Environmental microorganism nucleic acid electrochemical biosensing quantitative detection method based on nano-channel

Abstract

The invention relates to the technical field of electrochemical biosensors, and discloses an environmental microorganism nucleic acid electrochemical biosensing quantitative detection method based on a nanochannel, which comprises the following steps: filtering at normal pressure to obtain a MoS2-nanochannel, designing a target nucleic acid specific recognition probe, and preparing a biological recognition element and a carbon nanotube modified electrode. And constructing the electrochemical biosensor. In the electrochemical detection process, the distinguishable single-chain and double-chain nucleic acid characteristic of MoS2 is combined with a specific recognition probe, so that the anti-interference capability and selectivity of the detection method are improved; the sensitivity of the detection method is improved by utilizing the diffusion process of signal molecules in the nano-channel porous vertical array and a dual signal amplification strategy of the carbon nanotube modified electrode; the detection method provided by the invention overcomes the defects of expensive instrument and equipment, tedious operation process and high sample purity requirement of a traditional environmental microorganism nucleic acid quantitative detection method, and realizes the effect of sensitively detecting trace nucleic acid in a field complex environmental sample.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and in particular to a nanochannel-based electrochemical biosensor rapid detection method for environmental microbial nucleic acids. Background Art

[0002] Environmental microbial nucleic acids can assess ecosystem microbial diversity, monitor target microbial groups, and directly reveal microbial metabolic functions. They are important targets for ecological and environmental research, public health, and microbial technology. Therefore, environmental nucleic acid detection technology has important applications in ecological monitoring and protection, environmental remediation, and other fields. However, nucleic acid content in environmental samples is typically extremely low, environmental RNA is easily degraded, and the presence of complex interfering substances such as humic acid, proteins, and polysaccharides places extremely high demands on the sensitivity, specificity, and anti-interference capabilities of detection technologies. While traditional detection methods can achieve high sensitivity for specific nucleic acids under laboratory conditions, they rely on sophisticated instrumentation and complex sample pretreatment steps, making them difficult to meet the requirements of rapid field testing and may fail to detect trace amounts of target nucleic acids. Furthermore, quantitative analysis based on nucleic acid copy number is susceptible to environmental factors (such as temperature, pH fluctuations, and ionic strength), leading to biased quantitative results. Therefore, the development of new rapid field nucleic acid detection methods with high sensitivity and environmental stability is needed to overcome the limitations of existing technologies in field application and nucleic acid quantification.

[0003] Electrochemical biosensing technology, with its advantages of high sensitivity, rapid response, and low cost, has shown great potential in the field of nucleic acid detection. This technology typically utilizes probes binding to target nucleic acids, converting the biorecognition process into a measurable electrochemical signal (such as current, voltage, or impedance change), enabling quantitative detection of nucleic acids without the need for real-time fluorescence quantitative polymerase chain reaction (qPCR). Traditional electrochemical biosensing technology faces two technical challenges when detecting complex environmental samples: first, interfering substances in the sample easily bind to nonspecific probes, generating significant background signal interference; second, the limited mass transfer efficiency of the biosensor interface based on the working electrode makes it difficult to achieve ultra-high sensitivity detection.

[0004] Nanochannel technology is a novel detection method based on nanoscale pores. It offers advantages such as high resolution, no labeling, and real-time monitoring, making it suitable for quantitative analysis of nucleic acids in complex environmental samples. Two-dimensional nanomaterials such as graphene and transition metal dihalides are widely used in biosensing analysis due to their large surface area and high electrical conductivity. Transition metal dihalides such as MoS2 nanosheets also have the ability to distinguish between single-stranded and double-stranded nucleic acids, exhibiting strong adsorption for single-stranded nucleic acids and very weak adsorption for double-stranded nucleic acids. Combining nanochannels with MoS2 nanosheets to create nanochannels can imbue the nanochannels with the ability to distinguish between single-stranded and double-stranded nucleic acids, further improving their detection performance.

[0005] Many existing detection methods rely on qPCR for quantitative detection. Current mainstream high-sensitivity detection methods, such as sequencing technology and mass spectrometry, require expensive equipment and specialized operating procedures, making them difficult to promote and apply in resource-limited areas or for on-site testing. Furthermore, existing detection methods are mostly designed for specific target nucleic acids and lack the flexibility to achieve multi-target detection and improve methods by simply replacing recognition elements. Therefore, it is extremely necessary to find a method that can still accurately detect microbial nucleic acids in the environment in resource-limited areas or for on-site testing. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acids that overcomes the limitations of a single technology, combines electrochemical biosensor technology with nanochannels, and gives full play to the advantages of both. This method can not only significantly improve the detection sensitivity, but also effectively reduce the influence of complex interfering substances in environmental samples, thereby improving the selectivity and accuracy of detection.

[0007] In order to solve the above technical problems, the technical solution of the present invention is: The nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acids comprises the following steps: Step 1, preparing MoS2-nanochannels; Step 2: preparing a biorecognition element and biorecognition element samples with different copy numbers of target nucleic acid; Step 3, constructing an electrochemical biosensor; Step 4: applying voltage through an electrochemical workstation, and electrochemically detecting and recording the current response I0 of the electrochemical biosensor when detecting the biorecognition element, as well as the current response I when detecting biorecognition element specimens with different copy numbers of the target nucleic acid; and plotting a standard curve with the Lg value of the target nucleic acid copy number as the horizontal axis and the current change value ΔI=I-I0 as the horizontal axis; Step 5: Take the target nucleic acid sample to be detected in the field and perform simple cell lysis to prepare the biorecognition element sample to be detected. Use an electrochemical method to detect and record the current response I1 of the electrochemical biosensor when detecting the biorecognition element sample, and substitute ΔI=I1-I0 into the standard curve in step 4 to calculate the copy number of the target nucleic acid to be detected.

[0008] As a preferred technical solution, the step 1 is specifically as follows: Step 1a, preparing a MoS2 nanosheet dispersion; Step 1b, washing the nanochannel with ethanol and deionized water in sequence, and drying; Step 1c: Take 40 mg of chitosan and 8 mL of 0.1 mol·L -1 Acetic acid was stirred in the dark at room temperature for 1 h to obtain a 5% chitosan dispersion; Step 1d, take 100 to 500 μL of the MoS2 nanosheet dispersion in step 1a and the chitosan dispersion in step 1c, mix the two in a volume ratio of 20:1, and rapidly oscillate to obtain a mixed solution; fix the nanochannel on filter paper, drop the mixed solution on the surface of the nanochannel, and use a normal pressure filtration process to retain the MoS2 nanosheets in the mixed solution on the outer surface of the nanochannel to achieve solid-liquid separation; after normal pressure filtration, dry at room temperature to obtain the MoS2-nanochannel.

[0009] As a preferred technical solution, the step 1a is specifically as follows: In step 1aa, thiourea and ammonium molybdate powders were weighed, with a final atomic ratio of Mo:S of 1:4, added to deionized water and vigorously stirred until a blue dispersion was obtained. The blue dispersion was poured into a polytetrafluoroethylene liner, which was then placed in a reactor and reacted at 200° C. for 12 hours. Step 1ab: After the reaction is completed, the product is centrifuged and washed with ethanol and deionized water, respectively, and dried at 70° C. for 8 h to obtain a black solid, which is ground into a black powder with crystals, wherein the black powder is MoS2 nanosheets; Step 1ac: add the MoS2 nanosheets to N,N-dimethylformamide and ultrasonicate for 30 min until the MoS2 nanosheets are uniformly dispersed to obtain the MoS2 nanosheet dispersion. The concentration of the MoS2 nanosheet dispersion is 1 mg mL -1 .

[0010] As a preferred technical solution, the step 2 is specifically as follows: Preparation of biorecognition elements: The DNA probe solution that is complementary to the target nucleic acid was centrifuged appropriately, and the DNA probe solution was added to TE buffer for dilution and mixed thoroughly to obtain 1µmol·L -1 The DNA probe dilution solution is directly drop-coated on the outer surface of the MoS2-nanochannel with 100 μL of the DNA probe dilution solution, and the mixture is incubated at 4°C for 12 hours to obtain the biorecognition element; Preparation of biorecognition element specimens: 5µL of target gene standard solution was taken respectively, mixed with TE buffer solution to prepare multiple standard solutions with different concentration gradients, and placed in a 95°C environment for 2 minutes for denaturation, then cooled to 65°C, and drop-coated on the biorecognition element at 65°C for incubation. After incubation, it was gently rinsed with deionized water and dried at room temperature to obtain multiple biorecognition element specimens.

[0011] As a preferred technical solution, the sequence of the DNA probe is a partial complementary sequence of the target nucleic acid. The target nucleic acid sequence is searched on the NCBI website, and a series of sequences are compared in ClustalX2 software. The overlapping sequence parts are found upstream, downstream, and midstream of the target nucleic acid sequence, and the complementary sequence of the partially overlapping sequence is designed as the DNA probe. The number of DNA probes can be selected from 1 to 5.

[0012] As a preferred technical solution, the step three is as follows: Step 3a: N,N-dimethylformamide and the chitosan dispersion prepared in step 1c were mixed in a volume ratio of 4:1 to obtain a dispersion. The carbon nanotube powder was added to the dispersion and ultrasonicated until uniform dispersion was obtained. -1 A carbon nanotube modified material; drop-coating the carbon nanotube modified material on the surface of a glassy carbon electrode and drying at room temperature to obtain the carbon nanotube modified electrode; Step 3b: add 5 mmol·L -1 0.1 mol·L of K3[Fe(CN)6] -1 KCl solution, add 0.1 mol·L -1 The biorecognition element is fixed in the middle of the H-type electrolytic cell with one side of the MoS2-nanochannel-modified MoS2 nanosheet facing left; In step 3c, a three-electrode system is used as the detection system. A platinum wire electrode and a calomel electrode are placed in the left cell as the counter electrode and the reference electrode, respectively. The carbon nanotube-modified electrode is placed in the right cell as the working electrode. The electrochemical biosensor is obtained by free diffusion using the concentration difference of the electrolyte on both sides.

[0013] As a preferred technical solution, the steps for preparing the biometric recognition element sample in step 5 are as follows: When the target nucleic acid is a target DNA, an environmental sample is taken, the cells are lysed, and then centrifuged to obtain a supernatant, which is then collected into a centrifuge tube to obtain a mixed solution containing the target DNA. The mixed solution containing the target DNA is thoroughly mixed with TE buffer, first placed in a 95°C environment for 2 minutes to melt, then cooled to 65°C, and 100 μL is dropwise applied to the biorecognition element at 65°C for incubation. After the incubation is completed, it is gently rinsed with deionized water and dried at room temperature to obtain the biorecognition element specimen to be tested; When the target nucleic acid is target RNA, an environmental sample is taken, the cells are lysed, and the sample is centrifuged to obtain an upper aqueous phase, which is then collected into a centrifuge tube to obtain a mixed solution containing the target RNA. The mixed solution containing the target RNA is thoroughly mixed with TE buffer, and 100 μL is dropwise applied to the biorecognition element at 4°C for incubation. After the incubation is completed, the sample is gently rinsed with deionized water and dried at room temperature to obtain a specimen of the biorecognition element to be tested.

[0014] As a preferred technical solution, the electrochemical method in step 4 and step 5 includes differential pulse voltammetry, cyclic sweep voltammetry or impedance voltammetry.

[0015] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: MoS2-nanochannels are obtained by normal pressure filtration, specific recognition probes for target nucleic acids are designed to prepare biorecognition elements, which are then integrated with carbon nanotube modified electrodes to form electrochemical biosensors, thereby combining nanochannels with electrochemical biosensor technology to give full play to the advantages of both. In the electrochemical detection process, the ability of MoS2 to distinguish between single and double-stranded nucleic acids is combined with specific recognition probes, thereby improving the anti-interference ability and selectivity of the detection method; utilizing the diffusion process of signal molecules in the porous vertical array of nanochannels and the dual signal amplification strategy of carbon nanotube modified electrodes, it converts the changes generated by biorecognition into the free diffusion flux of electrochemical signal molecules, which can not only significantly improve the detection sensitivity, but also effectively reduce the influence of complex interfering substances in environmental samples, thereby improving the selectivity and accuracy of detection, and also improving the sensitivity of the detection method. The electrochemical biosensor detection method overcomes the shortcomings of traditional nucleic acid quantitative detection methods, such as expensive instruments and equipment, cumbersome operating procedures, and high sample purity requirements, thereby achieving the effect of rapid and sensitive detection of trace nucleic acids in complex environmental samples in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings are intended only to illustrate and explain the present invention and are not intended to limit the scope of the present invention. Figure 1It is a detection principle diagram of the present invention; Figure 2 This is a diagram showing the effect of the present invention using differential pulse voltammetry for detection; Figure 3 This is a diagram showing the effect of the present invention using cyclic sweep voltammetry for detection; Figure 4 This is a diagram showing the effect of the impedance method used in the present invention; Figure 5 This is a comparison chart of the volumes of materials modified with different carbon nanotubes used in the present invention; Figure 6 This is a comparison chart of different MoS2 nanosheet dispersion volumes used in the present invention; Figure 7 This is a comparison chart of different target nucleic acid incubation times used in the present invention; Figure 8 This is a comparison diagram of different numbers of DNA probes used in the present invention; Figure 9 is the current response curve of the biological recognition element specimens of the present invention with different copy numbers of the target nucleic acid; Figure 10 is the standard curve of differential pulse voltammetry for target nucleic acid established by the present invention; Figure 11 This is the result of the electrochemical biosensing method of the present invention detecting a simple cleavage DNA solution; Figure 12 This is a comparison chart of the time and cost of detecting target DNA using the electrochemical biosensor method of the present invention and the qPCR method; Figure 13 This is the result of the electrochemical biosensing method of the present invention detecting a simple cleavage RNA solution; Figure 14 This is a comparison chart of the time and cost of detecting target RNA using the electrochemical biosensor method of the present invention and the qPCR method. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the accompanying drawings and examples. In the following detailed description, certain exemplary embodiments of the present invention are described by way of illustration only. It is understood that those skilled in the art will recognize that the described embodiments may be modified in various ways without departing from the spirit and scope of the present invention. Therefore, the drawings and description are illustrative in nature and are not intended to limit the scope of the claims.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] Example 1: The target nucleic acid in this example is target DNA, with cytochrome cd1 type nitrite reductase gene ( nirS ) as an example to illustrate; Nanochannel-based electrochemical biosensing quantitative detection method for environmental microbial nucleic acids, such as Figure 1 As shown, the following steps are included: Step 1, preparing MoS2-nanochannels; The step 1 is specifically as follows: Step 1a, preparing a MoS2 nanosheet dispersion; The step 1a is specifically as follows: In step 1aa, thiourea and ammonium molybdate powders were weighed, with the final atomic ratio of thiourea and ammonium molybdate being 1:4, and added to deionized water to obtain 0.065 g mL -1 Thiourea and 0.035 g·mL -1 The mixed solution of ammonium molybdate was vigorously stirred until a blue dispersion was obtained, and the blue dispersion was poured into a polytetrafluoroethylene liner, and then the polytetrafluoroethylene liner was placed in a reactor and reacted at 200°C for 12 hours; Step 1ab: After the reaction is completed, the product is centrifuged and washed with ethanol and deionized water, respectively, and dried at 70° C. for 8 h to obtain a black solid, which is ground into a black powder with crystals, wherein the black powder is MoS2 nanosheets; Step 1ac: add the MoS2 nanosheets to N,N-dimethylformamide and ultrasonicate for 30 min until the MoS2 nanosheets are uniformly dispersed to obtain the MoS2 nanosheet dispersion. The concentration of the MoS2 nanosheet dispersion is 1 mg mL -1 .

[0021] Step 1b, washing the nanochannel with ethanol and deionized water in sequence, and drying; Step 1c: Take 40 mg of chitosan and 8 mL of 0.1 mol·L -1 Acetic acid was stirred in the dark at room temperature for 1 h to obtain a 5% chitosan dispersion; Step 1d: Take 300 µL of the MoS2 nanosheet dispersion in step 1a, and then take 15 µL of the chitosan dispersion in step 1c, mix the two, and rapidly oscillate to obtain a mixed solution; fix the nanochannel on filter paper, drop the mixed solution on the surface of the nanochannel, and use a normal pressure filtration process to retain the MoS2 nanosheets in the mixed solution on the outer surface of the nanochannel to achieve solid-liquid separation; after normal pressure filtration, dry at room temperature to obtain the MoS2-nanochannel.

[0022] Step 2: preparing a biorecognition element and biorecognition element samples with different copy numbers of target nucleic acid; The step 2 is specifically as follows: Preparation of biorecognition elements: Target DNA nirS For example, search for genes in the NCBI database nirS Gene sequences were obtained, and a series of sequences (such as MK178639.1, MF773686.1, MF776399.1, MF776495.1, KT934768.1, MF776458.1, MF178650.1, and MF776464.1) were compared in ClustalX2 software. nirS The overlapping sequence parts are found in the upstream, midstream and downstream of the gene sequence, and the complementary sequences of the partially overlapping sequences are designed as DNA probes. The number of DNA probes can be 1 to 5. In this embodiment, the number of DNA probes is 2, and the sequences are: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' 100µmol·L -1 The DNA probes P1 and P2 solutions were centrifuged appropriately and 1 μL of each solution was added to 98 μL of TE buffer for dilution and mixed thoroughly to obtain 1 μmol·L -1 The DNA probe dilution solution was directly drop-coated on the outer surface of the MoS2-nanochannel with 100 μL of the DNA probe dilution solution, and the mixture was incubated at 4° C. for 12 h to obtain the biorecognition element.

[0023] Preparation of biorecognition element specimens: Take 5µL of nirS The gene standard solution, mixed with TE buffer solution, has a copy number of 6 copies ~ 1.31 × 10 10 This process is conventional. In this example, 12 standard solutions of different copy numbers were prepared, each of which was placed in a 95°C environment for 2 minutes to melt, then cooled to 65°C. Each solution was drop-coated on the electrochemical biorecognition element and incubated at 65°C for 10 minutes. After incubation, the solution was gently rinsed with deionized water and dried at room temperature to obtain 12 biorecognition element specimens. The number of biorecognition element specimens is not limited to 12, and other numbers can also be selected.

[0024] Step 3, constructing an electrochemical biosensor; The step three is as follows: Step 3a: N,N-dimethylformamide and the chitosan dispersion prepared in step 1c were mixed in a volume ratio of 4:1 to obtain a dispersion. The carbon nanotube powder was added to the dispersion and ultrasonicated until uniform dispersion was obtained. -1 The carbon nanotube modified material is drop-coated on the surface of the glassy carbon electrode with 2 μL of the carbon nanotube modified material, and dried at room temperature to obtain the carbon nanotube modified electrode; Step 3b: add 5 mmol·L -1 0.1 mol·L of K3[Fe(CN)6] -1 KCl solution, add 0.1 mol·L -1 The biorecognition element is fixed in the middle of the H-type electrolytic cell with one side of the MoS2-nanochannel-modified MoS2 nanosheet facing left; In step 3c, a three-electrode system is used as the detection system. A platinum wire electrode and a calomel electrode are placed in the left cell as the counter electrode and the reference electrode, respectively. The carbon nanotube-modified electrode is placed in the right cell as the working electrode. The electrochemical biosensor is obtained by free diffusion using the concentration difference of the electrolyte on both sides.

[0025] Step 4: applying voltage through an electrochemical workstation, and electrochemically detecting and recording the current response I0 of the electrochemical biosensor when detecting the biorecognition element, as well as the current response I when detecting biorecognition element specimens with different copy numbers of the target nucleic acid; and plotting a standard curve with the Lg value of the target nucleic acid copy number as the horizontal axis and the current change value ΔI=I-I0 as the horizontal axis; According to step 2, biological recognition element samples with different copy numbers of target nucleic acid are prepared respectively, such as Figure 9 As shown, with nirS As the number of gene copies increases, the peak current I gradually increases, showing a good regularity. nirS The relationship between the copy number of the gene standard solution and the peak current change value is plotted as nirS The Lg value of the gene standard solution copy number, and the vertical axis is the standard curve of the current change value ΔI=I-I0; Figure 10 As shown, the standard curve has a good linear relationship, and its equation is: ΔI=0.608Lg(N / copies)+4.026, (R 2 =0.9997).

[0026] Step 5: Take 0.4g of the sample to be tested in the field nirSGene sediment samples were used to prepare biorecognition element samples to be tested. In this embodiment, four biorecognition element samples were prepared, with six samples in parallel for each sample. The DNA was purified by simple lysis and centrifugation. The supernatant was transferred to a centrifuge tube to obtain the sample containing nirS Gene mixture (if not tested immediately, store the mixture at -80℃ or in liquid nitrogen). Take 5µL of nirS The gene mixture is thoroughly mixed with 95 μL of TE buffer, first placed in a 95°C environment for 2 minutes for denaturation, then cooled to 65°C, and drop-coated on the electrochemical biorecognition element at 65°C for incubation for 10 minutes. After the incubation, it is gently rinsed with deionized water and dried at room temperature to obtain the biorecognition element sample to be detected; the biorecognition element samples are sequentially placed in the electrochemical biosensor. The electrochemical method selected in this embodiment is differential pulse voltammetry. The current response I1 of the electrochemical biosensor when detecting the biorecognition element sample is detected and recorded by differential pulse voltammetry, and ΔI=I1-I0 is substituted into the standard curve in step 4 to calculate the copy number of the target nucleic acid to be detected.

[0027] According to the standard curve, the four biorecognition element samples were calculated. nirS Gene copy number N / copies (N=10 (ΔI-4.026) / 0.608 ), thereby achieving nirS Quantitative detection of genes.

[0028] The test results are as follows Figure 11 As shown in Figure 2, the final copy number calculation results were well consistent with those of traditional qPCR, with an average relative deviation of 5.67%. The results of the two methods were highly correlated (R 2 =0.9970), which shows that the electrochemical detection method of this embodiment is comparable to the traditional qPCR detection method. Figure 12 As shown, the DNA solution obtained by simple lysis and centrifugation of the environmental sample can be directly incubated on the biorecognition element and the target DNA can be electrochemically detected. Figure 12 Figure A uses the traditional qPCR detection method, which takes 5 hours and 42 minutes and costs 210 yuan. Figure 12 Figure B in the figure uses the electrochemical detection method of the present invention, which takes 56 minutes and costs 90 yuan. This shows that the detection method of this embodiment greatly shortens the complex operation process of qPCR and reduces costs.

[0029] Example 2: The target nucleic acid in this example is target RNA. nirS Let’s take genes as an example to illustrate; Nanochannel-based electrochemical biosensing quantitative detection method for environmental microbial nucleic acids, such as Figure 1 As shown, the following steps are included: Step 1, preparing MoS2-nanochannels; The step 1 is specifically as follows: Step 1a, preparing a MoS2 nanosheet dispersion; The step 1a is specifically as follows: In step 1aa, thiourea and ammonium molybdate powders were weighed, with the final atomic ratio of thiourea and ammonium molybdate being 1:4, and added to deionized water to obtain 0.065 g mL -1 Thiourea and 0.035 g·mL -1 The mixed solution of ammonium molybdate was vigorously stirred until a blue dispersion was obtained, and the blue dispersion was poured into a polytetrafluoroethylene liner, and then the polytetrafluoroethylene liner was placed in a reactor and reacted at 200°C for 12 hours; Step 1ab: After the reaction is completed, the product is centrifuged and washed with ethanol and deionized water, respectively, and dried at 70° C. for 8 h to obtain a black solid, which is ground into a black powder with crystals, wherein the black powder is MoS2 nanosheets; Step 1ac: add the MoS2 nanosheets to N,N-dimethylformamide and ultrasonicate for 30 min until the MoS2 nanosheets are uniformly dispersed to obtain the MoS2 nanosheet dispersion. The concentration of the MoS2 nanosheet dispersion is 1 mg mL -1 .

[0030] Step 1b, washing the nanochannel with ethanol and deionized water in sequence, and drying; Step 1c: Take 40 mg of chitosan and 8 mL of 0.1 mol·L -1 Acetic acid was stirred in the dark at room temperature for 1 h to obtain a 5% chitosan dispersion; Step 1d: Take 300 µL of the MoS2 nanosheet dispersion in step 1a, and then take 15 µL of the chitosan dispersion in step 1c, mix the two, and rapidly oscillate to obtain a mixed solution; fix the nanochannel on filter paper, drop the mixed solution on the surface of the nanochannel, and use a normal pressure filtration process to retain the MoS2 nanosheets in the mixed solution on the outer surface of the nanochannel to achieve solid-liquid separation; after normal pressure filtration, dry at room temperature to obtain the MoS2-nanochannel.

[0031] Step 2: preparing a biorecognition element and biorecognition element samples with different copy numbers of target nucleic acid; The step 2 is specifically as follows: Preparation of biorecognition elements: Target RNA nirS For example, search for genes in the NCBI database nirSGene sequences were obtained, and a series of sequences (MK178639.1, MF773686.1, MF776399.1, MF776495.1, KT934768.1, MF776458.1, MF178650.1, MF776464.1) were compared in ClustalX2 software. nirS The overlapping sequence parts are found in the upstream, midstream and downstream of the gene sequence, and the complementary sequences of the partially overlapping sequences are designed as DNA probes. The number of DNA probes can be 1 to 5. In this embodiment, the number of DNA probes is 2, and the sequences are: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' 100µmol·L -1 The DNA probes P1 and P2 were centrifuged appropriately, and 1 μL of each solution was added to 98 μL of TE buffer for dilution and mixed thoroughly to obtain 1 μmol·L -1 The DNA probe dilution solution was directly drop-coated on the outer surface of the MoS2-nanochannel with 100 μL of the DNA probe dilution solution, and the mixture was incubated at 4° C. for 12 h to obtain the biorecognition element.

[0032] Preparation of biorecognition element specimens: Take 5µL of nirS The gene standard solution, mixed with TE buffer solution, has a copy number of 6 copies ~ 1.31 × 10 10 This process is conventional. In this example, 12 standard solutions of different copy numbers were prepared, each of which was placed in a 95°C environment for 2 minutes to melt, then cooled to 65°C. Each solution was drop-coated on the electrochemical biorecognition element and incubated at 65°C for 10 minutes. After incubation, the solution was gently rinsed with deionized water and dried at room temperature to obtain 12 biorecognition element specimens. The number of biorecognition element specimens is not limited to 12, and other numbers can also be selected.

[0033] Step 3, constructing an electrochemical biosensor; The step three is as follows: Step 3a: N,N-dimethylformamide and the chitosan dispersion prepared in step 1c were mixed in a volume ratio of 4:1 to obtain a dispersion. The carbon nanotube powder was added to the dispersion and ultrasonicated until uniform dispersion was obtained. -1The carbon nanotube modified material is drop-coated on the surface of the glassy carbon electrode with 2 μL of the carbon nanotube modified material, and dried at room temperature to obtain the carbon nanotube modified electrode; Step 3b: add 5 mmol·L -1 0.1 mol·L of K3[Fe(CN)6] -1 KCl solution, add 0.1 mol·L -1 The biorecognition element is fixed in the middle of the H-type electrolytic cell with one side of the MoS2-nanochannel-modified MoS2 nanosheet facing left; In step 3c, a three-electrode system is used as the detection system. A platinum wire electrode and a calomel electrode are placed in the left cell as the counter electrode and the reference electrode, respectively. The carbon nanotube-modified electrode is placed in the right cell as the working electrode. The electrochemical biosensor is obtained by free diffusion using the concentration difference of the electrolyte on both sides.

[0034] Step 4: applying voltage through an electrochemical workstation, and electrochemically detecting and recording the current response I0 of the electrochemical biosensor when detecting the biorecognition element, as well as the current response I when detecting biorecognition element specimens with different copy numbers of the target nucleic acid; and plotting a standard curve with the Lg value of the target nucleic acid copy number as the horizontal axis and the current change value ΔI=I-I0 as the horizontal axis; According to step 2, biological recognition element samples with different copy numbers of target nucleic acid are prepared respectively, such as Figure 9 As shown, with nirS As the number of gene copies increases, the peak current I gradually increases, showing a good regularity. nirS The relationship between the copy number of the gene standard solution and the peak current change value is plotted as nirS The Lg value of the gene standard solution copy number, and the vertical axis is the standard curve of the current change value ΔI=I-I0; Figure 10 As shown, the standard curve has a good linear relationship, and its equation is: ΔI=0.608Lg(N / copies)+4.026, (R 2 =0.9997).

[0035] Step 5: Take 5g of the sample to be tested in the field nirS The gene sediment sample was simply lysed and centrifuged using an RNA extraction kit, and the upper aqueous phase was transferred to a centrifuge tube to obtain the RNA containing nirS Gene mixture (if not tested immediately, it will contain nirSThe mixture is stored frozen at -80°C or in liquid nitrogen. After thoroughly mixing 5µL of the target RNA mixture with 95µL of TE buffer at 4°C, four biorecognition element samples are prepared, with six replicates of each sample. Each sample is drop-coated onto the biorecognition element and incubated at 4°C for 10 minutes. After the incubation period, the sample is gently rinsed with deionized water and dried at room temperature to obtain the biorecognition element samples to be tested. The biorecognition element samples are then sequentially placed into the electrochemical biosensor. In this embodiment, differential pulse voltammetry is used as the electrochemical method. The current response I1 of the electrochemical biosensor when testing the biorecognition element samples is sequentially detected and recorded using differential pulse voltammetry. ΔI = I1 - I0 is then substituted into the standard curve in step 4 to calculate the copy number of the target RNA to be tested.

[0036] According to the standard curve, the four biorecognition element samples were calculated. nirS Gene copy number N / copies (N=10 (ΔI-4.026) / 0.608 ), thereby achieving nirS Quantitative detection of genes.

[0037] Environmental samples were simply lysed and centrifuged, and electrochemical biosensing methods were able to detect nirS The electrochemical signal changes of genes. Test results are as follows Figure 13 As shown in Figure 2, the final copy number calculation results were highly consistent with those of traditional qPCR, with an average relative deviation of 3.91%. The results of the two methods showed a significant correlation (R 2 =0.9994), which shows that the electrochemical detection method of this embodiment is comparable to the traditional qPCR detection method. Figure 14 As shown, the RNA solution obtained by simple lysis and centrifugation of environmental samples can be directly incubated on the biorecognition element and electrochemically detected. Figure 14 Figure A uses the traditional qPCR detection method, which takes 8 hours and 42 minutes and costs 375 yuan. Figure 14 Figure B in the figure uses the electrochemical detection method of the present invention, which takes 61 minutes and costs 125 yuan. The detection method of this embodiment greatly shortens the complex operation process of qPCR and reduces costs.

[0038] Example 3: Selection of electrochemical method This embodiment is basically the same as the technical solution of the first embodiment, except that the electrochemical method is different. The details are as follows: (1) The electrochemical method selected differential pulse voltammetry, the nanochannel, MoS2-nanochannel, biorecognition element, and biorecognition element specimen were fixed in the middle of an H-type electrolytic cell, and the current response of the electrochemical biosensor was detected and recorded in sequence; the results were as follows: Figure 2 As shown in FIG, as the process of functionalizing the nanochannel and preparing the biorecognition element progresses, the current response of the differential pulse voltammetry gradually decreases, indicating that the biorecognition element is successfully prepared; nirS After the gene is detected, the differential pulse voltammetry signal response of the biorecognition element sample is increased compared with that of the biorecognition element, indicating that the DNA probe has successfully recognized nirS Gene, the complementary duplex desorbs away from the biological recognition element.

[0039] (2) The electrochemical method selected cyclic sweep voltammetry, the nanochannel, MoS2-nanochannel, biorecognition element, and biorecognition element specimen were fixed in the middle of an H-type electrolytic cell, and the current response of the electrochemical biosensor was detected and recorded in turn; the results were as follows: Figure 3 As shown in FIG, as the process of functionalizing the nanochannel and preparing the biorecognition element proceeds, the current peak of the cyclic sweep voltammetry gradually decreases, indicating that the biorecognition element is successfully prepared; nirS After the gene is generated, the peak current of the cyclic scanning voltammetry of the biorecognition element sample obtained is increased compared with that of the biorecognition element, indicating that the DNA probe has successfully recognized nirS Gene, the complementary duplex desorbs away from the biological recognition element.

[0040] (3) The electrochemical method selected the impedance method, and the nanochannel, MoS2-nanochannel, biorecognition element, and biorecognition element specimen were fixed in the middle of the H-type electrolytic cell, and the current response of the electrochemical biosensor was detected and recorded in turn; the results were as follows: Figure 4 As shown in FIG, as the process of functionalizing the nanochannel and preparing the biorecognition element proceeds, the steric hindrance effect of the outer surface of the nanochannel gradually increases, so the impedance value of the impedance method gradually increases, indicating that the biorecognition element is successfully prepared; nirS After the gene, the impedance value of the biological recognition element sample obtained is lower than that of the biological recognition element, indicating that the DNA probe has successfully recognized nirS The complementary double strands of genes are desorbed and leave the biorecognition element, and the steric hindrance on the nanochannel surface is correspondingly reduced.

[0041] A preliminary feasibility analysis of the electrochemical biosensor revealed that several conventional electrochemical methods are capable of quantitatively detecting nucleic acids. In contrast, differential pulse voltammetry (DPV) maintains high sensitivity while maintaining a relatively fast detection speed, producing more distinct and easily discernible changes in the current response before and after biorecognition. This results in a higher signal-to-noise ratio and more accurate quantitative analysis capabilities when detecting target nucleic acids at varying concentrations. Therefore, DPV is preferred as the core detection method in the present invention.

[0042] Example 4: Selection of Volume of Carbon Nanotube Modified Material This embodiment is basically the same as the technical solution of the first embodiment, the difference being the volume of the carbon nanotube modified material. The details are as follows: In step 3a, 0 µL, 1 µL, 2 µL, 4 µL, and 6 µL of the carbon nanotube modified material were drop-coated on the surface of the glassy carbon electrode, respectively. The remaining steps were the same as in Example 1, and the current change values of different modified electrode volumes were obtained.

[0043] The results are as follows Figure 5 As shown in the figure, as the volume of the carbon nanotube-modified material gradually increases, the current peak change ΔI before and after the biorecognition process first increases and then decreases, which indicates that the high-intensity accumulation of carbon nanotubes is not conducive to electron transfer on the electrode surface. Therefore, 2µL is the optimal modification volume.

[0044] Example 5: Selection of MoS2 nanosheet dispersion volume This embodiment is essentially the same as the first embodiment, except that the volume of the MoS2 nanosheet dispersion is different. The details are as follows: The volumes of the MoS2 nanosheet dispersion in step 1d were 100µL, 200µL, 300µL, 400µL and 500µL respectively; correspondingly, the volumes of the 5% chitosan dispersion were 5µL, 10µL, 15µL, 20µL and 25µL respectively. The remaining steps were the same as in Example 1, and the current peak values of different volumes were obtained. Figure 6 As shown in the figure, as the functionalized volume of MoS2 nanosheet dispersion gradually increases, there is no obvious pattern in the current peak change ΔI before and after the biorecognition process. Except for 100µL, the current peak changes obtained in the other four volumes do not increase or decrease significantly. Finally, 300µL was selected as the nanochannel functionalized volume.

[0045] Example 6: Selection of target nucleic acid incubation time This embodiment is essentially the same as the first embodiment, except for the target nucleic acid incubation time during the preparation of the biorecognition element sample in step 5. The incubation times were 5 min, 10 min, 20 min, 30 min and 40 min respectively, and the remaining steps were the same as those in Example 1, and the current peak values at different incubation times were obtained.

[0046] The results are as follows Figure 7 As shown in Figure 2, with the gradual extension of the incubation time, the current peak change ΔI before and after the biorecognition process first increases, then decreases, and then tends to be stable. This shows that the extension of the incubation time causes a large number of single and double strands in the solution to remain on the surface of the biorecognition element, which is not conducive to the interaction between the probe and the nirS After gene complementation, the double-stranded DNA is desorbed, so 10 minutes is the optimal incubation time.

[0047] Example 7: Selection of the number of DNA probes This embodiment is basically the same as the technical solution of embodiment 1, except for the number of DNA probes. The details are as follows: The number of DNA probes is 1; Target genes nirS For example, search for genes on the NCBI website nirS Gene sequences were obtained, and a series of sequences (MK178639.1, MF773686.1, MF776399.1, MF776495.1, KT934768.1, MF776458.1, MF178650.1, MF776464.1) were compared in ClustalX2 software. nirS Find the overlapping sequence upstream of the gene sequence, and design the complementary sequence of the partially overlapping sequence as a DNA probe. The number of DNA probes is 1, and the corresponding sequence is: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' 100µmol·L -1 The DNA probe P1 solution was centrifuged appropriately and 1 μL was added to 99 μL of TE buffer for dilution and mixed thoroughly to obtain 1 μmol·L -1 The probe dilution solution was prepared, and 100 μL of the dilution solution was directly drop-coated on the outer surface of the MoS2-nanochannel, and the mixture was incubated at 4°C for 12 hours to obtain the biorecognition element.

[0048] The number of DNA probes is 2; exist nirS The overlapping sequence parts are found upstream and downstream of the gene sequence, and the complementary sequences of the overlapping sequences are designed as DNA probes. There are 2 DNA probes with the following sequences: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' 100µmol·L -1 The DNA probes P1 and P2 solutions were centrifuged appropriately and 1 μL of each solution was added to 98 μL of TE buffer for dilution and mixed thoroughly to obtain a concentration of 1 μmol·L -1 The DNA probe dilution solution was added, and the remaining steps were the same, and the corresponding current response change value was obtained.

[0049] The number of DNA probes is 3; exist nirS The overlapping sequence parts are found upstream and downstream of the gene sequence, and the complementary sequences of the partially overlapping sequences are designed as DNA probes. The number of DNA probes is 3, and the sequences are: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' P3: 5'-GACTCGATGGTGGTGGTCTTCGAGTC-3' 100µmol·L -1 The DNA probes P1, P2, and P3 solutions were centrifuged appropriately and 1 μL of each solution was added to 97 μL of TE buffer for dilution and mixed thoroughly to obtain a concentration of 1 μmol·L -1 The DNA probe dilution solution was added, and the remaining steps were the same, and the corresponding current response change value was obtained.

[0050] The number of DNA probes is 4; exist nirS The overlapping sequence parts were found in the upstream, midstream and downstream of the gene sequence, and the complementary sequences of the overlapping sequences were designed as DNA probes. There were 4 DNA probes with the following sequences: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' P3: 5'-GACTCGATGGTGGTGGTCTTCGAGTC-3' P4: 5'-ATCAGCGCGGCCAGCTTGCCGCTGAT-3' 100µmol·L -1 The DNA probes P1, P2, P3, and P4 were centrifuged appropriately and 1 µL of each solution was added to 96 µL of TE buffer for dilution and mixed thoroughly to obtain a concentration of 1 µmol·L-1 The remaining steps are the same as those for the DNA probe dilution, and the corresponding current response change values are obtained.

[0051] The number of DNA probes was changed to 5; exist nirS The overlapping sequence parts were found in the upstream, midstream and downstream of the gene sequence, and the complementary sequences of the overlapping sequences were designed as DNA probes. The number of DNA probes was 5, and the sequences were: P1: 5'-CCSGTYTCCTTSACGTTSACRACSGG-3' P2: 5'-TCAAGAGASTTCGGRTGSGTCTTGA-3' P3: 5'-GACTCGATGGTGGTGGTCTTCGAGTC-3' P4: 5'-ATCAGCGCGGCCAGCTTGCCGCTGAT-3' P5: 5'-GTCGTGAGATCAGCGTCACGAC-3' 100µmol·L -1 The DNA probes P1, P2, P3, P4, and P5 were centrifuged appropriately, and 1 μL of each solution was added to 95 μL of TE buffer for dilution and mixed thoroughly to obtain a concentration of 1 μmol·L -1 The remaining steps are the same as those for the DNA probe dilution, and the corresponding current response change values are obtained.

[0052] The results are as follows Figure 8 As shown in the figure, as the number of DNA probes increases, the biosensor's current response first increases and then decreases. This may be because increasing the number of DNA probes within a certain range provides more recognition sites for the target nucleic acid, thereby improving the sensor's selectivity and sensitivity. However, an excessive number of DNA probes may lead to competitive binding, reducing the probability of target nucleic acid recognition and affecting the sensor's detection performance. Therefore, two DNA probes were selected as the optimal number of probes.

[0053] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acids, characterized in that: The steps include: Step 1, preparing MoS2-nanochannels; Step 2: preparing a biorecognition element and biorecognition element samples with different copy numbers of target nucleic acid; Step 3, constructing an electrochemical biosensor; Step 4: applying voltage through an electrochemical workstation, and electrochemically detecting and recording the current response I0 of the electrochemical biosensor when detecting the biorecognition element, as well as the current response I when detecting biorecognition element specimens with different copy numbers of the target nucleic acid; and plotting a standard curve with the Lg value of the target nucleic acid copy number as the horizontal axis and the current change value ΔI=I-I0 as the horizontal axis; Step 5: Take the target nucleic acid sample to be detected in the field and perform simple cell lysis to prepare the biorecognition element sample to be detected. Use an electrochemical method to detect and record the current response I1 of the electrochemical biosensor when detecting the biorecognition element sample, and substitute ΔI=I1-I0 into the standard curve in step 4 to calculate the copy number of the target nucleic acid to be detected.

2. The method for quantitative detection of environmental microbial nucleic acid by electrochemical biosensing based on nanochannels according to claim 1, characterized in that: The step 1 is specifically as follows: Step 1a, preparing a MoS2 nanosheet dispersion; Step 1b, washing the nanochannel with ethanol and deionized water in sequence, and drying; Step 1c: Take 40 mg of chitosan and 8 mL of 0.1 mol·L -1 Acetic acid was stirred in the dark at room temperature for 1 h to obtain a 5% chitosan dispersion; Step 1d, take 100 to 500 μL of the MoS2 nanosheet dispersion in step 1a and the chitosan dispersion in step 1c, mix the two in a volume ratio of 20:1, and rapidly oscillate to obtain a mixed solution; fix the nanochannel on filter paper, drop the mixed solution on the surface of the nanochannel, and use a normal pressure filtration process to retain the MoS2 nanosheets in the mixed solution on the outer surface of the nanochannel to achieve solid-liquid separation; after normal pressure filtration, dry at room temperature to obtain the MoS2-nanochannel.

3. The method for quantitative detection of environmental microbial nucleic acid by electrochemical biosensing based on nanochannels according to claim 2, characterized in that: The step 1a is specifically as follows: In step 1aa, thiourea and ammonium molybdate powders were weighed, with a final atomic ratio of Mo:S of 1:4, added to deionized water and vigorously stirred until a blue dispersion was obtained. The blue dispersion was poured into a polytetrafluoroethylene liner, which was then placed in a reactor and reacted at 200° C. for 12 hours. Step 1ab: After the reaction is completed, the product is centrifuged and washed with ethanol and deionized water, respectively, and dried at 70° C. for 8 h to obtain a black solid, which is ground into a black powder with crystals, wherein the black powder is MoS2 nanosheets; Step 1ac: add the MoS2 nanosheets to N,N-dimethylformamide and ultrasonicate for 30 min until the MoS2 nanosheets are uniformly dispersed to obtain the MoS2 nanosheet dispersion. The concentration of the MoS2 nanosheet dispersion is 1 mg·mL -1 .

4. The nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acid according to claim 1, characterized in that: The step 2 is specifically as follows: Preparation of biorecognition elements: The DNA probe solution that is complementary to the target nucleic acid was centrifuged appropriately, and the DNA probe solution was added to TE buffer for dilution and mixed thoroughly to obtain 1µmol·L -1 The DNA probe dilution solution is directly drop-coated on the outer surface of the MoS2-nanochannel with 100 μL of the DNA probe dilution solution, and the mixture is incubated at 4°C for 12 hours to obtain the biorecognition element; Preparation of biorecognition element specimens: 5µL of target gene standard solution was taken respectively, mixed with TE buffer solution to prepare multiple standard solutions with different concentration gradients, and placed in a 95°C environment for 2 minutes for denaturation, then cooled to 65°C, and drop-coated on the biorecognition element at 65°C for incubation. After incubation, it was gently rinsed with deionized water and dried at room temperature to obtain multiple biorecognition element specimens.

5. The nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acid according to claim 4, characterized in that: The sequence of the DNA probe is a partial complementary sequence of the target nucleic acid. The target nucleic acid sequence is searched on the NCBI website, and a series of sequences are compared in ClustalX2 software. The overlapping sequence parts are found upstream, downstream, and midstream of the target nucleic acid sequence, and the complementary sequence of the partially overlapping sequence is designed as the DNA probe. The number of the DNA probes can be selected from 1 to 5.

6. The nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acid according to claim 2, characterized in that: The step three is as follows: Step 3a: N,N-dimethylformamide and the chitosan dispersion prepared in step 1c were mixed in a volume ratio of 4:1 to obtain a dispersion. The carbon nanotube powder was added to the dispersion and ultrasonicated until uniform dispersion was obtained. -1 A carbon nanotube modified material; drop-coating the carbon nanotube modified material on the surface of a glassy carbon electrode and drying at room temperature to obtain the carbon nanotube modified electrode; Step 3b: add 5 mmol·L -1 0.1 mol·L of K3[Fe(CN)6] -1 KCl solution, add 0.1 mol·L -1 The biorecognition element is fixed in the middle of the H-type electrolytic cell with one side of the MoS2-nanochannel-modified MoS2 nanosheet facing left; In step 3c, a three-electrode system is used as the detection system. A platinum wire electrode and a calomel electrode are placed in the left cell as the counter electrode and the reference electrode, respectively. The carbon nanotube-modified electrode is placed in the right cell as the working electrode. The electrochemical biosensor is obtained by free diffusion using the concentration difference of the electrolyte on both sides.

7. The nanochannel-based electrochemical biosensor quantitative detection method for environmental microbial nucleic acid according to claim 1, characterized in that: The steps for preparing the biometric recognition element sample in step 5 are as follows: When the target nucleic acid is a target DNA, an environmental sample is taken, the cells are lysed, and then centrifuged to obtain a supernatant, which is then collected into a centrifuge tube to obtain a mixed solution containing the target DNA. The mixed solution containing the target DNA is thoroughly mixed with TE buffer, first placed in a 95°C environment for 2 minutes to melt, then cooled to 65°C, and 100 μL is dropwise applied to the biorecognition element at 65°C for incubation. After the incubation is completed, it is gently rinsed with deionized water and dried at room temperature to obtain the biorecognition element specimen to be tested; When the target nucleic acid is target RNA, an environmental sample is taken, the cells are lysed, and the sample is centrifuged to obtain an upper aqueous phase, which is then collected into a centrifuge tube to obtain a mixed solution containing the target RNA. The mixed solution containing the target RNA is thoroughly mixed with TE buffer, and 100 μL is dropwise applied to the biorecognition element at 4°C for incubation. After the incubation is completed, the sample is gently rinsed with deionized water and dried at room temperature to obtain a specimen of the biorecognition element to be tested.

8. The method for quantitative detection of environmental microbial nucleic acid by electrochemical biosensing based on nanochannels according to any one of claims 1 to 7, characterized in that: The electrochemical method in step 4 and step 5 includes differential pulse voltammetry, cyclic sweep voltammetry or impedance spectroscopy.

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