing a proton sensitive membrane can be used to detect the change of proton concentration during nucleic acid amplification to achieve real-time detection of nucleic acid.

[0055] In a sixth aspect, the present invention protects a kit for detecting nucleic acid, which contains any of the modified electrodes for electrochemical nucleic acid detection described above.

[0056] In the modified electrode containing the proton sensitive membrane of the present invention, the protons generated during the amplification process are converted into electrons through the proton sensitive membrane, thereby forming an electric potential, the conversion process is rapid, and the response to the generated protons is sensitive. Compared with the prior art, the advantages of the present invention are:

[0057] (1) Silver paste is used to print the reference electrode to avoid interference from impurities and improve the efficiency of nucleic acid amplification.

[0058] (2) The electrochemical electrode for nucleic acid detection based on the proton sensitive membrane has a simple manufacturing process and the prepared electrochemical electrode has good stability and reproducibility, providing a new idea for future nucleic acid detection.

[0059] (3) The proton sensitive membrane is directly drop-coated on the working electrode of the electrochemical electrode, and the electrode modification process is simple;

[0060] (4) Real-time detection of nucleic acid is achieved by detecting changes in proton concentration during nucleic acid amplification without inhibiting the nucleic acid amplification reaction.

[0061] In the modified electrode containing polyaniline and having proton exchange, the non-covalent functionalization of the polyaniline film provides abundant binding sites and makes the polyaniline film and the substrate of the electrochemical chip more tightly bound; the polyaniline film and the proton exchange membrane play a synergistic role, which helps the electronic transfer ability of the sensor. Compared with the prior art, the advantages of the present invention are:

[0062] (1) Silver paste is used to print the reference electrode to avoid interference from impurities and improve the efficiency of nucleic acid amplification.

[0063] (2) The electrochemical electrode for nucleic acid detection based on polyaniline-proton exchange membrane improves the sensitivity of the electrode. The prepared electrochemical electrode has good stability and reproducibility, providing new ideas for future nucleic acid detection.

[0064] (3) Real-time detection of nucleic acid is achieved by detecting changes in proton concentration during nucleic acid amplification without inhibiting the nucleic acid amplification reaction.

[0065] The present invention can integrate nucleic acid extraction, amplification, and detection, and combined with rapid amplification means, a proton exchange membrane is added to the working electrode deposited with polyaniline, which can improve the stability of the electrochemical electrode. The directly drop-coated proton-sensitive membrane electrode is sensitive to protons, and the detection limit is consistent with the real-time fluorescent nucleic acid. The detection time is shortened, the cost is low, and the equipment size is small. It is suitable for home use and point-of-care nucleic acid testing in the community. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a schematic diagram of the structure of the screen-printed rectangular electrodes in Example 1 and Example 2 of the present invention, wherein 1 is a working electrode; 2 is a reference electrode; and 3 is a counter electrode.

[0067] Figure 2 This is a schematic diagram of the structure of the screen-printed circular electrode in Example 1 and Example 2 of the present invention, wherein 1 is a working electrode; 2 is a reference electrode; and 3 is a counter electrode.

[0068] Figure 3 This is a linear relationship diagram of the proton concentration in the solution detected by using the modified electrode for electrochemical detection in Example 1 in Example 3 of the present invention.

[0069] Figure 4This is a linear relationship diagram of the proton concentration in the solution detected by using the modified electrode for electrochemical detection in Example 2 in Example 4 of the present invention.

[0070] Figure 5 In Example 5 of the present invention, the modified electrode of Comparative Example 1 is used to detect nucleic acid, and 10 7 Electrochemical nucleic acid detection results for targets with 0 copies / mL and 0 copies / mL.

[0071] Figure 6 In Example 5 of the present invention, the modified electrode of Example 1 is used for nucleic acid detection, and 10 7 Electrochemical nucleic acid detection results for targets with 0 copies / mL and 0 copies / mL.

[0072] Figure 7 In Example 5 of the present invention, 10 7 Fluorescent nucleic acid detection results of targets with copies / mL and 0 copies / mL.

[0073] Figure 8 In Example 6 of the present invention, the modified electrode of Example 2 is used for nucleic acid detection, and 10 7 Electrochemical nucleic acid detection results for targets with 0 copies / mL and 0 copies / mL.

[0074] Fig. 9 In Example 6 of the present invention, 10 7 Fluorescent nucleic acid detection results of targets with copies / mL and 0 copies / mL.

[0075] Fig.10 The electrochemical nucleic acid detection results of nucleic acid detection using the modified electrode of Comparative Example 2 in Example 5 of the present invention with the addition of targets of different concentrations.

[0076] Fig.11 The electrochemical nucleic acid detection results of Example 7 of the present invention using the modified electrode of Example 1 with the addition of nucleic acids of different concentrations are shown.

[0077] Fig.12 These are the fluorescent nucleic acid detection results of adding different concentrations of targets in Example 7 of the present invention. DETAILED DESCRIPTION

[0078] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary persons in the art without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used in the present invention should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0079] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0080] The Nafion in the following examples is purchased from DuPont, the product name is Nafion solution, and the product catalog number is D520. The electrochemical workstation is a Shanghai Chenhua electrochemical workstation, model CHI660.

[0081] Example 1. Preparation of polyaniline Nafion electrochemically modified electrode

[0082] The screen-printed electrodes for electrochemical detection were prepared as follows:

[0083] (1) The epoxy resin substrate was rinsed with deionized water to clean the surface and dried with nitrogen gas.

[0084] (2) A carbon paste (purchased from Jingze Electronic Technology (Qingdao) Co., Ltd., model number CCP-JM100) was printed on an epoxy resin substrate by screen printing. The thickness of the carbon paste was 10 μm to form a working electrode and a counter electrode, and then dried at 130° C. for 30 minutes.

[0085] (3) A silver paste (the composition of the silver paste, in terms of mass percentage, is as follows: 40% silver powder (purity greater than or equal to 99.95%), 15% chloroacetic acid resin + polyester resin, 40% divalent acid ester, 5% isocyanate crosslinking agent) is printed on an epoxy resin substrate by screen printing. The silver paste thickness is 10 μm to form a reference electrode, which is then dried at 130° C. for 30 minutes.

[0086] (4) The insulating ink was fixed on the electrode layer by screen printing to form an insulating layer, and the working areas of the reference electrode, the working electrode and the counter electrode were left outside, and then cured at 100°C for 30 minutes.

[0087] like Figure 1 and Figure 2As shown, the shapes of the reference electrode and the counter electrode can be arbitrarily adjusted, and can be rectangular or semicircular. A semicircular shape is used here. The shape of the working electrode can be arbitrarily adjusted, and can be circular or rectangular. The arrangement order of the reference electrode, the working electrode and the counter electrode can be arranged in a transverse regular pattern or intertwined in the form of concentric circles. A transverse regular pattern is used here. That is, in the following embodiments, Figure 1 The screen-printed electrode shown is a modified electrode prepared from a composite electrode substrate. Figure 2 Or modified electrodes prepared from composite electrode substrates of other shapes and nucleic acid detection methods are also within the protection scope of the present invention. Figure 1 The specific dimensions are as follows: L1 = 38.5mm, W1 = 27.4mm, L2 = 5mm, L3 = 2.5mm, W2 = 1.5mm, W3 = 1.7mm, W4 = 1mm; Figure 2 The specific dimensions are as follows: L1=38.5mm, W1=27.4mm, L3=2.5mm, W4=1mm, D1=3.5mm, D2=6.9mm.

[0088] The modified electrode containing polyaniline and having proton exchange was prepared as follows:

[0089] (1) Modification of polyaniline film

[0090] a. Take 20uL of 99% aniline (purchased from MacLean) and mix with 20ml of 0.5M hydrochloric acid, and mix using a vortex for 10 minutes;

[0091] b. Take 50uL of the mixed solution in step a and drip it onto the electrochemical electrode, covering the working electrode, reference electrode, and counter electrode at the same time. Set the corresponding parameters in the electrochemical workstation for CV scanning, with an initial potential of -0.2V, a termination potential of 1.0V, and a scan rate of 0.1V s -1 , the rest time was 2 s, and the number of scans was 20. The exhausted liquid was removed.

[0092] c. Gently rinse the working electrode with distilled water (purchased from Shanghai Shenggong Company).

[0093] (2) Modification of proton exchange membrane

[0094] 5 ul of 2% Nafion solution (solvent: anhydrous ethanol) was dripped onto the working electrode modified with the polyaniline film, and after being naturally air-dried for 0.5 h, the working electrode was gently rinsed with distilled water.

[0095] In the modified electrode containing polyaniline and having proton exchange prepared in this example, the thickness of the Nafion membrane is 100 μm.

[0096] Example 2: Preparation of proton sensitive membrane electrochemically modified electrode

[0097] (1) Prepare screen-printed electrodes according to the steps in Example 1

[0098] (2) Preparation of proton sensitive membrane solution

[0099] a. Add 8% (w / v) polyvinyl chloride and 0.4M di-n-octyl sebacate to cyclohexanone, stir until completely dissolved, and let stand for 4 hours.

[0100] b. Add 4.6 mM trilaurylamine and 2.5 mM potassium borate to the above solution and mix well.

[0101] (3) Modification of proton sensitive membrane

[0102] Take 2 μL of proton sensitive membrane solution, evenly apply it on the working electrode of the composite electrode, and dry it at 37°C for 15 min.

[0103] The proton sensitive membrane electrochemically modified electrode prepared in this embodiment has a thickness of 100 μm after film formation.

[0104] Comparative Example 1

[0105] A polyaniline Nafion electrochemically modified electrode was prepared according to the steps in Example 1, except that the reference electrode in the composite electrode substrate was modified to a silver / silver chloride electrode, wherein the mass ratio of Ag to AgCl in the silver / silver chloride slurry was 3:2 (the other components in the slurry were the same as in Example 1).

[0106] Comparative Example 2

[0107] The polyaniline electrochemically modified electrode was prepared according to the steps in Example 1, except that the Nafion membrane was not modified.

[0108] Example 3: Performance Characterization of the Electrochemically Modified Electrode Prepared in Example 1

[0109] Take the modified electrode containing polyaniline and having proton exchange prepared in Example 1, and characterize the electrode performance with solutions of different pH values. The specific steps are as follows:

[0110] Use pH buffer solution (TE buffer solution of different pH) for testing. Take 50 μL of pH buffer solution and drop it on the electrochemical modified electrode prepared in Example 1, covering the working electrode and the reference electrode at the same time. After waiting for 100 seconds, use the electrochemical workstation to test the open circuit voltage between the working electrode and the reference electrode of the electrochemical modified electrode. The test results are as follows:

[0111] Table 1. pH standard solution test results

[0112]

[0113] The results in Table 1 show that the electrochemical electrode prepared in Example 1 can detect the change of proton concentration in the detection solution with high sensitivity.

[0114] Multiple tests were performed using multiple electrochemical electrodes prepared in Example 1. TE buffer with the same pH (pH 6, 6.5, 7, 7.5, 8, 8.5) was tested each time. A total of 3 tests were performed. The linear fitting equation was: y = -37.507x + 257.973, R 2 =0.992, the result is Figure 3 shown. Figure 3 The results show that the electrochemical electrode prepared in Example 1 has high repeatability and strong reliability.

[0115] Performance Characterization of the Electrochemically Modified Electrodes Prepared in Example 4 and Example 2

[0116] Take the proton sensitive membrane modified electrode prepared in Example 2, and characterize the electrode performance with solutions of different pH values. The specific steps are as follows:

[0117] Use pH buffer solution (TE buffer solution of different pH) for testing. Take 50 μL of pH buffer solution and drop it on the electrochemical modified electrode prepared in Example 2, covering the working electrode and the reference electrode at the same time. After waiting for 100 seconds, use the electrochemical workstation to test the open circuit voltage between the working electrode and the reference electrode of the electrochemical modified electrode. The test results are as follows:

[0118] Table 2. pH standard solution test results

[0119]

[0120] The results in Table 2 show that the electrochemical electrode prepared in Example 2 can detect the change of proton concentration in the detection solution with high sensitivity.

[0121] Multiple tests were performed using multiple electrochemical electrodes prepared in Example 2. TE buffer with the same pH (pH 6, 6.5, 7, 7.5, 8, 8.5) was tested each time. A total of 3 tests were performed. The linear fitting equation was: y = -51.594x + 381.609, R 2 =0.999, the result is Figure 4 shown. Figure 4 The results show that the electrochemical electrode prepared in Example 2 has high repeatability and strong reliability.

[0122] Example 5: The electrochemically modified electrode prepared in Example 1 is used to detect nucleic acid

[0123] The electrochemically modified electrode prepared in Example 1 and the reference electrode prepared in Comparative Example 1 were silver / silver chloride electrochemically modified electrodes for nucleic acid detection, and the specific steps were as follows:

[0124] The positive quality control target (concentration of 10 6 copies / μL), 2.5μL of Staphylococcus aureus solution was added to 22.5μL of nucleic acid amplification solution (the nucleic acid amplification solution in the Staphylococcus aureus kit of Qingdao Jianma Gene Technology Co., Ltd.), and no target nucleic acid (NTC) was added as a negative control. Therefore, the final concentration of the target in the reaction solution is: 10 7 copies / mL of Staphylococcus aureus genomic DNA, 0 copies / mL of Staphylococcus aureus genomic DNA.

[0125] There are three groups configured in total;

[0126] A group was added to an electrochemically modified electrode whose reference electrode was silver / silver chloride (the rest of the steps were the same as in Example 1), heated at 65°C for 40 minutes, and the voltage of the working electrode VS the reference electrode was monitored in real time by an electrochemical workstation. The results were as follows: Figure 5 As shown, the data on the vertical axis have been normalized.

[0127] A group of them were added to the electrochemically modified electrode based on polyaniline-proton exchange membrane in which the reference electrode prepared in Example 1 was silver, and heated at 65°C for 40 minutes. The voltage of the working electrode VS the reference electrode was monitored in real time by an electrochemical workstation. The results are as follows: Figure 6 As shown, the data on the vertical axis have been normalized.

[0128] Another group used the ND2600 fluorescence quantitative PCR instrument purchased from Qingdao Jianma to test and determine the amplification effect. The results are as follows Figure 7 shown.

[0129] like Figure 5 As shown in the figure, when the silver / silver chloride reference electrode is used for nucleic acid detection, the two curves overlap, indicating that the silver / silver chloride reference electrode inhibits nucleic acid amplification, so it is impossible to qualitatively analyze the nucleic acid through the change time of the electrical signal. However, when the silver reference electrode is used for nucleic acid detection, the electrochemically modified electrode can qualitatively analyze the nucleic acid through the change time of the electrical signal. Figure 6 The electrical signal change time and Figure 7 The peak time of the two detectors remains consistent.

[0130] Example 6: Electrochemically modified electrode prepared in Example 2 is used to detect nucleic acid

[0131] The electrochemically modified electrode prepared in Example 2 was used for nucleic acid detection, and the specific steps were as follows:

[0132] The positive quality control target (concentration of 10 6 copies / μL), 2.5μL of Staphylococcus aureus solution was added to 22.5μL of nucleic acid amplification solution (the nucleic acid amplification solution in the Staphylococcus aureus kit of Qingdao Jianma Gene Technology Co., Ltd.), and no target nucleic acid (NTC) was added as a negative control. Therefore, the final concentration of the target in the reaction solution is: 10 7 copies / mL of Staphylococcus aureus genomic DNA, 0 copies / mL of Staphylococcus aureus genomic DNA.

[0133] There are two groups configured in total;

[0134] A group of them were added to the reference electrode prepared in Example 2, which was a silver electrochemical modified electrode based on a proton sensitive membrane, and heated at 65°C for 40 minutes. The voltage of the working electrode VS the reference electrode was monitored in real time using an electrochemical workstation. The results were as follows: Figure 8 shown.

[0135] Another group used the ND2600 fluorescence quantitative PCR instrument purchased from Qingdao Jianma to test and determine the amplification effect. The results are as follows Fig. 9 shown.

[0136] like Figure 8 As shown in the figure, when the proton sensitive membrane electrochemical modified electrode is used for nucleic acid detection, the negative control's electrical signal remains basically stable within 40 minutes, and the positive control's electrical signal shows an upward trend within 40 minutes. The distinction between positive and negative is obvious, and the nucleic acid can be qualitatively analyzed by the change time of the electrical signal. Figure 8 The electrical signal change time and Fig. 9 The peak onset time of the electrode electrochemically modified with a proton sensitive membrane will be delayed by 5 minutes.

[0137] This shows that the electrochemical electrode based on the proton sensitive membrane provided by the embodiment of the present invention has the advantages of high response degree and high repeatability in the process of detecting nucleic acids.

[0138] Example 7: Electrochemically modified electrode prepared in Example 1 for nucleic acid detection

[0139] The electrochemical electrode prepared in Example 1 and the polyaniline electrochemical electrode without a proton exchange membrane prepared in Comparative Example 2 were used for nucleic acid detection, and the specific steps were as follows:

[0140] The positive quality control target (concentration of 106 copies / μL), the concentration was 10 6 After mixing the genomic DNA of Staphylococcus aureus with deionized water, the concentration was 10 5 copies / μL Staphylococcus aureus genomic DNA, 10 4 copies / μL Staphylococcus aureus genomic DNA, 10 3 copies / μL Staphylococcus aureus genomic DNA, 10 2 copies / μL Staphylococcus aureus genomic DNA.

[0141] Take 2.5 μL of the above Staphylococcus aureus solutions of different concentrations and add them to 22.5 μL of nucleic acid amplification solution (the nucleic acid amplification solution in the Staphylococcus aureus kit of Qingdao Jianma Gene Technology Co., Ltd.), and use the target nucleic acid (NTC) not added as a negative control. Therefore, the final concentration of the target in the reaction solution is: 10 7 copies / mL Staphylococcus aureus genomic DNA, 10 6 copies / mL Staphylococcus aureus genomic DNA, 10 5 copies / mL Staphylococcus aureus genomic DNA, 10 4 copies / mL Staphylococcus aureus genomic DNA, 0 copies / mL Staphylococcus aureus genomic DNA. A total of three groups are configured;

[0142] One group was added to an electrochemical electrode without a proton exchange membrane (the rest of the steps were the same as in Example 1), heated at 65°C for 40 minutes, and the voltage of the working electrode VS the reference electrode was monitored in real time using an electrochemical workstation. The results were as follows: Fig.10 As shown, the data on the vertical axis have been normalized.

[0143] A group was added to the electrochemical electrode based on polyaniline-proton exchange membrane prepared in Example 1, heated at 65°C for 40 minutes, and the voltage of the working electrode VS the reference electrode was monitored in real time using an electrochemical workstation. The results are as follows: Fig.11 As shown, the data on the vertical axis have been normalized.

[0144] Another group used the ND2600 fluorescence quantitative PCR instrument purchased from Qingdao Jianma to test and determine the amplification effect. The results are as follows Fig.12 shown.

[0145] Fig.10 The electrical signal curve is not smooth and often has transitions, which makes it very difficult to conduct qualitative and quantitative analysis of nucleic acids. Fig.10 and Fig.11 The electrical signal curve, Fig.11 The curve is smoother and more stable, and the nucleic acid can be qualitatively and quantitatively analyzed by the change time of the electrical signal. Fig.11 The electrical signal change time and Fig.12 The peak time of the two detectors remains consistent.

[0146] This shows that the electrochemical electrode based on polyaniline-proton exchange membrane provided by the embodiment of the present invention has the advantages of high sensitivity, short time consumption, high repeatability, etc. in the process of detecting nucleic acid.

Claims

1. A modified electrode for electrochemical nucleic acid detection, characterized in that: It comprises a composite electrode substrate and a) or b) modified on the surface of the working electrode in the composite electrode substrate: a) Proton sensitive membrane; b) polyaniline membrane and proton exchange membrane modified on the surface of polyaniline membrane; The composite electrode substrate comprises a working electrode, a reference electrode and a counter electrode.

2. The modified electrode for electrochemical nucleic acid detection according to claim 1, characterized in that: The reference electrode is a silver electrode or a silver / silver chloride electrode; The working electrode is a carbon working electrode; The counter electrode is a carbon counter electrode.

3. The modified electrode for electrochemical nucleic acid detection according to claim 1 or 2, characterized in that: The proton sensitive membrane is made of a proton sensitive substance and a plasticizer; The proton sensitive substance is one or more of trilaurylamine, 4-nonadecanylpyridine, and potassium borate; The plasticizer is one or more of polyvinyl chloride, cyclohexanone, and di-n-octyl sebacate; The mass ratio of the proton sensitive substance to the plasticizer is 1:(10000-20000); The thickness of the proton sensitive membrane is 50 μm to 200 μm.

4. The modified electrode for electrochemical nucleic acid detection according to claim 1 or 2, characterized in that: The proton exchange membrane is a Nafion membrane; The thickness of the proton exchange membrane is 5 μm to 500 μm.

5. The method for preparing a modified electrode for electrochemical nucleic acid detection according to any one of claims 1 to 4, characterized in that: It is the following method A) or method B): The method A) comprises: Performing a film-forming treatment on the surface of the working electrode in the composite electrode using a proton sensitive membrane solution to obtain the modified electrode for electrochemical nucleic acid detection; The method B) comprises: B1) electrochemically depositing a solution containing aniline on the composite electrode substrate to obtain the polyaniline film; B2) forming a film on the surface of a working electrode modified with a polyaniline membrane by subjecting a solution of a proton exchange membrane to obtain the modified electrode for electrochemical nucleic acid detection.

6. The method for preparing a modified electrode for electrochemical nucleic acid detection according to claim 5, characterized in that: The proton sensitive membrane solution is composed of trilaurylamine, potassium borate, polyvinyl chloride, di-n-octyl sebacate and cyclohexanone; The concentration of trilaurylamine in the ion-sensitive membrane solution is 3 mM to 6 mM; The concentration of potassium borate in the ion-sensitive membrane solution is 2 mM to 4 mM; In terms of g / mL, the concentration of the polyvinyl chloride in the ion-sensitive membrane is 5% to 10%; The concentration of di-n-octyl sebacate in the ion-sensitive membrane is 0.1M to 0.6M; The proton sensitive membrane is formed by drop coating method.

7. The method for preparing a modified electrode for electrochemical nucleic acid detection according to claim 5, characterized in that: The solution containing aniline consists of an aqueous solution of aniline and an inorganic acid; The concentration of the aniline is 0.01M to 4M, and the concentration of the inorganic acid is 0.1M to 3M; The electrochemical deposition was carried out by cyclic voltammetry under the following conditions: The initial potential was -0.5 V to 0 V, the end potential was 0.8 V to 1.5 V, and the scan rate was 0.01 V s -1 ~0.5V s -1 , the static time is 1s to 10s, and the number of scans is 5 to 50 circles; The solution of the proton exchange membrane is a Nafion solution; The proton exchange membrane is formed by using a drop coating method; The mass concentration of the Nafion solution is 0.5% to 5%.

8. Use of the modified electrode for electrochemical nucleic acid detection according to any one of claims 1 to 4 in detecting proton concentration and / or detecting nucleic acid.

9. A method for electrochemical detection of nucleic acid, characterized in that: The following method P1 and / or method P2: Method P1 comprises the following steps: Amplifying the nucleic acid, and simultaneously monitoring the voltage of the working electrode VS the reference electrode in the amplification system using the modified electrode for electrochemical nucleic acid detection according to any one of claims 1 to 4, and monitoring the amplification reaction in real time according to the change of the voltage over time; Method P2 comprises the following steps: Amplify nucleic acids of different concentrations respectively, and simultaneously monitor the voltage of the working electrode VS the reference electrode in the amplification system using the modified electrode for electrochemical nucleic acid detection according to any one of claims 1 to 4, and record the curve of the voltage change over time in the nucleic acid amplification reaction of different concentrations; The nucleic acid is quantitatively analyzed according to the curve of voltage variation over time in the nucleic acid amplification reaction of different concentrations.

10. A kit for detecting nucleic acid, characterized in that: It contains the modified electrode for electrochemical nucleic acid detection described in any one of claims 1-4.

Images