Protein marker detection platform of DNA nanoflower cascade CRISPR system as well as construction method and application of protein marker detection platform

Abstract

The invention belongs to the technical field of protein detection, and particularly relates to a protein marker detection platform based on a DNA nanoflower cascade CRISPR system as well as a construction method and application of the protein marker detection platform. The protein marker detection platform based on the DNA nanoflower cascade CRISPR system comprises an upstream module for amplifying a protein marker input signal based on DNA nanoflowers and a downstream module for amplifying an output signal based on a CRISPR / Cas system. The upstream module based on the DNA nanoflower can convert a protein marker into hundreds of DNA signals, and the DNA signals are recognized by a Cas-crRNA compound and activate Cas protein cis-and trans-cleavage activity, so that an output signal is amplified, and high sensitivity and rapid detection of the protein marker are realized; especially in the detection of neutrophil gelatinase lipocalin as a kidney injury disease marker, the detection limit is 500 fg / ml.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and specifically relates to a protein marker detection platform based on a DNA nanoflower cascade CRISPR system, and a construction method and application thereof. Background Art

[0002] Protein markers in clinical tissue and fluid samples are important indicators of disease progression. Sensitive and accurate detection of protein markers is crucial for timely intervention and prognosis in disease management. To date, protein markers are usually analyzed and quantified by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and immunoblotting techniques. However, in real clinical samples with complex components, accurate, sensitive, and rapid detection of protein biomarkers remains a major challenge due to the limitations of efficient amplification of protein marker signals. At the same time, with the increasing demand for health management, there is an urgent need to develop methods for amplifying protein markers in real samples for early diagnosis and prognosis assessment of clinical diseases.

[0003] The CRISPR / Cas system, which has emerged in the past decade, has been widely used in the detection of nucleic acid, protein and small molecule markers due to its specific recognition of nucleic acids and cis- and trans-cleavage activities, opening the door for CRISPR / Cas biotechnology to enter clinical applications. Although the CRISPR / Cas system has the characteristics of multiple flip-flops and trans-cleavage, which can amplify the output signal, the current CRISPR-based detection platform still needs to be combined with pre-amplification technologies such as non-isothermal polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) to achieve high sensitivity and specificity in detecting target nucleic acids. However, for non-nucleic acid markers (such as protein markers), there is currently no direct pre-amplification technology, which hinders the transformation and application of the CRISPR system in protein markers. Therefore, the development of efficient protein marker pre-amplification technology and coupling it with the CRISPR / Cas system is crucial for sensitive and accurate detection of protein markers in real clinical samples with complex components. Summary of the invention

[0004] One of the purposes of the present invention is to solve the above-mentioned technical problems and to provide a protein marker detection platform (DNF-CRISPR) based on a DNA nanoflower cascade CRISPR system. The detection platform utilizes DNA nanoflowers to perform upstream amplification of protein marker input signals (efficiently realizing the pre-amplification technology of protein markers), and then utilizes the CRISPR system to perform downstream amplification of output signals, thereby realizing cascade amplification of biological signal input and output. Through this upstream and downstream cascade amplification, ultra-sensitive and highly specific detection of protein markers can be achieved. The platform is a universal protein marker detection platform and has broad application prospects in the fields of medical diagnosis and food safety.

[0005] The second object of the present invention is to provide a method for constructing the above-mentioned protein marker detection platform based on the DNA nanoflower cascade CRISPR system.

[0006] The third object of the present invention is to provide an application method of the above-mentioned protein marker detection platform based on the DNA nanoflower cascade CRISPR system in protein marker detection. Through this application method, ultra-sensitive and highly specific detection of protein markers can be achieved, and the detection limit can reach 500 fg / mL.

[0007] Technical principle of the present invention Hundreds of nucleic acids on the DNA nanoflowers designed based on protein markers can be recognized by the Cas-crRNA complex and activate the cis- and trans-cutting activities of the Cas protein, cis-cutting DNF and trans-cutting the reporter chain marked with fluorescence quenching pairs at both ends to release fluorescent signals.

[0008] Technical solution of the present invention A protein marker detection platform based on a DNA nanoflower cascade CRISPR system, comprising an upstream module that utilizes DNA nanoflowers to amplify protein marker input signals, and a downstream module that utilizes a CRISPR system to amplify protein marker output signals; The upstream module for amplifying the protein marker input signal using DNA nanoflowers includes: an antibody pair, a single-stranded DNA, a coupling agent, a template strand and a primer strand for preparing a DNA nanoflower with repeated amplification strands; The antibody pair is a capture antibody and a detection antibody for a protein marker; The sequence of the single-stranded DNA is SEQ ID NO.1; The amplified chain sequence on the DNA nanoflower is SEQ ID NO.2; The sequence of the primer strand is SEQ ID NO.3; The sequence of the template strand is SEQ ID NO.4; The coupling agent is sulfo-SMCC; The aforementioned downstream modules for amplifying the output signal using the CRISPR system include: crRNA, Cas protein, and a reporter chain with fluorescent and quenching groups modified at both ends respectively; The sequence of the crRNA is SEQ ID NO.5; The sequence of the reporter strand is TTTTTT; Preferably, the upstream module for amplifying the protein marker input signal using DNA nanoflowers also includes T4 DNA ligase, EquiPhi29 DNA polymerase, dNTP, DTT, and EquiPhi29 buffer for preparing DNA nanoflowers; Preferably, in the downstream module that amplifies the output signal using the CRISPR system, the Cas protein is Cas12a or Cas14; In the reporter chain with fluorescent and quenching groups modified at both ends respectively, the fluorescent group is 5-carboxyfluorescein (FAM), anthocyanin 3 (Cy3) or anthocyanin 5 (Cy5), and the quenching group is black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2) or black hole quencher 3 (BHQ3).

[0009] A method for constructing a protein marker detection platform based on a DNA nanoflower cascade CRISPR system comprises the following steps: (1) The upstream module for amplifying the input signal of the protein marker based on the DNA nanoflower includes the following construction steps: ①. According to the protein marker to be detected, select a specific binding antibody pair (capture antibody and detection antibody); the capture antibody is used to coat the microplate to specifically capture and bind the protein marker; the detection antibody is coupled to single-stranded DNA through a coupling agent to form an antibody-DNA conjugate (Ab-oligo); The sequence of the single-stranded DNA is SEQ ID NO.1; The coupling agent is sulfo-SMCC; The capture antibody coating concentration was 8 μg / mL, and the coating was carried out overnight at 4°C; The molar concentration ratio of the detection antibody to the sulfo-SMCC coupling agent was 1:100, the reaction concentration was 6 μg / mL, and the reaction was carried out at 4°C for 30 min. The molar concentration ratio of the detection antibody to the single-stranded DNA was 1:4, and the reaction was carried out at 37°C for 4 h. ②. Design amplification chains and corresponding primer chains and template chains that can hybridize and complement single-stranded DNA. The template chain and primer chain undergo rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase to generate DNA nanoflowers with repeated amplification chain sequences. The synthesized DNA nanoflowers serve as upstream modules; The sequence of the amplified strand on the DNA nanoflower is SEQ ID NO.2; The sequence of the primer strand is SEQ ID NO.3; The sequence of the template strand is SEQ ID NO.4; The reaction time of the rolling circle amplification is 2-6h, the reaction temperature is 37-42°C, and the working concentration of EquiPhi29 DNA polymerase is 10 U / μL; After the rolling circle amplification is terminated, the reaction solution is centrifuged at 10,000 rpm for 4 minutes, the lower precipitate is retained, and resuspended in Milli-Q water. After repeating 3 times, DNA nanoflowers are obtained, and the DNA nanoflowers are dispersed in Milli-Q water and stored at 4°C for later use; The DNA nanoflowers obtained above are spherical flower-like in shape, with a diameter of about 200-800 nm, preferably 200-650 nm; ③. After the antibody of the antibody-DNA conjugate detects the target protein, the single-stranded DNA at the other end hybridizes with the DNA nanoflower (the hybridization sequence is: SEQ ID NO.1 hybridizes with SEQ ID NO.2), thereby converting the protein biological signal into a repeated amplification chain signal on the DNA nanoflower, realizing upstream amplification of the input signal; The hybridization concentration of the DNA nanoflower and the antibody-DNA conjugate is 1.5 μg / mL, the reaction is carried out at room temperature or 37°C, and the reaction time is 10-30 minutes; (2) Construct a downstream module for amplifying the output signal based on the CRISPR / Cas system, including the following construction steps: ① Design crRNA that hybridizes with the DNA nanoflower sequence, incubate it with Cas protein in advance to obtain the Cas-crRNA complex and use it as a downstream module; The sequence of the crRNA is SEQ ID NO.5; The Cas protein is Cas12a or Cas14; The molar concentration ratio of the Cas protein to crRNA is 1:1, the reaction is carried out at room temperature, and the reaction time is 10-30 min; ② Hundreds of repeated amplification chains on the DNA nanoflower can be recognized by the Cas-crRNA complex and activate the cis- and trans-cleavage activities of the Cas protein, cis-cleaving the DNA nanoflower and trans-cleaving the reporter chain modified with fluorescent quenching groups at both ends, releasing the fluorescent signal and amplifying the output signal; The sequence of the reporter strand is TTTTTT; The fluorescent group is 5-carboxyfluorescein (FAM), anthocyanin 3 (Cy3) or anthocyanin 5 (Cy5), and the quenching group is black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2) or black hole quencher 3 (BHQ3); The Cas-crRNA complex and the DNA nanoflower are co-incubated and cut for 30-60 minutes.

[0010] The above-mentioned protein marker detection platform based on the DNA nanoflower cascade CRISPR system is used in the detection of protein markers, wherein the protein markers are kidney injury disease markers such as neutrophil gelatinase lipocalin (NGAL), kidney injury molecule-1 (kim-1), interleukin-18 (IL-18) or cystatin C (CysC), etc.

[0011] Beneficial technical effects of the present invention The protein marker detection platform based on the DNA nanoflower cascade CRISPR system of the present invention contains an upstream module for amplifying the protein marker input signal based on the DNA nanoflower, which can convert the protein marker into DNA signals, and hundreds or thousands of DNA signals; in addition, the DNA nanoflower has the ability to resist nonspecific degradation in complex biological matrices, thereby being able to efficiently amplify the protein marker input signal in complex biological samples, providing a basis for the sensitive and accurate detection of protein markers in actual complex clinical samples.

[0012] Furthermore, the protein marker detection platform based on the DNA nanoflower cascade CRISPR system of the present invention, because it contains a downstream module for output signal amplification based on the CRISPR / Cas system, can specifically identify the DNA sequence on the DNA nanoflower, activate its own Cas protein activity, cis-cut the DNA nanoflower, and at the same time can multiple flip trans-cut signal reporting chains to achieve downstream amplification of the output signal.

[0013] Furthermore, the protein marker detection platform based on the DNA nanoflower cascade CRISPR system of the present invention is a universal detection platform. Since neither the DNA nanoflower nor the CRISPR system needs to be changed, when used for the detection of any protein marker, it is only necessary to select specific antibody pairs according to the protein marker. Other DNA, crRNA and other sequences, construction conditions, etc. of the detection platform do not need to be changed, and the detection of protein markers can be achieved.

[0014] The present invention discloses a method for constructing a protein marker detection platform based on a DNA nanoflower cascade CRISPR system, which has a simple construction process and is easy to operate.

[0015] Furthermore, the application of a protein marker detection platform based on a DNA nanoflower cascade CRISPR system in protein marker detection can achieve high sensitivity and rapid detection of protein markers, especially in the detection of neutrophil gelatinase lipocalin (NGAL), a biomarker of kidney injury, with a sensitivity of 500 fg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a polyacrylamide gel electrophoresis characterization diagram of DNA nanoflowers.

[0017] Figure 2 These are scanning electron microscopy and transmission electron microscopy characterization images of the DNA nanoflower before and after being cut by the Cas12a-crRNA complex.

[0018] Figure 3 This is a polyacrylamide gel electrophoresis characterization diagram of the detection antibody coupled to single-stranded DNA.

[0019] Figure 4 This is an absorbance characterization graph that verifies the specificity of antibody pairing.

[0020] Figure 5 This is a fluorescence signal diagram generated by single-stranded DNA activating Cas12a.

[0021] Figure 6 This is a graph of the fluorescent signal generated by DNA nanoflowers activating Cas12a.

[0022] Figure 7 The fluorescence signal intensity change curve of the DNF-CRISPR platform within a certain concentration range of the neutrophil gelatinase lipocalin (NGAL) marker (a) and the NGAL detection limit determination diagram (b). DETAILED DESCRIPTION

[0023] The present invention is further described below by specific examples in conjunction with the accompanying drawings. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are selected according to conventional methods and conditions, or according to the product specifications. Example 1

[0024] DNA and RNA sequence design for detection platform. The DNA sequences in this table can be replaced with other DNA sequences while ensuring that the template strand and primer can hybridize for subsequent amplification reaction.

[0025] For the renal injury protein biomarker neutrophil gelatinase lipocalin (NGAL: NO.D147611, purchased from Sangon Biotech Co., Ltd.), a highly specific binding antibody pair was selected (capture antibody: 10222-MM03, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; detection antibody: D261026, purchased from Sangon Biotech Co., Ltd.), and a single-stranded DNA sequence (SEQ ID NO.1) coupled to the detection antibody was designed (the 5' end of the single-stranded DNA sequence was thiolated). According to the single-stranded DNA sequence, the amplification chain (SEQ ID NO.2) and the corresponding primer chain (SEQ ID NO.3) and the template chain (SEQ ID NO.4) for the hybridization reaction with the single-stranded DNA were designed (the 5' end of the template chain sequence was phosphorylated); and the template chain was amplified by rolling circle amplification to generate DNA nanoflowers with repeated amplification chain fragments, and the crRNA (SEQ ID NO.5) corresponding to the DNA nanoflower was designed. In addition, the reporter chain with fluorescent and quenching groups modified at the ends was designed. The fluorescent group is Cy5 and the quenching group is BHQ2.

[0026] Table 1 DNA and RNA sequences and base sequence numbers involved in the examples . Example 2 Preparation and characterization of upstream module DNA nanoflowers

[0027] Preparation of circular template: The reaction mixture for synthesizing circular DNA templates consisted of 0.5 μM 5' phosphorylated template strand, 0.5 μM primer strand, and 1 × T4 ligation buffer. The reaction conditions were heating at 95°C for 2 min and 30 min at -1°C. -1 The rate was gradually cooled from 95 ℃ to 4 ℃; 10 U μL -1 Incubate with T4 DNA ligase at 4°C overnight to form a circular template (the sequence of the circular template is the same as that of the template chain); then place at 65°C for 10 minutes to terminate the reaction.

[0028] Preparation of DNA nanoflowers: Mix the circular template (10 μL), EquiPhi29 DNA polymerase (1 μL, 10 U / μL), dNTP (including dATP, dCTP, dGTP, dTTP, all 100 mM, 2.5 μL), DTT (100 mM, 2 μL), EquiPhi29 DNA polymerase buffer (10 ×, 10 μL), and Milli-Q water (67 μL), incubate at 37°C for 2 hours, and place at 65°C for 10 minutes to terminate the amplification reaction; mechanically break the formed hydrogel product and wash it three times with Milli-Q water by centrifugation, disperse the lower precipitate in Milli-Q water, and store it at 4°C for use in the final DNA nanoflowers. Polyacrylamide gel electrophoresis verifies the synthesis of DNA nanoflowers.

[0029] The experimental results are as follows Figure 1 As shown, the circular template chain undergoes a rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase to generate DNA nanoflowers with repeated amplification chain sequences. The product DNA nanoflowers are blocked in the gel pores due to their large molecular weight, and their mobility is significantly reduced, proving that the synthesis of DNA nanoflowers was successful. Example 3

[0030] Preparation and activity verification of downstream module Cas12a-crRNA complex Cas12a protein was purchased from New England Biolabs (NEB) Technology Co., Ltd., USA. crRNA and Cas12a protein were mixed in 1×NEB buffer r2.1 at a molar concentration ratio of 1:1 and incubated at room temperature for 20 minutes to form a Cas12a-crRNA complex; The pre-prepared DNA nanoflowers were added to the Cas12a-crRNA complex solution (molar concentration ratio of 1:1) and reacted at 37°C for 30 minutes to perform a cis-cleavage reaction. The morphology of the DNA nanoflowers before and after cleavage was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0031] The experimental results are as follows Figure 2 As shown in a, 2b, 2c, and 2d, DNA nanoflowers were co-incubated with Cas12a-crRNA complexes and efficiently cut, proving that the DNA nanoflowers before the reaction were spherical flower-like in shape with a diameter of approximately 200-650 nm; after co-incubation, Cas12a protein could be activated by DNA nanoflowers, cis-cutting the DNA nanoflowers and causing the DN nanoflowers to disintegrate. Example 4

[0032] Conjugation and purification of detection antibodies and single-stranded DNA The detection antibody and Sulfo-SMCC were reacted in phosphate buffered saline (PBS) at 4°C for 2 h, where the molar concentration ratio of the detection antibody to Sulfo-SMCC was 1:100, and excess sulfo-SMCC was removed by 3kd ultrafiltration; The single-stranded DNA was added to the above reaction solution and incubated at 37°C for 4 hours, wherein the molar concentration ratio of the detection antibody to the single-stranded DNA was 1:4. After the reaction, the antibody-single-stranded DNA conjugate was purified by high performance liquid chromatography, and the purification effect of the antibody-single-stranded DNA conjugate was verified by polyacrylamide gel electrophoresis.

[0033] The experimental results are as follows Figure 3 As shown, the final product expressed only a single band and exhibited a significantly reduced mobility, demonstrating the successful synthesis and purification of the antibody-DNA conjugate. Example 5

[0034] Verification of the matching effect of antibody pairs The capture antibody was coated on the microplate overnight at 4°C. After coating, each well was washed with washing solution (0.05% Tween + PBS). 5% bovine serum albumin (BSA) was used as blocking solution. After incubation at 37°C, the wells were washed with washing solution. Add 100 ng ml to each reaction well -1 NGAL or buffer, seal the plate and incubate at 37°C; discard the liquid, spin dry, add the detection antibody to the microplate, incubate at 37°C and then wash; Horseradish peroxidase-labeled goat anti-rabbit secondary antibody was diluted proportionally and added to each well at 100 μL. The color developing agent 3,3',5,5'-tetramethylbenzidine (TMB) was added to terminate the reaction at 37°C in the dark, and the absorbance was measured at 450 nm using a multifunctional microplate reader.

[0035] The experimental results are as follows Figure 4 As shown, the enzyme colorimetric signal was significantly enhanced when the target protein NGAL was present, proving that the antibody pair exhibited strong specificity in recognizing and binding to the protein marker (NGAL). Example 6

[0036] Effects of DNA nanoflowers on CRISPR / Cas cleavage activity The crRNA and Cas12a protein were mixed in a 1×NEB buffer r2.1 (purchased from New England Biolabs Technology Co., Ltd., USA) solution at a molar concentration ratio of 1:1, and incubated at room temperature for 20 minutes to form a Cas12a-crRNA complex; Single-stranded DNA or pre-prepared DNA nanoflowers of the same molar concentration were added to the Cas12a-crRNA complex solution (molar concentration ratio of 1:1) and incubated at room temperature for 60 minutes to activate the activity of the Cas12a-crRNA complex, trans-cleave the reporter chain, and release a readable fluorescent signal. The signal was collected every 10 minutes, and the fluorescence intensity was measured by a fluorescence spectrophotometer (excitation wavelength of 630 nm, emission wavelength range of 650 nm~670 nm).

[0037] The experimental results are as follows Figure 5 and Figure 6 As shown, when the molar concentration of DNA nanoflowers is the same as that of single-stranded DNA, the fluorescence intensity signal generated by the DNA nanoflowers activating the Cas12a-crRNA complex is 20-32 times that of the fluorescence intensity signal generated by single-stranded DNA, which is much higher than the fluorescence intensity signal generated by single-stranded DNA, indicating that DNA nanoflowers and the CRISPR / Cas system have a cascade signal amplification effect. Example 7

[0038] Construction of DNF-CRISPR protein detection platform and determination of detection limit 8 μg mL -1 The microplate was coated with NGAL capture antibody overnight at 4°C. After coating, each well was washed three times with 300 μL of washing solution (0.05% Tween+PBS); 200 μL of 5% BSA was added to each well as blocking solution, incubated at 37°C for 1 hour, and washed three times after incubation; 100 uL of different concentrations of protein marker NGAL (500 fg / mL, 2.5 pg / ml, 25 pg / ml, 250 pg / ml, 2.5 ng / ml, 25 ng / ml, 250 ng / ml, 2.5 μg / mL) was added to each reaction well, and the plates were sealed and incubated at 37°C for 30 min. The liquid was discarded and dried, and then 100 μL of antibody-DNA conjugate was added to each well, and the plates were incubated at 37°C for 30 min. After the incubation was completed, the plates were washed 3 times. Add 100 μL of DNA nanoflower to each well, and allow the DNA nanoflower to hybridize with the single-stranded DNA on the antibody-DNA conjugate for 20-30 minutes at room temperature; 100 μL of Cas12a-crRNA complex and reporter strand were added to each well, and the reaction was carried out at 37°C in the dark for 40 min. The fluorescence intensity was measured by a multifunctional microplate reader (excitation wavelength was 493 nm, emission wavelength was 520 nm).

[0039] The experimental results are as follows Figure 7As shown, within a certain concentration range of the protein marker NGAL (500 fg / mL~2.5 μg / mL), as the concentration increases, the intensity of the fluorescent signal output by the DNF-CRISPR platform increases; the detection limit of the protein marker NGAL is determined to be 500 fg / mL. This test result further illustrates that the protein detection platform based on the DNA nanoflower cascade CRISPR system proposed in the present invention can significantly improve the detection sensitivity of protein markers, thereby realizing how to sensitively and accurately detect protein markers in actual complex clinical samples.

[0040] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Anyone familiar with the technology can modify or change the above embodiment without violating the spirit and scope of the present invention. Therefore, all simple and equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A protein marker detection platform based on DNA nanoflower cascade CRISPR system, characterized in that: The protein marker detection platform based on the DNA nanoflower cascade CRISPR system includes an upstream module that uses the DNA nanoflower to amplify the protein marker input signal, and a downstream module that uses the CRISPR system to amplify the protein marker output signal; The upstream module for amplifying the protein marker input signal using DNA nanoflowers includes: an antibody pair, a single-stranded DNA, a coupling agent, a template strand and a primer strand for preparing a DNA nanoflower with repeated amplification strands; The antibody pair is a capture antibody and a detection antibody for a protein marker; The sequence of the single-stranded DNA is SEQ ID NO.1; The sequence of the amplified strand on the DNA nanoflower is SEQ ID NO.2; The sequence of the primer strand is SEQ ID NO.3; The sequence of the template strand is SEQ ID NO.4; The coupling agent is sulfo-SMCC; The downstream modules for amplifying the output signal using the CRISPR system include: crRNA, Cas protein, and a reporter chain with fluorescent and quenching groups modified at both ends respectively; The sequence of the crRNA is SEQ ID NO.5; The base sequence of the reporter strand is TTTTTT.

2. The protein marker detection platform based on the DNA nanoflower cascade CRISPR system according to claim 1, characterized in that: The upstream module for amplifying the protein marker input signal using DNA nanoflowers also includes T4 DNA ligase, EquiPhi29 DNA polymerase, dNTP, DTT, and EquiPhi29 buffer used for preparing DNA nanoflowers.

3. The protein marker detection platform based on the DNA nanoflower cascade CRISPR system according to claim 1, characterized in that: The protein markers are kidney injury disease markers such as neutrophil gelatinase lipocalin, kidney injury molecule-1, interleukin-18 or cystatin C; The Cas protein of the CRISPR system is Cas12a or Cas14; In the reporter chain with fluorescent and quenching groups modified at both ends respectively, the fluorescent group is 5-carboxyfluorescein, anthocyanin 3 or anthocyanin 5, and the quenching group is black hole quencher 1, black hole quencher 2 or black hole quencher 3.

4. A method for constructing a protein marker detection platform based on a DNA nanoflower cascade CRISPR system according to claim 1, 2 or 3, characterized in that The build steps include: (1) The upstream module for amplifying the input signal of the protein marker based on the DNA nanoflower includes the following construction steps: ①. According to the protein marker to be detected, a pair of specifically binding antibodies is selected; wherein the capture antibody is used to coat the microplate to specifically capture and bind the protein marker; wherein the detection antibody is coupled to the single-stranded DNA through a coupling agent to form an antibody-DNA conjugate; The sequence of the single-stranded DNA is SEQ ID NO.1; The coupling agent is sulfo-SMCC; ② Designing amplification chains that can hybridize and complement single-stranded DNA and corresponding primer chains and template chains. The template chains and primer chains undergo rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase to generate DNA nanoflowers with repeated amplification chain sequences. The synthesized DNA nanoflowers serve as upstream modules. The sequence of the amplified strand on the DNA nanoflower is SEQ ID NO.2; The sequence of the primer strand is SEQ ID NO.3; The sequence of the template strand is SEQ ID NO.4; ③. After the detection antibody in the antibody-DNA conjugate detects the protein marker, the single-stranded DNA at the other end hybridizes with the DNA nanoflower, converting the biological signal of the protein marker into a repeated amplification chain signal on the DNA nanoflower, thereby realizing upstream amplification of the input signal; The hybridization sequence is SEQ ID NO.1 hybridizing with SEQ ID NO.2; (2) Construct a downstream module for amplifying the output signal based on the CRISPR / Cas system, including the following construction steps: ① Design crRNA that hybridizes with the DNA nanoflower sequence, incubate it with Cas protein in advance to obtain the Cas-crRNA complex and use it as a downstream module; The sequence of the crRNA is SEQ ID NO.5; The Cas protein is Cas12a or Cas14; ②. The repeated amplification chain on the DNA nanoflower is recognized by the Cas-crRNA complex and activates the cis- and trans-cutting activities of the Cas protein, cis-cutting the DNA nanoflower and trans-cutting the reporter chain modified with fluorescent quenching groups at both ends, releasing the fluorescent signal and amplifying the output signal.

5. The method for constructing a protein marker detection platform based on a DNA nanoflower cascade CRISPR system as claimed in claim 4, characterized in that: The DNA nanoflowers in step (1) are formed by rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase, with a reaction time of 2-6 hours and a reaction temperature of 37-42°C.

6. The method for constructing a protein marker detection platform based on a DNA nanoflower cascade CRISPR system as claimed in claim 4, characterized in that: The DNA nanoflowers described in step (1) are obtained by physical and mechanical destruction, repeated washing, centrifugation and resuspension after the rolling circle amplification reaction.

7. The method for constructing a detection platform for protein markers based on a DNA nanoflower cascade CRISPR system as claimed in claim 4, characterized in that: The DNA nanoflowers in step (1) are spherical in shape and have a diameter of about 200-800 nm.

8. Application of the protein marker detection platform based on the DNA nanoflower cascade CRISPR system according to claim 1, 2 or 3 in protein marker detection.

9. The use of the protein marker detection platform based on the DNA nanoflower cascade CRISPR system as claimed in claim 8 in protein marker detection, characterized in that: The resulting DNA nanoflower serves as an upstream module for amplification of the input signal, and the Cas-crRNA complex serves as a downstream module.

10. Application of the protein marker detection platform based on the DNA nanoflower cascade CRISPR system as claimed in claim 9 in protein marker detection, characterized in that: After the Cas12a-crRNA complex is activated by the DNA nanoflower corresponding to the protein marker, it trans-cuts the reporter chain, and utilizes this cascade amplification principle to realize the detection application of the protein marker.

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