A virus RNA electrical detection module based on CRISPR-Cas13 protein and application

Abstract

The application relates to a virus RNA electrical detection module based on a CRISPR-Cas13 protein and application. The RNA electrical detection module comprises an insulating substrate, a source electrode and a drain electrode arranged on the insulating substrate, a semiconductor film arranged on the insulating substrate and located between the source electrode and the drain electrode, and a Cas13-gRNA probe connected with the semiconductor film, wherein the Cas13 protein is connected with the surface of the semiconductor film through a covalent bond or a van der Waals force, and the gRNA is combined with the Cas13 protein to form a complex; wherein the semiconductor film forms a conductive channel between the source electrode and the drain electrode, and a sample inlet groove is arranged on the insulating substrate and contains the surface of the semiconductor film in the sample inlet groove. During detection, the detection module is connected with an electrical testing instrument, sample solution to be detected is added dropwise in the sample inlet groove, and whether the target RNA molecule exists in the sample is judged through electrical signal change. The application combines the CRISPR-Cas13 protein with the transistor technology, and successfully realizes instant and accurate feedback on the target RNA molecule.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, and in particular relates to a viral RNA electrophysiological detection module based on CRISPR-Cas13 protein and its application. Background Technology

[0002] Accurate identification of base sequences and their mutations is one of the core elements of precision medicine. For example, in epidemic control, nucleic acid sequence analysis of pathogens in respiratory extracts remains the most effective method for diagnosing virus types in the population. Furthermore, over time, viral strains will develop into different subspecies due to base mutations, leading to significant differences in their transmission speed and immune evasion capabilities. Therefore, rapid and accurate detection of target nucleic acid sequences is particularly important for virus control and clinical medicine.

[0003] CRISPR (Regularly Spaced Clustered Short Palindromic Repeats) originated from the bacterial immune system. Its Cas protein can bind to guide nucleotide chains, thereby achieving specific recognition and editing of target nucleic acid sequences. Currently, CRISPR-based nucleic acid detection platforms have been successfully developed. Most of these platforms utilize the synergistic cleavage effect of CRISPR-Cas proteins to cleave reporter molecules carrying fluorescent groups, thereby using the intensity of the fluorescence signal for indirect detection (CN 113373264 B). However, most of these methods require complex optical instruments and inevitably involve amplification techniques, making the operation process lengthy and increasing the risk of sample contamination. During the same period, the application of two-dimensional field-effect transistors (FETs) in the sensing field also developed rapidly. Utilizing the atomic-level thickness of the two-dimensional material in the conductive channel, these devices can provide real-time and sensitive feedback to changes in the external environment.

[0004] In recent years, some teams have modified graphene transistors with the Cas9 protein to identify base mutations in human genomic DNA (Nat. Biomed. Eng. 2021, 5, 713-725). However, the Cas9 protein can only specifically target double-stranded DNA, and this type of transistor cannot directly detect viral RNA molecules. Furthermore, its detection limit is 1.7 femtomolar, and its sensitivity needs improvement. In addition, there are reports of binding Cas13-gRNA probes to target nucleic acid molecules in test tubes to activate the co-cleavage effect of proteins, and immobilizing negatively charged oligoRNA molecules on the channel surface of graphene transistors. When an activated protease solution is added, the modified negatively charged RNA molecules are cleaved and diffuse into the solution, generating an electrical signal response (Angew. Chem. Int. Ed. 2022, e202203826). However, this strategy of not directly modifying the Cas13 protein onto the transistor surface does not fully utilize the potential of transistor devices in terms of sensitivity. Meanwhile, this strategy requires the introduction of other oligoRNA molecules in addition to the guide RNA. When untargeted nucleic acid molecules accidentally activate the protein's cleavage effect, the device can still produce a large response, posing a significant challenge to its specificity. To our knowledge, there are currently no reports of recognizing RNA sequences by directly modifying the Cas13 protein onto the surface of a graphene transistor. This is because, on the one hand, the Cas13 protein cleaves the target RNA while targeting it, making it difficult to achieve effective aggregation of the target on the device surface. Furthermore, if the Cas13 protein is modified with a cleavage-deficient cas13 protein (dCas13), the device's RNA capture rate will be adversely affected (Adv. Sci. 2021, 8, 2004685). On the other hand, after directly modifying the Cas13 protein onto the graphene surface, a series of modifications to the protein-graphene complex within the transistor device are required while ensuring the activity of the Cas13 protein. Therefore, this strategy places strict requirements on the type of Cas13 protein and the modification conditions at each stage.

[0005] In summary, although CRISPR-Cas technology has led to the development of numerous detection methods for RNA, these methods either require amplification or reverse transcription processes that can easily lead to sample contamination, or they necessitate the introduction of additional fluorescent reporter molecules or oligoRNA molecules. Therefore, developing a simple, rapid, cost-effective RNA detection platform that can detect target RNA molecules without amplification would have significant clinical implications. Summary of the Invention

[0006] In order to overcome the problems existing in the prior art, the present invention aims to provide a viral RNA electrophysiological detection module based on CRISPR-Cas13 protein and its application.

[0007] The method provided by this invention combines CRISPR-Cas13 protein and transistor technology, using changes in transistor electrical signals to determine whether a sample contains the target viral nucleic acid. This method requires no fluorescent labeling, no introduction of other oligoRNA molecules, and no amplification. The average detection time is 5–15 minutes, and the detection limit is 1 atmolar per liter (1 × 10⁻⁶). -18 (moles / liter)

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention first provides a viral RNA electrophysiological detection module based on the CRISPR-Cas13 protein, the module comprising:

[0010] Insulating substrate;

[0011] The source and drain electrodes are disposed on the insulating substrate;

[0012] A semiconductor thin film disposed on the insulating substrate and located between the source and drain electrodes;

[0013] The Cas13-gRNA probe is attached to a semiconductor thin film, wherein the Cas13 protein is attached to the surface of the semiconductor thin film by covalent bonds or van der Waals forces, and the guide RNA (gRNA) binds to the Cas13 protein to form a complex.

[0014] In this process, a conductive channel is formed between the source and drain of the semiconductor thin film, and a sample inlet is provided on the insulating substrate, with the surface of the semiconductor thin film contained within the sample inlet.

[0015] In one embodiment of the present invention, the insulating substrate is selected from silicon dioxide / silicon substrate, quartz or polymer film.

[0016] In one embodiment of the present invention, the source and drain electrodes are metallic materials selected from one or more of gold, copper, silver, chromium, aluminum, etc., and the thickness of the source and drain electrodes is 10 to 100 nanometers.

[0017] In one embodiment of the present invention, the semiconductor thin film is selected from graphene, two-dimensional transition metal dichalcogenides, silicon, or black phosphorus.

[0018] In one embodiment of the present invention, the Cas13 protein is a class VI protein in the CRISPR / Cas system, which can bind to guide RNA to achieve specific recognition of nucleic acids. The Cas13 protein is derived from the following species: *Leptotrichia wadei*, *Porphyromonas gulae*, *Lachnospiraceae bacterium*, *Ruminoccocus flavefaciens*, *Eubacterium siraeum*, and *Prevotella bacterium*.

[0019] In one embodiment of the present invention, the gRNA is an oligonucleotide chain of 35 to 60 bases in length, which can bind to the Cas13 protein to form a binary complex and guide the Cas13 protein to target the target RNA molecule.

[0020] In one embodiment of the present invention, the virus is an RNA virus selected from influenza virus, hepatitis virus, rhinovirus, coronavirus, HIV and 2019 novel coronavirus.

[0021] This invention further provides a method for preparing the CRISPR-Cas13 protein-based viral RNA electrochemical detection module, comprising the following steps:

[0022] Step 1: Fabricate electrodes on an insulating substrate, the electrodes including a source electrode and a drain electrode;

[0023] Step 2: Deposit a semiconductor thin film on an insulating substrate, and use photolithography to etch the semiconductor thin film material so that the patterned semiconductor thin film serves as a conductive channel to connect the source and drain, thereby obtaining the device to be modified.

[0024] Step 3: Modify the semiconductor thin film channel surface of the device to be modified with connecting molecules;

[0025] Step 4: Attach Cas13 protein to the linker molecule to form a semiconductor thin film surface modified with Cas13 protein;

[0026] Step 5: Introduce gRNA targeting the analyte onto the surface of a semiconductor thin film modified with Cas13 protein, so that the gRNA is transferred to the Cas13 protein to prepare a Cas13-gRNA probe.

[0027] Step 6: Install the sample inlet on the insulating substrate and make it contain the surface of the semiconductor thin film to obtain the viral RNA electrical detection module based on CRISPR-Cas13 protein.

[0028] In one embodiment of the present invention, the principle of modifying the linking molecules in step 3 includes: π-π stacking, hydrogen bonding, electrostatic attraction, and covalent bonding.

[0029] In one embodiment of the present invention, the linker molecule modified in step 3 is selected from pyrene / anthracene derivatives with large conjugated planes, transition metal ligands, or gold nanoparticles.

[0030] In one embodiment of the present invention, the method for linking the Cas13 protein in step 4 is to immerse the semiconductor thin film modified with the linking molecule in a Cas13 protein solution of 1 to 100 micromoles per liter for at least 15 minutes.

[0031] In one embodiment of the present invention, the specific method for preparing the Cas13-gRNA probe in step 5 is to soak a semiconductor film modified with Cas13 protein in a 0.1 to 10 μmol / L gRNA solution for at least 15 minutes.

[0032] This invention further provides an electrochemical detection method for viral RNA based on CRISPR-Cas13 protein, the method comprising the following steps:

[0033] Step A: Connect the CRISPR-Cas13 protein-based viral RNA electrical detection module to the electrical testing system;

[0034] Step B: Heat-treat the RNA sample or treat it with viral lysis buffer to obtain the sample to be tested;

[0035] Step C: Add the sample to be tested into the sample inlet of the viral RNA electrophysiological detection module based on CRISPR-Cas13 protein, and start the test.

[0036] In one embodiment of the present invention, the heat treatment in step B is to place the sample in a constant temperature shaker with a temperature setting range of 55 to 95°C and shake for at least 30 minutes; the virus lysis buffer treatment is to place the sample in an inactivated virus preservation solution and treat for 5 to 15 minutes.

[0037] In one embodiment of the present invention, in step C, the ambient temperature required for RNA sample testing is 15–60°C.

[0038] In one embodiment of the present invention, in step C, RNA sample detection is performed in either current-time detection mode or current-gate voltage detection mode.

[0039] In one embodiment of the present invention, the current-time mode is specifically as follows:

[0040] (a) Add a certain volume of background sample to the sample inlet of the CRISPR-Cas13 protein-based viral RNA electrical detection module, adjust the electrical testing system to keep the source and drain current of the detection module constant, and when the percentage change of the source and drain current within 5 minutes is less than 0.3%, record the initial current between the source and drain electrodes to prepare for the detection of the sample to be tested.

[0041] (b) Take a certain volume of background sample from the sample inlet of the RNA electrical detection module, add the same volume of the inactivated test sample, and record the change in the current between the source and drain electrodes after the current reading stabilizes. When the ratio of the change in the current between the source and drain electrodes to the initial value of the current is less than three times the sample threshold, the test sample is considered negative; when the ratio is greater than five times the sample threshold, the test sample is considered positive; when the ratio is between three and five times the threshold, it is recommended to verify it as a positive result.

[0042] (c) The sample threshold is defined as follows: a certain volume of negative standard sample is added to the electrical detection module containing the background sample in the sample inlet, and the initial and change values ​​of the current between the source and drain electrodes are recorded after a period of time. The experiment is repeated three times, and the average of the three current changes is taken as the sample threshold.

[0043] In one embodiment of the present invention, the current-gate voltage detection mode is specifically as follows:

[0044] (a) Add a certain volume of background sample to the sample inlet of the viral RNA electrical detection module based on CRISPR-Cas13 protein, and give the source drain voltage and gate voltage scan range in the electrical test system. Start the measurement when the threshold voltage change value is less than 1 mV.

[0045] (b) Take a certain volume of background sample from the sample inlet of the CRISPR-Cas13 protein-based viral RNA electrical detection module, add the same volume of inactivated test sample, and obtain the change value of the threshold voltage; when the voltage change value is less than three times the sample threshold, the test sample is considered negative; when the voltage change value is greater than five times the sample threshold, the test sample is considered positive; when the change value is between three and five times the threshold, it is recommended to verify it as a positive result.

[0046] (c) The sample threshold is defined as follows: a certain volume of negative standard sample is added to the electrical detection module containing the background sample in the sample inlet, and the change in threshold voltage is recorded after a period of time. The experiment is repeated three times, and the average of the three threshold voltages is taken to obtain the sample threshold.

[0047] This invention achieves direct detection of RNA molecules by combining regularly spaced clustered short palindromic repeats (CRISPR)-Cas13 protein with a two-dimensional field-effect transistor (FET). The CRISPR-Cas13 protein has the ability to specifically bind RNA. When target RNA is present in the system, the Cas13 protein-gRNA complex targets the target RNA and undergoes conformational inversion, thereby activating it as a ribonuclease with non-specific degradation function. The two-dimensional FET device possesses an exposed semiconductor thin-film channel. Thanks to the atomic-level thickness and ultra-large specific surface area unique to two-dimensional materials, when the Cas13-gRNA complex captures negatively charged RNA molecules and undergoes conformational inversion, the surface potential energy of the semiconductor thin film in the transistor changes, thereby affecting the conductivity of the channel and enabling the device to provide timely and sensitive feedback.

[0048] This invention involves directly modifying the surface of a semiconductor thin film at the transistor channel with Cas13 protein. When the target molecule is present in the system, the Cas13-gRNA complex specifically binds to the target molecule and undergoes a conformational inversion. This influences the surface potential energy of the semiconductor thin film through intermolecular forces and charge effects, thereby altering the electrical performance of the field-effect transistor. Although Cas13 tends to cleave the bound RNA molecule, due to the fast response advantage of two-dimensional field-effect transistors, the targeted negatively charged RNA molecule can still accumulate on the device channel surface under suitable temperature and liquid conditions, causing changes in the channel electrical signal.

[0049] During detection, the detection module is connected to an electrical testing instrument. The sample solution is added to the injection chamber, and the presence of target RNA is determined by changes in the electrical signal. On the one hand, the detection module involved in this method has an average detection time of 5–15 minutes for RNA molecules, and a detection limit of 1 atmolar per liter (1 × 10⁻⁶). -18 (Moles per liter). On the other hand, this method does not require amplification or the use of reporter molecules with fluorescent groups, showing promising application prospects in clinical medicine. This invention successfully achieves immediate and accurate feedback on target RNA molecules.

[0050] The advantages and beneficial effects of this invention are as follows:

[0051] (1) This invention combines CRISPR-Cas13 protein with a two-dimensional field-effect transistor to obtain a novel RNA detection module. Unlike traditional Cas13 fluorescence detection, this detection strategy does not require amplification or an optical testing platform; it directly reads the detection information of RNA molecules based on electrical signals.

[0052] (2) The detection module involved in this invention can be fabricated using only a CRISPR-Cas13 system and graphene transistor devices. It possesses specific recognition capabilities and can stably bind to target RNA molecules. Compared with other electrochemical detection platforms, this invention eliminates the need for reverse transcription of RNA into DNA. The capture of RNA molecules by the Cas13 protein and the electrical signal response occur in the same system, enabling one-step sample addition and detection. Unlike other nucleic acid detection technologies that take several hours, this invention greatly simplifies the operation steps of RNA detection and shortens the time required for signal reading. These innovations facilitate low-cost, rapid, and convenient detection of target RNA, providing a new and powerful tool for epidemic prevention and control and medical clinical research. Attached Figure Description

[0053] Figure 1 This is a three-dimensional structural diagram of the viral RNA electrical detection module based on CRISPR-Cas13 protein in Example 1, where 1 represents the insulating substrate, 2 represents the electrode, 3 represents the semiconductor thin film, and 4 represents the sample inlet.

[0054] Figure 2 This is a schematic diagram of the main structure of the viral RNA electrical detection module based on CRISPR-Cas13 protein in Example 1, where 1 represents the insulating substrate, 2 represents the electrode, 3 represents the semiconductor thin film, and 4 represents the sample inlet.

[0055] Figure 3 The results of the current-gate voltage test for detecting SARS-CoV-2 (wild-type) RNA using the CRISPR-Cas13 protein-based viral RNA electrical detection module in Example 1 are shown, where the RNA concentration was increased from 0.001 copies / µL to 1000 copies / µL.

[0056] Figure 4 This is the current-gate voltage test result of the viral RNA electrophysiological detection module based on CRISPR-Cas13 protein in Example 2, which detects SARS-CoV-2 (alpha) mutant RNA. The RNA concentration was increased from 0.001 copies / µL to 1000 copies / µL.

[0057] Figure 5 The results of the current-gate voltage test for detecting different concentrations of SARS-CoV-2 (Omicron) mutant RNA using the CRISPR-Cas13 protein-based viral RNA electrical detection module in Example 3 are shown.

[0058] Figure 6 This is the relationship between the device threshold voltage shift and the sample RNA concentration obtained from three influenza A virus RNA detections using the CRISPR-Cas13 protein-based viral RNA electrical detection module in Example 4. Detailed Implementation

[0059] To enhance understanding of the present invention, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] Example 1

[0061] A method for RNA detection based on CRISPR-Cas13 protein-specific nucleic acid targeting. In this embodiment, the viral nucleic acid to be detected is SARS-CoV-2 (wild-type) RNA, wherein the designed 5' phosphorylated RNA guide strand sequence (5' phosphorylated RNA guide strand sequence, the sequence is shown in SEQ ID NO.1) is 5'-GACCA CCCCA AAAAU GAAGGGGACU AAAAC ACGUA GUCGC AACAG UUCAA-3'.

[0062] Electrode patterns are obtained by etching photoresist spin-coated onto the surface of a SiO2 / Si substrate (with SiO2 as the top layer) using a photolithography machine. A 5-nanometer chromium layer is then deposited at the electrode pattern, followed by a 20-nanometer gold layer to obtain a composite metal electrode. The electrode structure includes a source and a drain.

[0063] A semiconductor thin film is grown on a metal substrate, and 8% by weight of polymethyl methacrylate (PMMA) is spin-coated onto the surface. The PMMA layer is then separated from the copper foil using an electrochemical bubbling method. The PMMA layer is subsequently transferred to a SiO2 / Si substrate and connected between the source and drain electrodes. The semiconductor thin film is preferably graphene. The film is sequentially rinsed with acetone, ethanol, and deionized water. The semiconductor thin film is then etched into channels of a specific shape using photolithography and oxygen plasma etching to obtain the device to be modified.

[0064] The semiconductor film on the device to be modified was immersed in an acetone solution of 5 mmol / L 1-pyrenebutyrate N-hydroxysuccinimide (PASE) for 5 hours. It was then washed with anhydrous ethanol and deionized water. The semiconductor film was then immersed in a Cas13 protein solution of 30 μmol / L using a magnesium chloride solution of 2 mmol / L for 30 minutes. After immersion, it was rinsed with a pure magnesium chloride solution of 5 mmol / L. The semiconductor film was then incubated in a double-distilled aqueous solution of gRNA of 1 μmol / L for 30 minutes. After incubation, it was rinsed twice with a magnesium chloride solution of 5 mmol / L.

[0065] A polydimethylsiloxane (PDMS) injection cell was fabricated and placed in a graphene channel to obtain an RNA detection module, in which the injection cell can hold approximately 60 microliters of liquid.

[0066] At this point, the viral RNA electrophysiological detection module based on the CRISPR-Cas13 protein has been fabricated, with the structure shown below. Figure 1 , Figure 2 As shown, Figure 1 In the diagram, 1 represents the insulating substrate, 2 represents the electrode, 3 represents the semiconductor thin film, and 4 represents the sample inlet.

[0067] The RNA detection module is connected to the electrical testing system, with the drain of the detection module connected to the negative terminal of the electrical testing system and the source terminal connected to the positive terminal of the electrical testing system.

[0068] Preliminary experiments revealed that when the ambient temperature is too low, the activity of the Cas13 protein is weak, making it difficult to efficiently capture RNA chains; while when the temperature is too high, the cleavage activity of the Cas13 protein on the target RNA chain is activated, weakening the change in the transistor's electrical signal. Therefore, the recommended temperature range for testing is 15–60°C, with the temperature range of 25–35°C used in the examples.

[0069] When RNA samples are directly added to the detection module, the presence of the RNA secondary stem-loop structure reduces the capture efficiency of the Cas13-gRNA probe. To address this issue, a heat treatment process for RNA is introduced in this embodiment. By placing the RNA sample in a constant-temperature shaker and oscillating at 56°C for 20 minutes, the stem-loop structure is broken down to obtain long-chain RNA, effectively improving the transistor's signal response.

[0070] During testing, 60 μL of artificial saliva (neutral) background buffer was first added to the sample inlet. Current-gate voltage detection was used as the selected mode in the electrical testing system, with the source-drain voltage set at 0.05 volts and the gate voltage scan range at 0–0.5 volts. Testing of the RNA sample began when the threshold voltage or Dirac point change was less than the instrument's voltage resolution. The RNA sample used in this example was a standard sample extracted from the nucleic acid of the novel coronavirus SARS-CoV-2 (wild type). During sample addition, the concentration of the test sample was increased in a gradient from 0.01 copies / µL to 1000 copies / µL. After each addition of a higher concentration of RNA sample, the sample was allowed to stand for 15 minutes before acquiring the signal. The absolute value of the Dirac point change before and after sample addition was used as the read signal. Specifically, 6 μL of artificial saliva (neutral) buffer was first drawn from the sample inlet, followed by the same volume of RNA sample solution.

[0071] Figure 3 The current-gate voltage response curve of the CRISPR-Cas13 protein-based viral RNA electrical detection module in Example 1 for sample RNA shows that the sensor still has a sensitive response for low concentrations of sample RNA, with a detection limit of 1 atmolar per liter (1 × 10⁻⁶). -18 (moles / liter)

[0072] Example 2

[0073] The difference between this embodiment and Embodiment 1 is that the viral nucleic acid detected is the RNA of the novel coronavirus SARS-CoV-2 mutant (alpha), and the designed guide RNA sequence (guide RNA sequence 1, as shown in SEQ ID NO.2) is 5'-GACCA CCCCA AAAAU GAAGG GGACU AAAAC CCAAC ACCAU AAGUG GGUUG-3'.

[0074] The modification of the linker molecules involved immersing the graphene on the device to be modified in a dimethyl sulfoxide solution of 1-pyrene butyrate N-hydroxysuccinimide ester (PASE) at 5 mmol / L for 2 hours; the modification parameters for the Cas13 protein were immersion in a Cas13 protein solution at 50 μmol / L for 15 minutes at room temperature; and the modification parameters for the guide RNA were immersion in a gRNA solution at 5 μmol / L for 15 minutes at room temperature.

[0075] The current-gate voltage detection mode was selected as the electrical testing system mode, and the source-drain voltage value was set to 0.05 volts, with a gate voltage scan range of 0–0.5 volts. Testing of the RNA sample began when the threshold voltage or Dirac point variation was less than 1 mV, the instrument's voltage resolution. The RNA sample used in this example was a standard sample extracted from the nucleic acid of the SARS-CoV-2 mutant (alpha).

[0076] Figure 4 The current-gate voltage response curve of the field-effect transistor sensor in Example 2 for the novel coronavirus mutant (alpha) RNA shows that the sensor still has a sensitive response even at low concentrations of sample RNA. For a viral sample solution of 10 copies / µL, the threshold voltage offset is 30 mV, and the detection limit is 0.1 copies / µL.

[0077] Example 3

[0078] A method for RNA detection based on CRISPR-Cas13 protein-specific nucleic acid targeting. In this embodiment, the viral nucleic acid to be detected is the RNA of the novel coronavirus SARS-CoV-2 mutant (Omecron). The designed guide RNA sequence (guide RNA sequence 2, as shown in SEQ ID NO.3) is 5'-GACCA CCCCA AAAAU GAAGG GGACU AAAACUUGUU UUUGU GGUAA ACACC-3'.

[0079] Electrode patterns were obtained by etching photoresist spin-coated onto a SiO2 / Si substrate (with SiO2 as the top layer) using a photolithography machine. A 20 nm aluminum layer was first deposited onto the electrode pattern, followed by a 10 nm gold layer to obtain metal electrodes. The electrode structure included a source and a drain. Two-dimensional graphene crystal material was then transferred onto the channel to obtain the modified graphene transistor device.

[0080] The graphene film on the device to be modified was immersed in a 5 mM / L solution of 1-pyrenebutyrate N-hydroxysuccinimide (PASE) for 6 hours. It was then rinsed twice with anhydrous ethanol and deionized water, respectively. The semiconductor film was then immersed in a 50 μM / L solution of Cas13 protein (using a 2 mM / L magnesium chloride solution) at 4°C for 12 hours. After immersion, it was rinsed twice with a 5 mM / L magnesium chloride solution. Finally, the graphene was immersed in a 5 μM / L double-distilled gRNA aqueous solution at 4°C for 12 hours, and then rinsed twice with a 2 mM / L magnesium chloride solution.

[0081] A polydimethylsiloxane (PDMS) injection chamber was fabricated and placed on a graphene channel. The injection chamber could hold approximately 40 microliters of liquid, resulting in an RNA detection module. The RNA detection module was then connected to an electrical testing system, with the drain of the detection module connected to the negative electrode of the electrical testing system and the source of the detection module connected to the positive electrode of the electrical testing system.

[0082] 40 μL of artificial saliva (neutral) background buffer was added to the PDMS tank. Current-gate voltage detection was used as the selected mode in the electrical testing system to determine the source-drain voltage values ​​and the gate voltage scan range. Testing of the RNA sample began when the threshold voltage or Dirac point change was less than 1 mV of the instrument voltage resolution. The RNA sample used in this example was a standard sample extracted from the nucleic acid of the SARS-CoV-2 mutant (Omeprone).

[0083] During testing, the sample concentration was increased in a gradient from 0.001 copies / µL to 1000 copies / µL. After each addition of a higher concentration of RNA sample to the injection chamber, a 15-minute wait was required before acquiring the signal. The absolute value of the change in the Dirac point before and after injection (|ΔV) was recorded. Dirac |) serves as the read signal. The sample loading procedure is as follows: first, draw 5 μL of artificial saliva (neutral) buffer from the sample inlet, and then add the same volume of RNA sample solution.

[0084] Figure 5 The current-gate voltage response curve of the field-effect transistor sensor for sample RNA in Example 3 shows that the detection module has a high sensitivity response for the SARS-CoV-2 Omeprone mutant, with a detection limit of 1 atmolar per liter (1×10⁻⁶). -18 (moles / liter)

[0085] Example 4

[0086] The difference between this embodiment and Embodiment 1 is that the viral nucleic acid detected is influenza A virus, and the designed guide RNA sequence (guide RNA sequence 3, as shown in SEQ ID NO.4) is 5'-AAUUU CUACU GUUGU AGAUU CACAACAUUU GCCAG U-3'.

[0087] The linker molecule modification method involved immersing the graphene on the device to be modified in an acetone solution of 1-pyrenebutyrate N-hydroxysuccinimide ester (PASE) at 5 mmol / L for 6 hours; the Cas13 protein modification parameters were immersion in a Cas13 protein solution at 20 μmol / L for 1 hour at room temperature; and the guide RNA modification parameters were immersion in a gRNA solution at 1 μmol / L for 1 hour at room temperature.

[0088] Current-gate voltage sensing was selected as the mode in the electrical testing system, with the source-drain voltage value set at 0.1 volts and the gate voltage scan range from -0.2 to 0.4 volts. The RNA sample used in this embodiment was a standard influenza A virus sample.

[0089] Figure 6 This is a summary of the results of the three-stage field-effect transistor sensor experiments for influenza A virus RNA in Example 4. It can be found that the sensor still has a sensitive response to low concentrations of sample RNA and has a good positive correlation, with a detection limit of 0.1 copies / µL.

[0090] Example 5

[0091] The difference between this embodiment and Embodiment 1 is that this embodiment tests upper respiratory tract pharyngeal swab samples of the novel coronavirus SARS-CoV-2 (wild type). Pharyngeal swab samples were obtained from a designated hospital, totaling 5 positive samples and 3 negative samples, all of which were verified by PCR. Eight samples were tested separately. Before testing, the pharyngeal swabs were placed in a virus sampling tube containing 3.5 mL of sampling solution and directly heated at 56°C for 30 minutes to induce viral lysis and release RNA. During testing, 40–50 μL of artificial saliva (neutral) buffer was first added to the sample inlet, followed by 5–6 μL of the heat-treated sampling solution. The signal was read after 5–10 minutes, and the signal was read as the change in threshold voltage.

[0092] The test results of 8 pharyngeal swab samples in this embodiment are compared with those of PCR detection, as shown in Table 1. It can be seen that the viral RNA detection module has good consistency with PCR detection.

[0093] Table 1 shows the test results of 8 pharyngeal swab samples in the examples and the comparison results with PCR detection.

[0094]

[0095] Example 6

[0096] The difference between this embodiment and Embodiment 1 is that this embodiment designs four types of probes to detect artificially synthesized RNA samples of Zika virus (ZIKV), Ebola virus (Ebola), human papillomavirus (HPV), and rhvirus (RhV). The corresponding probes are as follows:

[0097] ZIKV (probe 1, sequence as shown in SEQ ID NO. 5): 5'-GGAUA CUUCA UGAUG CCAGC UGCUGUUC-3'

[0098] Ebola (probe 2, sequence as shown in SEQ ID NO. 6): 5'-GUACA UGCAG AGCAA GGACU GAUACAAU-3'

[0099] HPV (probe 3, sequence as shown in SEQ ID NO.7): 5'-UCAAA GACUU CAUCU AUACC GUCGUGUAUU ACUGU AUAA-3'

[0100] RhV (probe 4, sequence as shown in SEQ ID NO. 8): 5'-AAGUC CUGCC UGCCU GCCUG UGUGUAUGGA-3'

[0101] In this embodiment, a molybdenum disulfide (MoD) field-effect transistor is used. The MoD is obtained by mechanical exfoliation, and a polypropylene carbonate (PC) film is spin-coated onto the surface of the MoD. Through physical adhesion, the MoD is successfully bonded to the transistor channel with the support of the PC film. The PC film is then dissolved with acetone to obtain a field-effect transistor with MoD as the channel.

[0102] During testing, 40–50 μL of artificial saliva neutral buffer was added to the sample inlet. After the threshold voltage stabilized, non-target background RNA nucleic acid strands (negative samples) were added first, and the threshold voltage change was read after 5–15 minutes. Then, targeted target RNA nucleic acid samples (positive samples) were added, and the threshold voltage change was read a second time after the same time interval. The sample threshold used for positive / negative determination was obtained as described in the instruction manual. Table 2 summarizes the test results of negative and positive samples for the four viral probes in this embodiment. It can be seen that this viral RNA detection module has good applicability to different virus types and can significantly distinguish target virus species.

[0103] Table 2 shows the positive and negative test results of the four viral probes in the examples.

[0104]

[0105] Example 7

[0106] The difference between this embodiment and Embodiment 1 is that this embodiment uses six Cas13 proteins—LwaCas13a, LbaCas13a, PguCas13b, PspCas13b, EsCas13d, and RfxCas13d—to detect the RNA of the wild-type SARS-CoV-2 novel coronavirus. The common guide RNA sequence used is (5' phosphorylated RNA guide strand sequence, as shown in SEQ ID NO. 1): 5'-GACCA CCCCA AAAAU GAAGG GGACU AAAAC ACGUA GUCGC AACAG UUCAA-3'

[0107] The channel portion of the transistor device is made of two-dimensional black phosphorus material, obtained by mechanically exfoliating the black phosphorus and then thinning it with argon ions. The protein modification strategy involves immersing the two-dimensional semiconductor film in a 30 μmol / L Cas13 protein solution for 30 minutes, using a 2 mM / L magnesium chloride solution as the solvent. After immersion, it is rinsed with a 5 mM / L pure magnesium chloride solution. The semiconductor film is then incubated in a 1 μmol / L gRNA double-distilled aqueous solution for 30 minutes. After incubation, it is rinsed twice with a 5 mM / L magnesium chloride solution.

[0108] During testing, add 40–50 μL of artificial saliva neutral buffer to the sample inlet. After the threshold voltage stabilizes, add 100 copies / μL of the target RNA nucleic acid strand (positive sample). After 15 minutes, read the change in threshold voltage. When the voltage change is less than three times the sample threshold, the sample is considered negative; when the voltage change is greater than five times the sample threshold, the sample is considered positive; when the change is between three and five times the threshold, it is recommended to confirm the result as positive.

[0109] The test results of different types of Cas13 proteins are shown in Table 3, which confirms that under the protein modification and test conditions, Cas13 proteins can still maintain their activity in transistor devices and achieve targeted recognition of target nucleic acid molecules.

[0110] Table 3 shows the test results of six different Cas13 proteins on positive samples in the examples.

[0111]

[0112] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A viral RNA electrophysiological detection module based on CRISPR-Cas13 protein, characterized in that, This module includes: Insulating substrate; The source and drain electrodes are disposed on the insulating substrate; A semiconductor thin film disposed on the insulating substrate and located between the source and drain electrodes; A Cas13-gRNA probe is attached to a semiconductor thin film, wherein the Cas13 protein is attached to the surface of the semiconductor thin film via covalent bonds or van der Waals forces, and the gRNA binds to the Cas13 protein to form a complex. In this process, a conductive channel is formed between the source and drain of the semiconductor thin film, and a sample inlet is provided on the insulating substrate, with the surface of the semiconductor thin film contained within the sample inlet. The Cas13 protein is a class VI protein in the interstitial palindromic repeat cluster / interstitial palindromic repeat cluster associated protein system, which can bind to guide RNA to achieve specific recognition of nucleic acids. The gRNA is a guide RNA, which is an oligonucleotide chain of 35-60 bases in length. It can bind to the Cas13 protein to form a binary complex and guide the Cas13 protein to target the target RNA molecule. The virus is selected from one of the following: influenza virus, hepatitis virus, rhinovirus, coronavirus, HIV, or 2019 novel coronavirus; The detection limit of this detection module is 1 atmolar per liter.

2. The viral RNA electrophysiological detection module based on CRISPR-Cas13 protein according to claim 1, characterized in that, The insulating substrate is selected from silicon dioxide / silicon substrate, quartz or polymer film; The source and drain electrodes are metallic materials, selected from one or a combination of gold, copper, silver, chromium, and aluminum. The thickness of the source and drain electrodes is 10~100 nanometers; The semiconductor thin film is selected from graphene, two-dimensional transition metal chalcogenides, silicon, or black phosphorus.

3. The viral RNA electrophysiological detection module based on CRISPR-Cas13 protein according to claim 1, characterized in that, The Cas13 protein is derived from the following species: Leptotrichia wadei , Porphyromonas gulae , Lachnospiraceae bacterium , Ruminoccocus flavefaciens , Eubacterium siraeum , Prevotella bacterium .

4. The method for preparing the viral RNA electrophysiological detection module based on CRISPR-Cas13 protein according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Fabricate electrodes on an insulating substrate, the electrodes including a source electrode and a drain electrode; Step 2: Deposit a semiconductor thin film on an insulating substrate, and use photolithography to etch the semiconductor thin film material so that the patterned semiconductor thin film serves as a conductive channel to connect the source and drain, thereby obtaining the device to be modified. Step 3: Modify the semiconductor thin film channel surface of the device to be modified with connecting molecules; Step 4: Attach Cas13 protein to the linker molecule to form a semiconductor thin film surface modified with Cas13 protein; Step 5: Introduce gRNA targeting the analyte onto the surface of a semiconductor thin film modified with Cas13 protein, so that the gRNA is transferred to the Cas13 protein to prepare a Cas13-gRNA probe. Step 6: Install the sample inlet on the insulating substrate and make it contain the surface of the semiconductor thin film to obtain the viral RNA electrical detection module based on CRISPR-Cas13 protein.

5. The method for preparing the viral RNA electrophysiological detection module based on CRISPR-Cas13 protein according to claim 4, characterized in that, The principles of modifying and linking molecules in step 3 include: π-π stacking, hydrogen bonding, electrostatic attraction, and covalent bonding; The linker molecules modified in step 3 are selected from pyrene / anthracene derivatives with large conjugated planes, transition metal ligands, or gold nanoparticles. The method for linking the Cas13 protein in step 4 is to immerse the semiconductor film modified with the linker molecules in a Cas13 protein solution of 1 to 100 mol per liter for at least 15 minutes. The specific method for preparing the Cas13-gRNA probe in step 5 is to immerse the semiconductor film modified with Cas13 protein in a 0.1~10 μmol / L gRNA solution for at least 15 minutes.

6. A method for the electrochemical detection of viral RNA based on CRISPR-Cas13 protein, characterized in that, The method includes the following steps: Step A: Connect the viral RNA electrical detection module based on CRISPR-Cas13 protein as described in any one of claims 1-3 to the electrical testing system; Step B: Heat-treat the RNA sample or treat it with viral lysis buffer to obtain the sample to be tested; Step C: Add the sample to be tested into the sample inlet of the viral RNA electrophysiological detection module based on CRISPR-Cas13 protein, and start the test.

7. The method for electrochemical detection of viral RNA based on CRISPR-Cas13 protein according to claim 6, characterized in that, In step B, the heat treatment method is to place the sample in a constant temperature shaker with a temperature setting range of 55~95 ℃ and shake for at least 30 minutes; the virus lysis buffer treatment method is to place the sample in an inactivated virus preservation solution and treat for 5~15 minutes.

8. The method for electrochemical detection of viral RNA based on CRISPR-Cas13 protein according to claim 6, characterized in that, In step C, the ambient temperature required for RNA sample testing is 15~60 ℃.

9. The method for electrochemical detection of viral RNA based on CRISPR-Cas13 protein according to claim 6, characterized in that, In step C, RNA sample detection is performed in either current-time detection mode or current-gate voltage detection mode; The current-time mode is as follows: (a) Add a certain volume of background sample to the sample inlet of the viral RNA electrical detection module based on CRISPR-Cas13 protein, adjust the electrical testing system to keep the source and drain current of the detection module constant, and when the percentage change of the source and drain current within 5 minutes is less than 0.3%, record the initial current between the source and drain electrodes to prepare for the detection of the sample to be tested. (b) Take a certain volume of background sample from the sample inlet of the RNA electrical detection module, add the same volume of the inactivated test sample, and record the change in the current between the source and drain electrodes after the current reading stabilizes. When the ratio of the change in the current between the source and drain electrodes to the initial value of the current is less than three times the sample threshold, the test sample is considered negative; when the ratio is greater than five times the sample threshold, the test sample is considered positive; when the ratio is between three and five times the threshold, it is recommended to verify it as a positive result. (c) The sample threshold is defined as follows: In the electrical detection module containing the background sample in the sample inlet, a certain volume of negative standard sample is added. After a period of time, the initial and change values ​​of the current between the source and drain electrodes are recorded. The experiment is repeated three times. The change value of the current each time is divided by the initial value and the average of the three data is taken to obtain the sample threshold. The current-gate voltage detection mode is as follows: (a) Add a certain volume of background sample to the sample inlet of the viral RNA electrical detection module based on CRISPR-Cas13 protein, and give the source drain voltage and gate voltage scan range in the electrical test system. Start the measurement when the threshold voltage change value is less than 1 mV. (b) Take a certain volume of background sample from the sample inlet of the CRISPR-Cas13 protein-based viral RNA electrical detection module, add the same volume of inactivated test sample, and obtain the change value of the threshold voltage; when the voltage change value is less than three times the sample threshold, the test sample is considered negative; when the voltage change value is greater than five times the sample threshold, the test sample is considered positive; when the change value is between three and five times the threshold, it is recommended to verify it as a positive result. (c) The sample threshold is defined as follows: a certain volume of negative standard sample is added to the electrical detection module containing the background sample in the sample inlet, and the change value of the threshold voltage is recorded after a period of time. The experiment is repeated three times, and the average value of the three threshold voltages is taken to obtain the sample threshold.

Images