Protein detection system and method based on single-droplet friction nano-generator and CRISPR-Cas trans-cleavage system

Abstract

The invention discloses a protein detection system and method based on a single-droplet friction nano-generator and a CRISPR-Cas trans-cutting system.The detection scheme provided by the invention is simple and convenient in design process, firstly, a gold wire electrode is fixed to the surface of a FEP friction layer of a dielectric layer of the friction nano-generator, a DNA tetrahedron is assembled, and a single-droplet friction nano-generator is obtained; three vertexes at the bottom are modified with sulfydryl and fixed with a gold wire electrode, and single-stranded DNA extends from the vertexes and is used for capturing PDA signal molecules and changing triboelectricity output; after the target protein is subjected to a binding reaction with dumbbell-shaped DNA with 5 'flap in a microcentrifuge tube, a CRISPR / Cas trans-cleavage function is activated, so that PDA cannot be fixed, finally, high-sensitivity quantitative detection of the target protein can be realized through friction electric signal detection, and the detection range is 0.001-1000 mU.

Description

Technical Field

[0001] The present invention relates to the field of high-sensitivity nanoanalysis technology, and in particular to a protein detection system and method based on a single-droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system. Background Art

[0002] Protein detection is an important field in life science research, which involves the identification, quantification and functional analysis of proteins in organisms. With the development of science and technology, protein detection methods are also constantly improving. Researchers have developed a series of protein detection methods, such as Western Blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), etc. These detection technologies have their own advantages and disadvantages. For example, the immunoblotting method is time-consuming, the operation steps are cumbersome, it is easy to have false positive or false negative results, and it is difficult to achieve high-throughput detection; the sensitivity of the enzyme-linked immunosorbent assay is limited by the antibodies used, it is not suitable for detecting low-abundance proteins in complex samples, and the cost is relatively high; the mass spectrometry equipment is expensive, and professional technicians are required for operation and data analysis, and the processing time and cost are high; the surface plasmon resonance instrument is expensive and has limited detection capabilities for certain types of protein interactions; fluorescence resonance energy transfer requires fluorescent labeling of proteins, which may interfere with the natural state and function of proteins.

[0003] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a protein detection system and method based on a single-droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system in view of the deficiencies in the above-mentioned prior art.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: In the first aspect of the present invention, a protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system is provided, comprising: CRISPR / Cas12a trans-cleavage system; DNA tetrahedron; it is formed by hybridization reaction of four single-stranded DNA sequences: TA, TB, TC, and TD. Each of the four single-stranded DNA sequences contains three functional regions, which form six sides of the DNA tetrahedron through complementary pairing. The TA chain on the DNA tetrahedron is a protruding long chain, which serves as a substrate for the CRISPR / Cas12a trans-cutting system and is also used to adsorb PDA; Dumbbell-shaped DNA; it can bind to the target protein to be detected to form a double-stranded structure, activate the CRISPR / Cas12a trans-cutting system, and thus cut the single-stranded DNA sequence on the DNA tetrahedron; and triboelectric nanogenerators; DNA tetrahedrons are pre-modified on the gold wire electrode of the friction nanogenerator. In the presence of target protein, the TA chain on the DNA tetrahedron is cut by CRISPR / Cas, and the polydopamine particles cannot bind to the DNA tetrahedron, so that the gold wire electrode will not be covered by PDA and the friction electric signal of the friction nanogenerator remains unchanged. When the target protein is not present, the TA chain on the DNA tetrahedron binds to the polydopamine particles, which weakens the friction electric signal of the friction nanogenerator, thereby achieving quantitative detection of the target protein by analyzing the friction electric signal.

[0006] Preferably, the DNA tetrahedron is denoted as TDNA, and the ends of TB, TC, and TD in TDNA are all modified with SH groups to form a triangular base of the DNA tetrahedron and connected to the gold wire electrode.

[0007] Preferably, the target protein is Flap endonuclease 1, denoted as FEN1.

[0008] Preferably, the dumbbell-shaped DNA has a 5' flap structure.

[0009] Preferably, the sequences of TA, TB, TC and TD are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0010] Preferably, the dumbbell-shaped DNA is denoted as dbDNA, and its sequence is shown in SEQ ID NO.5.

[0011] Preferably, the friction nanogenerator comprises an inclined substrate, a copper foil electrode and a fluorinated ethylene propylene film stacked in sequence on the substrate, and a gold wire electrode disposed in the middle of the surface of the fluorinated ethylene propylene film.

[0012] In a second aspect of the present invention, a detection method of a protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system as described above is provided, comprising the following steps: S1. Preparation of DNA tetrahedron: TA, TB, TC, and TD were mixed in an equimolar ratio and added to a Tris-HCl buffer, incubated at 90-95°C for 2-10 minutes, and then cooled to room temperature to obtain a DNA tetrahedron: TDNA; S2, mixing the sample solution to be tested with FEN1, dbDNA, and ThermoPol reaction buffer, incubating at 37 °C for 0.5-2 hours, then adding T4 DNA ligase, reacting at room temperature for 30-90 minutes, to obtain a mixture 1; S3, LbaCas12a, crRNA and Mn 2+ Mix and react for 5-20 minutes to construct a CRISPR / Cas12a trans-cleavage system; S4, mixing the DNA tetrahedron obtained in step S1, the mixture 1 obtained in step S2, and the CRISPR / Cas12a trans-cleavage system obtained in step S3, and reacting at 37 ° C for 15-60 minutes to obtain CRISPR / Cas12a-treated TDNA; S5. Gold wire electrode treatment: soak the gold wire electrode in the CRISPR / Cas12a-treated TDNA obtained in step S4 overnight, take it out and rinse it with ultrapure water, blow it dry, then soak it in PDA for 1-6 hours, take it out and rinse it, and blow it dry; S6. Assembling the triboelectric nanogenerator: The substrate is tilted, a copper foil electrode is laid on the substrate, and then a fluorinated ethylene propylene film of the same size is laid on the copper foil electrode, and the treated gold wire electrode obtained in step S5 is fixed on the surface of the fluorinated ethylene propylene film to obtain a friction nanogenerator; S7. Connect an electrometer between the gold wire electrode and the copper foil electrode, output pure water droplets through the hanging bottle, and drip them onto the fluorinated ethylene propylene membrane of the friction nanogenerator, record the current signal detected by the electrometer at the moment of single droplet falling, and obtain the concentration of the target protein in the sample solution to be tested based on the current signal analysis.

[0013] Preferably, the detection method of the protein detection system based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system comprises the following steps: S1. Preparation of DNA tetrahedron: TA, TB, TC, and TD were mixed in equal molar amounts and added to a mixture containing TCEP and MgCl 2 Tris-HCl buffer, incubated at 95 °C for 5 minutes, and then cooled to room temperature to obtain DNA tetrahedron: TDNA; S2, the sample solution to be tested was mixed with FEN1, dbDNA, and ThermoPol reaction buffer, incubated at 37 °C for 1 hour, and then T4 DNA ligase was added and reacted at room temperature for 45 minutes to obtain a mixture 1; S3, LbaCas12a, crRNA and Mn 2+ Mix for 10 minutes to construct a CRISPR / Cas12a trans-cleavage system; S4, mixing the DNA tetrahedron obtained in step S1, the mixture 1 obtained in step S2, and the CRISPR / Cas12a trans-cleavage system obtained in step S3, and reacting at 37 ° C for 0.5 hour to obtain CRISPR / Cas12a-treated TDNA; S5. Gold wire electrode treatment: soak the gold wire electrode in the CRISPR / Cas12a-treated TDNA obtained in step S4 overnight, take it out and rinse it with ultrapure water, blow it dry, then soak it in PDA for 3 hours, take it out and rinse it, and blow it dry; S6. Assembling the triboelectric nanogenerator: The substrate is tilted, a copper foil electrode is laid on the substrate, and then a fluorinated ethylene propylene film of the same size is laid on the copper foil electrode, and the treated gold wire electrode obtained in step S5 is fixed on the surface of the fluorinated ethylene propylene film to obtain a friction nanogenerator; The substrate is a rectangular structure made of polyvinyl chloride; S7. Connect an electrometer between the gold wire electrode and the copper foil electrode, output pure water droplets through the hanging bottle, and drip them onto the fluorinated ethylene propylene membrane of the friction nanogenerator, record the current signal detected by the electrometer at the moment of single droplet falling, and obtain the concentration of the target protein in the sample solution to be tested based on the current signal analysis.

[0014] Preferably, the sequence of crRNA is as shown in SEQ ID NO.6.

[0015] The beneficial effects of the present invention are: The present invention provides a protein detection system and method based on a single-droplet friction nanogenerator and a CRISPR-Cas trans-cutting system. The detection scheme provided by the present invention has a simple design process. First, a gold wire electrode is fixed on the surface of the friction layer of the friction nanogenerator dielectric layer polyperfluoroethylene propylene (FEP), and a DNA tetrahedron is assembled. The three vertices at the bottom are modified with thiol groups and fixed with the gold wire electrode. The vertices are extended with single-stranded DNA to capture polydopamine (PDA) signal molecules and change the triboelectric output. After the target protein binds to the dumbbell-shaped DNA with a 5'flap in a microcentrifuge tube, the CRISPR / Cas trans-cutting function is activated, so that PDA cannot be fixed. Finally, through triboelectric signal detection, high-sensitivity quantitative detection of the target protein can be achieved, and the detection range is 0.001-1000 mU. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the principle of the protein detection method based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system of the present invention; Figure 2 It is a schematic diagram of the system structure of the protein detection system based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system of the present invention; Figure 3 The current output diagram (A) and the significant difference diagram (B) of the gold wire electrode modified by the reaction solution after being treated with TDNA, FEN1 and PDA respectively; Figure 4 Infrared spectra of dopamine and polydopamine (A), and DNA assembly electrophoresis analysis results (B); Figure 5 The output current of the target protein with a detection concentration of 0.001-1000 mU changes with time (A), and the relationship between the peak current and the logarithm of the target concentration (B). DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0018] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0019] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0020] The present invention provides a protein analysis system based on a triboelectric nanogenerator, which combines a DNA tetrahedral nanostructure and a CRISPR / Cas trans-cleavage system to enhance the detection signal, and quantitatively analyzes the target protein Flap endonuclease 1 (FEN1) through the triboelectric nanogenerator detection technology. FEN1 is a ubiquitous nuclease with structural selectivity and sequence independence, and can specifically recognize and cut the bifurcated DNA structure (5'flap) at the 5' end of double-stranded DNA, thereby generating a complete double-stranded DNA. At present, most triboelectric nanogenerators are in a vertical contact separation mode, with a single detection form and low detection sensitivity. The triboelectric nanogenerator designed in the present invention is in an independent layer mode, which generates electricity by friction between a single droplet and a gold wire electrode. The product of a series of related reactions of the target protein FEN1 pre-incubated on the gold wire electrode, and the high-intensity triboelectric signal collected can be used to characterize trace amounts of target proteins.

[0021] The above is the overall concept of the present invention, and detailed embodiments and comparative examples are provided below on the basis of the overall concept of the present invention to further illustrate the present invention.

[0022] The nucleic acid sequences involved in the following examples are shown in Table 1: Table 1 Example 1 A protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system, comprising: CRISPR / Cas12a trans-cleavage system; DNA tetrahedron; it is formed by hybridization reaction of four single-stranded DNA sequences: TA, TB, TC, and TD. The TA chain on the DNA tetrahedron is a protruding long chain, which serves as a substrate for the CRISPR / Cas12a trans-cutting system and is also used to adsorb PDA; Dumbbell-shaped DNA; it can bind to the target protein to be detected to form a double-stranded structure, activate the CRISPR / Cas12a trans-cutting system, and thus cut the single-stranded DNA sequence on the DNA tetrahedron; and triboelectric nanogenerators; DNA tetrahedrons are pre-modified on the gold wire electrode of the friction nanogenerator. In the presence of target protein, the TA chain on the DNA tetrahedron is cut by CRISPR / Cas, and the polydopamine particles cannot bind to the DNA tetrahedron, so that the gold wire electrode will not be covered by PDA and the friction electric signal of the friction nanogenerator remains unchanged. When the target protein is not present, the TA chain on the DNA tetrahedron binds to the polydopamine particles, which weakens the friction electric signal of the friction nanogenerator, thereby achieving quantitative detection of the target protein by analyzing the friction electric signal.

[0023] Reaction principle Figure 1 As shown, Figure 1 In the figure, Ligase means ligase; Cleavage means enzyme cleavage reaction; Dumbbell-structured means dumbbell-shaped structure; Tetrahedra means DNA tetrahedron. First, dumbbell-shaped DNA is designed. The target protein can bind to the dumbbell-shaped DNA. Further, a double-stranded structure is formed to activate the CRISPR / Cas trans-cutting system to cut the single-stranded DNA sequence on the tetrahedron. The DNA tetrahedron is composed of four single-stranded DNA sequences: TA, TB, TC, and TD. After hybridization reaction, tetrahedral DNA can be formed. In this nanostructure, the TA chain is a protruding long chain, and the other three chains are labeled with SH groups, which are located at the three vertices of the DNA tetrahedron. The friction probe Au wire has modified the DNA tetrahedron in advance. In the presence of the target protein, the single-stranded DNA on the DNA tetrahedron is cut by CRISPR / Cas, the polydopamine particles cannot bind to the single-stranded DNA on the DNA tetrahedron, the Au wire will not be covered by PDA, and the friction medium will not change. Correspondingly, when the target protein is not present, the single-stranded DNA on the DNA tetrahedron is intact, and can then adsorb PDA nanoparticles. The friction medium on the Au wire changes, causing the triboelectric signal to weaken. By analyzing the triboelectric signal response, the target protein can be highly sensitively quantitatively analyzed.

[0024] In this embodiment, the DNA tetrahedron is denoted as TDNA, and the ends of TB, TC, and TD in TDNA are all modified with SH groups to form a triangular base of the DNA tetrahedron and connected to the gold wire electrode.

[0025] In this example, the target protein is Flap endonuclease 1, denoted as FEN1.

[0026] In this embodiment, the dumbbell-shaped DNA has a 5'flap structure.

[0027] In this embodiment, the sequences of TA, TB, TC, and TD are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0028] In this embodiment, the dumbbell-shaped DNA is recorded as dbDNA, and its sequence is shown in SEQ ID NO.5.

[0029] In this embodiment, the friction nanogenerator includes an inclined substrate, a copper foil electrode and a fluorinated ethylene propylene film (FEP film) stacked in sequence on the substrate, and a gold wire electrode arranged in the middle of the surface of the fluorinated ethylene propylene film.

[0030] Reference Figure 2 A, TENG (friction nanogenerator) device is placed on the angle switching bracket, and the TENG device is connected to the 6514 electrometer for signal conversion. The droplet generating device is a hanging bottle fixed by an iron frame. The optimal angle, drop height and other parameters can be obtained by condition optimization. The detailed working mechanism of the single droplet generator (SDEG) is shown in Figure 1. Figure 2 B (Ⅰ-Ⅳ). When the droplet falls on the FEP film, the droplet spreads rapidly on the surface of the FEP film, thereby expanding the contact area of ​​the solid-liquid interface. In this process, due to the contact charging effect, the water droplet is positively charged and the FEP film is negatively charged. Then, as the droplet expands and slides, the droplet eventually contacts the gold wire electrode. Once the droplet contacts the gold wire electrode, an electrical connection is established between the surface of the FEP film and the gold wire electrode. In this case, the positive charge is attracted to the surface of the FEP film, while the negative charge flows to the gold wire electrode, forming an unbalanced potential at the contact position between the water and the electrode. Then, the negative charge flows from the gold wire electrode to the ground to maintain the overall charge balance in the system, generating a negative current. As the droplet continues to expand, the maximum surface area decreases due to the effect of surface tension, causing the droplet to shrink toward the center. At the same time, due to the effect of gravity, the droplet continues to slide downward, reducing the contact area of ​​the solid-liquid interface. This process continues until the droplet finally separates from the gold wire electrode. At this time, the negative charge that flows into the ground returns to the gold electrode. Then, the next droplet falls and SDEG prepares for the next power generation process.

[0031] This embodiment also uses TENG signal to detect current output. Figure 3 As shown in Figures 3A and 3B, it can be seen from the experimental results that DNA and FEN1 alone have no effect on the electrical signal. Only when the target protein does not exist and the single-stranded DNA in the DNA tetrahedron exists and captures the signal PDA (Au+TDNA+PDA), the output current of TENG will be significantly reduced (p<0.0001), indicating that the sensor has good feasibility for target protein detection.

[0032] In order to confirm the successful assembly of PDA and DNA tetrahedron, infrared test and polyacrylamide gel electrophoresis experiment were performed in the embodiment. Figure 4A shows that PDA has multiple characteristic peaks, indicating the successful preparation of PDA. Different combinations of DNA fuel chains were prepared: (1-7): TA, TB, TC, TD, TA+TB, TA+TB+TC, TA+TB+TC+TD mixtures. These single chains or hybridization products show different bands in PAGE gel. Figure 4 B shows that the more chains involved in the hybridization reaction, the larger the molecular weight, and thus the slower the movement of the band in the gel. The product after the four probes reacted showed the largest molecular weight, thus proving the formation of DNA tetrahedron.

[0033] This embodiment also provides a detection method of a protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system, comprising the following steps: S1. Preparation of DNA tetrahedron: TA, TB, TC, and TD were mixed in an equimolar ratio and added to a solution containing 10 mM TCEP and 50 mM MgCl 2 10 mM Tris-HCl buffer (pH 8.0), the concentrations of TA, TB, TC, and TD were all 4 μM, incubated at 95 °C for 5 minutes, and then cooled to room temperature to obtain DNA tetrahedron: TDNA; S2, the sample solution to be tested was mixed with FEN1, dbDNA, and ThermoPol reaction buffer, incubated at 37 °C for 1 hour, and then T4 DNA ligase was added and reacted at room temperature for 45 minutes to obtain a mixture 1; In this example, a standard FEN1 solution was used as the sample solution to be tested for the experiment. Specifically, the standard FEN1 solution was diluted to different concentrations, and 7 μL FEN1, 2 μL dbDNA (30 nM) and 1 μL 10×ThermoPol reaction buffer were mixed and incubated at 37 °C for 1 hour. Then, 10 μL of 2×T4 DNA ligase buffer containing 5 U T4 DNA ligase was added, and the reaction was carried out at room temperature for 45 minutes to obtain a mixture 1; S3, 20 nM LbaCas12a, 20 nM crRNA and 50 μM Mn 2+ Mix for 10 minutes to construct a CRISPR / Cas12a trans-cleavage system; The sequence of crRNA is shown in SEQ ID NO.6; S4, mixing the DNA tetrahedron obtained in step S1, the mixture 1 obtained in step S2, and the CRISPR / Cas12a trans-cleavage system obtained in step S3, and reacting at 37 ° C for 0.5 hour to obtain CRISPR / Cas12a-treated TDNA; S5. Gold wire electrode treatment: soak the gold wire electrode in the CRISPR / Cas12a-treated TDNA obtained in step S4 overnight, take it out and rinse it with ultrapure water, blow it dry, then soak it in PDA for 3 hours, take it out and rinse it, and blow it dry; S6. Assembling the triboelectric nanogenerator: The substrate is tilted, a copper foil electrode is laid on the substrate, and then a fluorinated ethylene propylene film of the same size is laid on the copper foil electrode, and the treated gold wire electrode obtained in step S5 is fixed on the surface of the fluorinated ethylene propylene film to obtain a friction nanogenerator; The substrate is a rectangular structure made of polyvinyl chloride with a size of 2.5 cm × 9 cm. The copper foil electrode is also 2.5 cm × 9 cm in size and 25 μm thick. It is connected to the 6514 electrometer as the bottom electrode for signal conversion. The fluorinated ethylene propylene film is 2.5 cm × 9 cm in size and 20 μm thick. It is placed on the copper foil electrode as a dielectric material. S7. An electrometer is connected between the gold wire electrode and the copper foil electrode. The hanging bottle is used as a droplet generator. The liquid in the hanging bottle is pure water. Pure water droplets are output through the hanging bottle and drip onto the fluorinated ethylene propylene membrane of the friction nanogenerator. The current signal detected by the electrometer at the moment when a single droplet falls is recorded. The concentration of the target protein in the sample solution to be tested is obtained based on the current signal analysis.

[0034] Since the tetrahedral DNA nanotag can adsorb PDA, the triboelectric nanogenerator output current is further used to detect the concentration of the target protein. Figure 5 As shown in A, as the concentration of the target protein (FEN1) increases, more dumbbell-shaped DNA forms double strands, the CRISPR / Cas trans-cleavage system is activated, and more single-stranded DNA on the DNA tetrahedral nanotags is sheared through the reaction. Therefore, PDA cannot be adsorbed on the gold wire electrode, and the peak current obtained is larger. The relationship between the peak current and the logarithm of the protein concentration is shown in Figure 5 As shown in B.

[0035] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system, characterized in that: include: CRISPR / Cas12a trans-cleavage system; DNA tetrahedron; It is formed by hybridization reaction of four single-stranded DNA sequences: TA, TB, TC, and TD. Each of the four single-stranded DNA sequences contains three functional regions, which form six sides of a DNA tetrahedron through complementary pairing. The TA chain on the DNA tetrahedron is a protruding long chain, which serves as a substrate for the CRISPR / Cas12a trans-cutting system and is also used to adsorb PDA. Dumbbell-shaped DNA; It can form a double-stranded structure after binding reaction with the target protein to be detected, activating the CRISPR / Cas12a trans-cutting system, thereby cutting the single-stranded DNA sequence on the DNA tetrahedron; and triboelectric nanogenerators; DNA tetrahedrons are pre-modified on the gold wire electrode of the friction nanogenerator. In the presence of target protein, the TA chain on the DNA tetrahedron is cut by CRISPR / Cas, and the polydopamine particles cannot bind to the DNA tetrahedron, so that the gold wire electrode will not be covered by PDA and the friction electric signal of the friction nanogenerator remains unchanged. When the target protein is not present, the TA chain on the DNA tetrahedron binds to the polydopamine particles, which weakens the friction electric signal of the friction nanogenerator, thereby achieving quantitative detection of the target protein by analyzing the friction electric signal.

2. The protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system according to claim 1, characterized in that: The DNA tetrahedron is denoted as TDNA. The ends of TB, TC, and TD in TDNA are all modified with SH groups to form the triangular base of the DNA tetrahedron and connect to the gold wire electrode.

3. The protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system according to claim 2, characterized in that: The target protein is Flap endonuclease 1, denoted as FEN1.

4. The protein detection system based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system according to claim 3, characterized in that: The dumbbell-shaped DNA contains a 5'flap structure.

5. The protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system according to claim 4, characterized in that: The sequences of TA, TB, TC and TD are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

6. The protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system according to claim 5, characterized in that: The dumbbell-shaped DNA is denoted as dbDNA, and its sequence is shown in SEQ ID NO.

5.

7. The protein detection system based on single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system according to claim 1, characterized in that: The friction nanogenerator comprises an inclined substrate, a copper foil electrode and a fluorinated ethylene propylene film which are sequentially stacked on the substrate, and a gold wire electrode which is arranged in the middle of the surface of the fluorinated ethylene propylene film.

8. A detection method for a protein detection system based on a single droplet friction nanogenerator and a CRISPR-Cas trans-cleavage system as described in any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Preparation of DNA tetrahedron: TA, TB, TC, and TD were mixed in an equimolar ratio and added to a Tris-HCl buffer, incubated at 90-95°C for 2-10 minutes, and then cooled to room temperature to obtain a DNA tetrahedron: TDNA; S2, mixing the sample solution to be tested with FEN1, dbDNA, and ThermoPol reaction buffer, incubating at 37 °C for 0.5-2 hours, then adding T4 DNA ligase, reacting at room temperature for 30-90 minutes, to obtain a mixture 1; S3, LbaCas12a, crRNA and Mn 2+ Mix and react for 5-20 minutes to construct a CRISPR / Cas12a trans-cleavage system; S4, mixing the DNA tetrahedron obtained in step S1, the mixture 1 obtained in step S2, and the CRISPR / Cas12a trans-cleavage system obtained in step S3, and reacting at 37 ° C for 15-60 minutes to obtain CRISPR / Cas12a-treated TDNA; S5. Gold wire electrode treatment: soak the gold wire electrode in the CRISPR / Cas12a-treated TDNA obtained in step S4 overnight, take it out and rinse it with ultrapure water, blow it dry, then soak it in PDA for 1-6 hours, take it out and rinse it, and blow it dry; S6. Assembling the triboelectric nanogenerator: The substrate is tilted, a copper foil electrode is laid on the substrate, and then a fluorinated ethylene propylene film of the same size is laid on the copper foil electrode, and the treated gold wire electrode obtained in step S5 is fixed on the surface of the fluorinated ethylene propylene film to obtain a friction nanogenerator; S7. Connect an electrometer between the gold wire electrode and the copper foil electrode, output pure water droplets through the hanging bottle, and drip them onto the fluorinated ethylene propylene membrane of the friction nanogenerator, record the current signal detected by the electrometer at the moment of single droplet falling, and obtain the concentration of the target protein in the sample solution to be tested based on the current signal analysis.

9. The detection method of the protein detection system based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system according to claim 8, characterized in that: The following steps are involved: S1. Preparation of DNA tetrahedron: TA, TB, TC, and TD were mixed in an equimolar ratio and added to a Tris-HCl buffer containing TCEP and MgCl2, incubated at 95°C for 5 minutes, and then cooled to room temperature to obtain a DNA tetrahedron: TDNA; S2, the sample solution to be tested was mixed with FEN1, dbDNA, and ThermoPol reaction buffer, incubated at 37 °C for 1 hour, and then T4 DNA ligase was added and reacted at room temperature for 45 minutes to obtain a mixture 1; S3, LbaCas12a, crRNA and Mn 2+ Mix for 10 minutes to construct a CRISPR / Cas12a trans-cleavage system; S4, mixing the DNA tetrahedron obtained in step S1, the mixture 1 obtained in step S2, and the CRISPR / Cas12a trans-cleavage system obtained in step S3, and reacting at 37 ° C for 0.5 hour to obtain CRISPR / Cas12a-treated TDNA; S5. Gold wire electrode treatment: soak the gold wire electrode in the CRISPR / Cas12a-treated TDNA obtained in step S4 overnight, take it out and rinse it with ultrapure water, blow it dry, then soak it in PDA for 3 hours, take it out and rinse it, and blow it dry; S6. Assembling the triboelectric nanogenerator: The substrate is tilted, a copper foil electrode is laid on the substrate, and then a fluorinated ethylene propylene film of the same size is laid on the copper foil electrode, and the treated gold wire electrode obtained in step S5 is fixed on the surface of the fluorinated ethylene propylene film to obtain a friction nanogenerator; The substrate is a rectangular structure made of polyvinyl chloride; S7. Connect an electrometer between the gold wire electrode and the copper foil electrode, output pure water droplets through the hanging bottle, and drip them onto the fluorinated ethylene propylene membrane of the friction nanogenerator, record the current signal detected by the electrometer at the moment of single droplet falling, and obtain the concentration of the target protein in the sample solution to be tested based on the current signal analysis.

10. The detection method of the protein detection system based on the single droplet friction nanogenerator and CRISPR-Cas trans-cleavage system according to claim 9, characterized in that: The sequence of crRNA is shown in SEQ ID NO.6.

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