CRISPR (clustered regularly interspaced short palindromic repeats) and light-operated electrochemistry based nucleic acid detection module and method

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a nucleic acid detection module and method based on CRISPR (clustered regularly interspaced short palindromic repeats) and light-operated electrochemistry. The nucleic acid detection module comprises an insulating substrate, metal electrodes arranged on the insulating substrate, a two-dimensional sensitive material arranged on the insulating substrate and located between the electrodes, and a sample groove covering the two-dimensional sensitive material between the electrodes, the surface of the two-dimensional sensitive material is modified with a single-chain biological probe through a chemical bond or an intermolecular force; complementary sequences of PC-Linker molecules uniformly modified with ultraviolet degradation characteristics are introduced into a CRISPR / Cas12a and crRNA compound system, the cleavage activity of CRISPR / Cas12a is effectively controlled through light-controlled electrochemistry under the conditions of no purple light illumination and existence of ultraviolet light illumination, the detection accuracy of 98% or above of nucleic acid markers of various cancers is achieved, and the method can be used for disease diagnosis, clinical diagnosis and the like. The method has potential social and economic values.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a nucleic acid detection module and method based on CRISPR and photocontrolled electrochemistry. Background Technology

[0002] Against the backdrop of the rapid development of digital healthcare technology, clinical diagnosis is placing higher demands on rapid and high-precision nucleic acid detection methods. Currently, molecular detection technologies are receiving widespread attention due to their ease of operation, rapid response, and cost advantages. However, existing mainstream detection methods (such as mass spectrometry, surface-enhanced Raman sensors, transistor sensors, and immunochromatographic sensors) generally suffer from problems such as single signal modulation mechanisms, complex operation processes, and fixed discrimination logic. When processing complex, untreated clinical samples, they often face challenges such as decreased sensitivity, insufficient accuracy, and complex signal interpretation. Therefore, there is an urgent need to construct a sensing system with multi-signal collaborative control capabilities to improve the efficiency and reliability of nucleic acid target identification in clinical samples.

[0003] Machine learning, as a data-driven intelligent decision-making tool, has demonstrated significant potential in various fields such as materials design, artificial intelligence, and clinical analysis. In sensor detection scenarios, by rationally selecting algorithm models and parameter configurations, efficient classification and accurate judgment of input signals can be achieved. Furthermore, by combining this with visualization analysis methods, systematic comparison and feature extraction of multidimensional signal responses can be performed, thus providing more accurate and robust technical support for the CRISPR / Cas12a system in nucleic acid detection of clinical samples. Summary of the Invention

[0004] To overcome the problems of existing molecular detection methods such as "single signal control method, complex sample addition process and insufficient sensitivity", this invention provides a nucleic acid detection module and method based on CRISPR and photoelectrochemistry.

[0005] This invention modifies an electrical sensing unit with a biological probe to obtain an electrical detection device, namely a nucleic acid detection module. The electrical sensing unit includes an insulating substrate, a working electrode, a counter electrode, a reference electrode, and a two-dimensional sensitive material. The nucleic acid detection module acquires electrical signals, and combines data feature extraction and machine learning algorithms for intelligent classification, achieving highly sensitive, highly specific, and rapid detection of multiple nucleic acids. This invention can complete detection within 5 minutes with an accuracy rate exceeding 98%, making it suitable for disease screening and prevention, and possessing potential social application value.

[0006] The present invention provides a nucleic acid detection module based on CRISPR and photocontrolled electrochemistry, comprising an insulating substrate, metal electrodes disposed on the insulating substrate, a two-dimensional sensitive material disposed on the insulating substrate and located between the electrodes, and a sample loading groove covering the two-dimensional sensitive material between the electrodes. The surface of the two-dimensional sensitive material is modified with CRISPR / Cas12a biological probes by chemical bonds or intermolecular forces. The electrodes include a working electrode, a reference electrode, and a counter electrode.

[0007] in:

[0008] The insulating substrate includes a polyimide film, a SiO2 / Si substrate, and a polyethylene terephthalate film.

[0009] The electrode material is chromium, titanium, silver, silver / silver chloride, and / or gold. Preferably, the working electrode and counter electrode are gold, and the reference electrode is a silver / silver chloride electrode.

[0010] The two-dimensional sensitive material is selected from graphene, laser-induced graphene, molybdenum disulfide, or thin films containing silicon, germanium, or organic semiconductors.

[0011] The CRISPR / Cas12a bioprobes include liver cancer markers, human papillomavirus markers, and amyotrophic lateral sclerosis (ALS) markers.

[0012] The present invention also provides a method for preparing a nucleic acid detection module based on CRISPR and photocontrolled electrochemistry, the specific steps of which are as follows:

[0013] Step 1: Fabricate a metal electrode on an insulating substrate, wherein the electrode includes a working electrode, a reference electrode, and a counter electrode;

[0014] Step 2: Transfer the two-dimensional sensitive material to an insulating substrate and fix it to the surface of the circular region of the working electrode. Use reactive ion etching technology to etch the two-dimensional sensitive material into a specific shape to obtain the device to be modified.

[0015] Step 3: Install polydimethylsiloxane sample slots on the two-dimensional sensitive material of the device to be modified, modify the linker molecules, and modify the linker molecules with single-chain biological probes with redox groups.

[0016] Step 4: Add a complex system consisting of CRISPR / Cas12a biological probes, CRISPR / Cas12a protein, and target material to the sample well to obtain a functionalized nucleic acid detection module. The crRNA recognition sequence of the CRISPR / Cas12a biological probes is introduced with a complementary sequence of a PC-Linker molecule modified with UV degradation properties.

[0017] Furthermore:

[0018] In step 2, the insulating substrate is a polyimide film, a SiO2 / Si substrate with a resistivity greater than 1 ohm·cm (preferably 5-10 ohm·cm), and a polyethylene terephthalate film.

[0019] In step 3, the linker molecule is 1-pyrenebutyrate-succinimide ester, and the single-chain biological probe sequence is:

[0020] Probe A: 5'-Methylene Blue-AAGAGCCGC-NH2-3' (SEQ.ID.NO.1);

[0021] Probe B: 5'-Methylene Blue-GAGAAGAGCCGCCATAGTA-NH2-3' (SEQ.ID.NO.2);

[0022] Probe C: 5'-Methylene Blue-GAGAAGAGCCGCCATAGAAGAGCCGCGTA-NH2-3' (SEQ.ID.NO.3);

[0023] In step 4, the crRNA recognition sequence of the CRISPR / Cas12a biological probe includes:

[0024] Sequence 1: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGUACACATCTGCCAAACUU-3' (SEQ.ID.NO.4);

[0025] Sequence 2: 5'-UAAUUUCUACUAAGUGUAGAUGGGACACAUCUGCCAAACU-3' (SEQ.ID.NO.5);

[0026] Sequence 3: 5'-UAAUUUCUACUAAGUGUAGAUGGCGGUACACATCTGCCAAACUU-3' (SEQ.ID.NO.6);

[0027] Sequence 4: 5'-UAAUUUCUACUAAGUGUAGAUAAGGCCAAACCAAAAUUUACA-3' (SEQ.ID.NO.7);

[0028] Sequence 5: 5'-UAAUUUCUACUAAGUGUAGAUGAUUCCAUGUUCAUGAGUUUG-3' (SEQ.ID.NO.8);

[0029] In step (4), the chemical formula of the PC-Linker molecule is C 39 H46 N3O7P.

[0030] The present invention provides a method for nucleic acid detection based on CRISPR and photocontrolled electrochemistry, comprising the following steps:

[0031] (S1) Connect the nucleic acid detection module to the electrochemical testing system, wherein the working electrode of the nucleic acid detection module is connected to the green alligator clip of the electrochemical testing system, the reference electrode of the nucleic acid detection module is connected to the white alligator clip of the electrochemical testing system, the reference electrode of the nucleic acid detection module is connected to the counter electrode of the electrochemical testing system, and the counter electrode is connected to the red alligator clip.

[0032] (S2) The sample to be tested is added to the sample cell. The UV light is turned on or off by controlling the UV light box. When there is no UV light, the CRISPR / Cas12a bioprobe is locked and cannot detect the target nucleic acid biomarker, and the control electrical response signal I1 is obtained. When UV light is applied, the PC-Linker molecule contained in the crRNA recognition sequence of the CRISPR / Cas12a bioprobe is rapidly degraded, releasing the cleavage activity of the CRISPR / Cas12a bioprobe, completing the detection of the target nucleic acid biomarker, and obtaining the electrical response signal I2.

[0033] (S3) The acquired electrical sensing unit signal responses I1 and I2 are processed to obtain normalized electrical sensing unit signal response characteristic values.

[0034] (S4) Train the machine learning model, use a portion of the normalized signal response data points of the electric sensing unit as the training set, and use the remaining data points as the test set to determine the classification threshold.

[0035] (S5) Evaluate the accuracy of the machine learning model and detect the sample to be tested after the model has sufficient accuracy.

[0036] Furthermore:

[0037] In step (S1), the electrical testing system is a CHI7600 electrochemical workstation;

[0038] In step (S2), the ultraviolet irradiation time is 10 to 30 seconds;

[0039] In step (S2), the concentration of the CRISPR protein is 70 nanomolars (10 -9 mol / L);

[0040] In step (S3), the normalized electrical sensing unit signal response characteristic value is calculated by comparing the deviation of the test value from the initial value with the initial value;

[0041] In step (S4), the machine learning model being trained is either a support vector machine algorithm or a decision tree model.

[0042] This invention introduces a complementary sequence of a PC-Linker molecule uniformly modified with UV-degradable properties into a CRISPR / Cas12a and crRNA complex system. Under UV light-free conditions, the locked PC-Linker molecule blocks the recognition of target DNA by Cas12 / crRNA and inhibits the cleavage activity of CRISPR / Cas12a. Under UV light, the PC-Linker molecule rapidly degrades and breaks down, detaching from the crRNA recognition sequence, allowing Cas12 / crRNA to recognize and recombine with the target DNA, thereby releasing the cleavage activity of CRISPR / Cas12a. Addressing the issues of random perturbations and low accuracy caused by non-specific substances in complex biological fluids faced by CRISPR-based electrochemical sensors, this invention effectively controls the cleavage activity of CRISPR / Cas12a through photo-controlled electrochemistry, achieving a detection accuracy of over 98% for nucleic acid markers (including markers for liver cancer, human papillomavirus, and amyotrophic lateral sclerosis), enabling disease diagnosis and possessing potential socio-economic value.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention innovatively establishes a multi-dimensional signal control strategy by integrating a front-end electrochemical nucleic acid sensing device with a back-end machine learning model. Through optimization of the classification algorithm, it overcomes the low clinical detection accuracy problem caused by the single control signal in existing molecular detection methods, providing a new direction for the practical application of molecular detection in complex clinical samples. The machine learning-based auxiliary algorithm constructs classification thresholds for different nucleic acids, providing a reliable tool for rapid screening of infectious diseases with a detection accuracy greater than 98%, demonstrating significant clinical translational value. Attached Figure Description

[0045] Figure 1 This is a flowchart of the CRISPR / Cas electrical detection method for nucleic acids according to the present invention.

[0046] Figure 2 This is a schematic diagram of the electrode modification of the present invention.

[0047] Figure 3 It is the electrical signal acquired by the machine learning-assisted electrical detection device of this invention.

[0048] Figure 4 This is the time optimization result of the machine learning-assisted electrical detection device of the present invention.

[0049] Figure 5 It is the cyclic voltammetric signal acquired by the machine learning-assisted electrical detection device of this invention.

[0050] Figure 6 This is the result of machine learning-assisted electrical detection of liver cancer virus in Embodiment 1 of the present invention.

[0051] Figure 7 These are the training and test set results of the SVM classification method used in the electrical detection nucleic acid device of this invention.

[0052] The labels in the figure are as follows: 1 is CRISPR / Cas12a protein; 2 is a PC-Linker-modified crRNA complementary probe; 3 is a PC-Linker molecule; 4 is the target of the CRISPR system; 5 is UV lamp on; 6 is UV lamp off; 7 is photolysis of PC-Linker molecule; 8 is an insulating substrate; 9 is the counter electrode; 10 is the working electrode; 11 is the reference electrode; 12 is the sample well; 13 is the nucleic acid probe; and 14 is the two-dimensional sensitive material. Detailed Implementation

[0053] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0054] The present invention relates to a method for preparing a nucleic acid detection module based on CRISPR technology and photocontrolled electrochemistry:

[0055] Step 1: The working electrode, counter electrode, and reference electrode are disposed above the insulating substrate, wherein the counter electrode and reference electrode are symmetrically arranged, and the working electrode is disposed between the counter electrode and the reference electrode; the sample cell is located above the working electrode, counter electrode, and reference electrode.

[0056] The two-dimensional sensitive material is disposed at the bottom of the working electrode near the end and on the upper surface of the insulating substrate, and a biological probe is attached to the surface of the two-dimensional sensitive material.

[0057] The working electrode, counter electrode, and reference electrode are made of gold;

[0058] The two-dimensional sensitive material is processed by photolithography to obtain a specific pattern, which covers the working electrode;

[0059] The sample cell is obtained by baking polydimethylsiloxane after removing air under vacuum, wherein the polydimethylsiloxane is mixed in a ratio of A:B = 1:10 (wt.%); during the baking process, the temperature is 70℃ and the baking time is 30 minutes.

[0060] The two-dimensional sensitive material and the biological probe binding solution is a 1× phosphate buffered saline (PBS) solution;

[0061] Step 2: The electrical sensing unit includes an insulating substrate, a working electrode, a counter electrode, a reference electrode, and a two-dimensional sensitive material. The electrical sensing unit is modified with a biological probe to obtain an electrical detection device, namely a nucleic acid detection module.

[0062] The two-dimensional sensitive material is graphene, and the insulating substrate is a PI film with a light transmittance greater than 90%.

[0063] The present invention provides a method for nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry, comprising the following steps:

[0064] (S1) Connect the liver cancer nucleic acid detection module to the electrochemical testing system, wherein the working electrode of the liver cancer nucleic acid detection module is connected to the green alligator clip of the electrochemical testing system, the reference electrode of the liver cancer nucleic acid detection module is connected to the white alligator clip of the electrochemical testing system, and the counter electrode of the liver cancer nucleic acid detection module is connected to the red alligator clip.

[0065] (S2) Add the sample to be tested into the sample cell and obtain the signal response I of the electrosensing unit;

[0066] (S3) Perform data processing on the acquired signal response of the electric sensing unit to obtain the characteristic value of the normalized signal response of the electric sensing unit.

[0067] (S4) Train the machine learning model by selecting the feature values ​​of the normalized signal response of the electrosensor unit as input and the interpretation result of the test sample as output to train the machine learning model and obtain the threshold D. cutoff ;

[0068] (S5) Evaluate the accuracy of the machine learning model and detect the sample to be tested after the model has sufficient accuracy.

[0069] The electrical testing system described in step (S1) is a CHI7600 electrochemical workstation;

[0070] The normalized signal response characteristic value of the electrical sensing unit mentioned in step (S3) is calculated by comparing the deviation of the test value from the initial value with the initial value;

[0071] The training machine learning model described in step (S4) is the support vector machine algorithm.

[0072] Unless otherwise specified, all reagents used in the following embodiments are commercially available; all detection methods and techniques used are conventional in the art. The liver cancer specimens used in the detection method of this invention are clinical serum samples. The specific steps for sample collection and processing should refer to the nucleic acid processing requirements of the "National Clinical Laboratory Operation Procedures" issued by the Chinese Center for Disease Control and Prevention. The specific method for obtaining the samples is as follows:

[0073] Serum: Venous blood was collected into sterile tubes and allowed to stand at room temperature for 10–20 minutes to coagulate naturally. Then, it was centrifuged at 2–8°C and 2000–3000 rpm for 20 minutes, and the supernatant was collected and aliquoted.

[0074] Example 1: Preparation of a nucleic acid detection module based on CRISPR technology and photocontrolled electrochemistry.

[0075] like Figure 1 and Figure 2 As shown, the electrical detection nucleic acid module includes an insulating substrate 8, a counter electrode 9, a sample cell 12, a working electrode 10, and a reference electrode 11. The counter electrode 9, working electrode 10, and reference electrode 11 are located above the insulating substrate 8, with the counter electrode 9 and reference electrode 11 arranged in parallel, and the working electrode 10 located between the counter electrode 9 and reference electrode 11. The sample cell 12 is disposed above the working electrode 10, reference electrode 11, and counter electrode 9. A two-dimensional sensitive material 14 is located in the circular region of the working electrode 10 and on the upper surface of the insulating substrate 8. The surface of the two-dimensional sensitive material is connected to the biological probe 13. The insulating substrate 8, counter electrode 9, working electrode 10, reference electrode 11, two-dimensional sensitive material 14, and biological probe 13 constitute an electrosensing unit.

[0076] The workstation used was a CHI7600 electrochemical workstation.

[0077] Among them, the insulating substrate 8 is a plastic film with a light transmittance of more than 90%, and the film includes one or more of polyester resin, polyimide or polymethyl methacrylate; the counter electrode 9, the working electrode 10 and the reference electrode 11 are all gold electrodes; the biological probe 13 is a nucleic acid probe; and the two-dimensional sensitive material 14 is a two-dimensional sensitive material film obtained by chemical deposition, mechanical exfoliation or liquid phase exfoliation.

[0078] Specifically, a crRNA was designed targeting the nucleic acid sequence of liver cancer biomarkers. This crRNA can form a Cas12a-crRNA complex with CRISPR / Cas12a at room temperature. The process involves mixing crRNA and CRISPR / Cas12a protein at a 1:1 concentration ratio in PBS and incubating at room temperature for 10 minutes. The ligation molecule used is probe A, and the crRNA used is sequence 1 (SEQ.ID.NO.4).

[0079] Example 2: Nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry, wherein the nucleic acid is derived from a liver cancer serum sample. The specific steps are as follows:

[0080] (1) A metal electrode (50 nanometer gold) is fabricated on a PI film with a thickness of 135 micrometers by printing electronics technology. The electrode includes a working electrode, a counter electrode and a reference electrode.

[0081] (2) Spin-coating 8 wt.% polymethyl methacrylate (PMMA) onto a two-dimensional sensitive material grown on a metal substrate, and transferring the two-dimensional sensitive material / PMMA film to the working electrode surface by electrochemical method; after soaking in acetone for 2 hours, it is washed with isopropanol and deionized water respectively, and the two-dimensional sensitive material / PMMA film is etched into a circle by photolithography to obtain the device to be modified.

[0082] (3) Modification of probes in the channel region of the two-dimensional sensitive material: The two-dimensional sensitive material / PMMA film was immersed in 5 mmol / L PASE solution for 2 hours; then immersed in 10 μmol / L nucleic acid B probe solution for 6 hours. After washing with 1×PBS buffer, the pretreatment device was obtained.

[0083] (4) Mix type 194 polydimethylsiloxane (PDMS) at A:B = 1:10 (wt.%), remove air under vacuum and bake at 70°C for 30 minutes to obtain PDMS. Punch holes in the PDMS to obtain sample cells and place them on top of the pretreatment device to obtain an electrical detection device for nucleic acid.

[0084] (5) Steps for machine learning-assisted CRISPR / Cas electrochemical nucleic acid detection using sequence 2 (SEQ.ID.NO.5):

[0085] (S1) Connect the working electrode, counter electrode, and reference electrode in the electrical detection nucleic acid device to the electrical testing system via alligator clips, wherein the working electrode is connected to the green unit, the reference electrode is connected to the white unit, and the counter electrode is connected to the red unit; ultraviolet light is applied by the ultraviolet lamp box.

[0086] (S2) Add the sample to be tested into the sample cell and obtain the signal response ΔI of the electrical sensing unit. Figure 3 ).

[0087] (S3) Use SVM to process the acquired signal response of the electrical sensing unit to obtain the threshold of the normalized signal response of the electrical sensing unit.

[0088] (S4) The accuracy of the machine learning model is evaluated, and the detection of liver cancer biomarkers in the test samples is achieved after the model has sufficient accuracy. The accuracy of machine learning-assisted electrical detection reaches 98%.

[0089] In this embodiment, the clinical test sample for liver cancer was provided by Shenzhen Hengsheng Hospital, and the concentration of the target nucleic acid contained therein was calibrated to 15 copies per milliliter.

[0090] Example 3: Nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry. The difference between this example and Example 2 is:

[0091] In step (1), the insulating substrate used is a polyester resin film (1000 micrometers thick);

[0092] In step (3), the probe used is probe B (SEQ.ID.NO.2);

[0093] In step (5), the crRNA used was from 37 clinical samples of liver cancer. The specific test results are as follows: Figure 7 As shown:

[0094] Machine learning-assisted CRISPR / Cas electro-nucleic acid detection of liver cancer biomarkers achieved an accuracy of 98%, demonstrating excellent detection performance and highlighting the value of this invention.

[0095] Example 4: Nucleic acid detection of liver cancer based on CRISPR technology and photocontrolled electrochemistry. The difference between this example and Example 2 is:

[0096] In step (1), the insulating substrate used is a polymethyl methacrylate film (1000 micrometers thick);

[0097] In step (5), the probe used is sequence 3 (SEQ.ID.NO.6);

[0098] In step (5), clinical samples from 20 liver cancer patients were tested. The specific test results are shown in Table 1:

[0099] Table 1

[0100]

[0101]

[0102] In this embodiment, the accuracy rate of liver cancer nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry was 100% in the clinical testing of 20 liver cancer patient samples.

[0103] Example 5: Human papillomavirus nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry. The difference between this example and Example 2 is:

[0104] In step (5), the probe used is sequence 4 (SEQ.ID.NO.7).

[0105] In step (5), clinical samples from 13 patients with human papillomavirus (HPV) infection were tested. The specific test results are shown in Table 2.

[0106] Table 2

[0107]

[0108]

[0109] In this embodiment, the accuracy rate of human papillomavirus nucleic acid detection based on CRISPR technology and photocontrolled electrochemistry was 100% in the clinical testing of 13 human papillomavirus patient samples.

[0110] Example 6: Nucleic acid detection for amyotrophic lateral sclerosis based on CRISPR technology and photocontrolled electrochemistry. The difference between this example and Example 2 is:

[0111] In step (5), the probe used is sequence 5 (SEQ.ID.NO.8).

[0112] In step (5), 10 serum simulants of amyotrophic lateral sclerosis (ALS) were tested. The specific test results are shown in Table 3.

[0113] Table 3

[0114] serial number clinical gold standard CRISPR / Cas Electron Nucleic Acid Detection consistency 1 Positive Positive yes 2 Negative Negative yes 3 Positive Positive yes 4 Positive Positive yes 5 Positive Positive yes 6 Positive Positive yes 7 Positive Positive yes 8 Positive Positive yes 9 Positive Positive yes 10 Positive Positive yes

[0115] In this embodiment, the accuracy of nucleic acid detection for amyotrophic lateral sclerosis (ALS) based on CRISPR technology and photocontrolled electrochemistry was 100% in the detection of 13 serum simulants of ALS.

[0116] The embodiments described in this specification are intended to enable those skilled in the art to fully understand and implement the present invention. Those skilled in the art can make various modifications to the embodiments without inventive effort, and extend the core principles disclosed herein to other embodiments. Therefore, the scope of protection of the present invention is not limited to the foregoing embodiments. Any equivalent modifications or adaptive adjustments made based on the essence of the present invention and within the scope defined by the claims of the present invention are covered by the patent rights of the present invention.

Claims

1. A nucleic acid detection module based on CRISPR and photocontrolled electrochemistry, characterized in that, The device includes an insulating substrate, metal electrodes disposed on the insulating substrate, a two-dimensional sensitive material disposed on the insulating substrate and located between the electrodes, and a sample groove covering the two-dimensional sensitive material between the electrodes. The surface of the two-dimensional sensitive material is modified with CRISPR / Cas12a biological probes by chemical bonds or intermolecular forces. The electrodes include a working electrode, a reference electrode, and a counter electrode.

2. The nucleic acid detection module according to claim 1, characterized in that, The CRISPR / Cas12a bioprobes include liver cancer markers, human papillomavirus markers, and amyotrophic lateral sclerosis (ALS) markers.

3. The nucleic acid detection module according to claim 1, characterized in that: The insulating substrate is a polyimide film, a SiO2 / Si substrate, or a polyethylene terephthalate film. The electrode material is chromium, titanium, silver, silver / silver chloride and / or gold; The two-dimensional sensitive material is selected from graphene, laser-induced graphene, molybdenum disulfide, or thin films containing silicon, germanium, or organic semiconductors.

4. The method for preparing the nucleic acid detection module as described in any one of claims 1-3, characterized in that, The specific steps are as follows: Step 1: Fabricate a metal electrode on an insulating substrate, wherein the electrode includes a working electrode, a reference electrode, and a counter electrode; Step 2: Transfer the two-dimensional sensitive material to an insulating substrate and fix it to the surface of the circular region of the working electrode. Use reactive ion etching technology to etch the two-dimensional sensitive material into a specific shape to obtain the device to be modified. Step 3: Install polydimethylsiloxane sample slots on the two-dimensional sensitive material of the device to be modified, modify the linker molecules, and modify the linker molecules with single-chain biological probes with redox groups. Step 4: Add a complex system consisting of CRISPR / Cas12a biological probes, CRISPR / Cas12a protein, and target material to the sample well to obtain a functionalized nucleic acid detection module. The crRNA recognition sequence of the CRISPR / Cas12a biological probes is introduced with a complementary sequence of a PC-Linker molecule modified with UV degradation properties.

5. The preparation method according to claim 4, characterized in that, In step 3, the linker molecule is 1-pyrenebutyrate-succinimide ester, and the single-chain biological probe sequence is: Probe A: SEQ.ID.NO.1; Probe B: SEQ.ID.NO.2; Probe C: SEQ.ID.NO.3; In step 4, the crRNA recognition sequence of the CRISPR / Cas12a biological probe is as follows: Sequence 1: SEQ.ID.NO.4; Sequence 2: SEQ.ID.NO.5; Sequence 3: SEQ.ID.NO.6; Sequence 4: SEQ.ID.NO.7; Sequence 5: SEQ.ID.NO.8; In step (4), the chemical formula of the PC-Linker molecule is C 39 H 46 N3O7P.

6. A method for nucleic acid detection based on CRISPR and photocontrolled electrochemistry, characterized in that, The specific steps are as follows: (S1) Connect the nucleic acid detection module to the electrochemical testing system, wherein the working electrode of the nucleic acid detection module is connected to the green alligator clip of the electrochemical testing system, the reference electrode of the nucleic acid detection module is connected to the white alligator clip of the electrochemical testing system, and the counter electrode of the nucleic acid detection module is connected to the red alligator clip of the electrochemical testing system. (S2) Add the sample to be tested into the sample holder. Control the UV light to turn on or off using the UV light box. When there is no UV light, the CRISPR / Cas12a bioprobe is locked and cannot detect the target nucleic acid biomarker, resulting in the control electrical response signal I1. When UV light is applied, the PC-Linker molecule contained in the crRNA recognition sequence of the CRISPR / Cas12a bioprobe is rapidly degraded, releasing the cleavage activity of the CRISPR / Cas12a bioprobe, completing the detection of the target nucleic acid biomarker, and resulting in the electrical response signal I2. (S3) The acquired electrical sensing unit signal responses I1 and I2 are processed to obtain normalized electrical sensing unit signal response characteristic values. (S4) Train the machine learning model, use a portion of the normalized signal response data points of the electric sensing unit as the training set, and use the remaining data points as the test set to determine the classification threshold. (S5) Evaluate the accuracy of the machine learning model and detect the sample to be tested after the model has sufficient accuracy.

7. The method for nucleic acid detection according to claim 6, characterized in that: In step (S1), the electrical testing system is a CHI7600 electrochemical workstation; In step (S2), the ultraviolet irradiation time is 10 to 30 seconds; In step (S2), the concentration of the CRISPR protein is 70 nanomolars; In step (S3), the normalized electrical sensing unit signal response characteristic value is calculated by comparing the deviation of the test value from the initial value with the initial value; 8. The method for nucleic acid detection according to claim 6, characterized in that, The training machine learning model mentioned in step (S4) is a support vector machine algorithm or a decision tree model.

Images