A protein marker detection platform for a DNA nanoflower cascade CRISPR system and a construction method and application thereof

Abstract

The application 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 and a construction method and application thereof. The protein marker detection platform based on the DNA nanoflower cascade CRISPR system comprises an upstream module of protein marker input signal amplification based on a DNA nanoflower and a downstream module of output signal amplification based on a CRISPR / Cas system. When the detection platform is used for protein marker detection, the upstream module based on the DNA nanoflower can convert the protein marker into hundreds or thousands of DNA signals, the DNA signals are recognized and activated by a Cas-crRNA compound to have Cas protein cis and trans cleavage activity, so that the output signal is amplified, high sensitivity and rapid detection of the protein marker are achieved, and in particular in detection of a neutrophil gelatinase lipocalin marker in a kidney injury disease, the detection limit is 500 fg / ml.

Description

TECHNICAL FIELD

[0001] The application 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 and a construction method and application thereof. BACKGROUND

[0002] Protein markers in clinical tissue and liquid samples are important indicators reflecting the degree of disease progression. Sensitively and accurately detecting protein markers is crucial for timely intervention and prognosis in disease management. So far, protein markers are usually analyzed and quantified by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and immunoblotting techniques. However, in complex real clinical samples, it is still a great challenge to accurately, sensitively, and rapidly detect protein biomarkers due to the limitation of efficient amplification of protein marker signals. At the same time, with the increasing demand for health management, it is urgent to develop amplification methods for protein markers in real samples for early diagnosis and prognosis evaluation of clinical diseases.

[0003] The CRISPR / Cas system, which has been widely used in the detection of nucleic acids, proteins, and small molecule markers due to its specific recognition of nucleic acids and cis and trans cleavage activity, has opened the door for the clinical application of CRISPR / Cas biotechnology in the past decade. Although the CRISPR / Cas system has multiple turnover trans cleavage properties that can amplify the output signal, the current CRISPR-based detection platform still needs to be combined with non-isothermal polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) and other pre-amplification techniques 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 conversion application of the CRISPR system in protein markers. Therefore, developing an efficient protein marker pre-amplification technology and coupling it with the CRISPR / Cas system is crucial for sensitively and accurately detecting protein markers in complex real clinical samples. SUMMARY

[0004] One of the purposes of the present application is to solve the above technical problems, provide a protein marker detection platform based on DNA nanoflower cascade CRISPR system (DNF-CRISPR), which realizes the upstream amplification of the protein marker input signal by using the DNA nanoflower (efficiently realizes the pre-amplification technology of the protein marker), and then uses the CRISPR system to amplify the output signal downstream, so as to realize the cascade amplification of the biological signal input and output, through the upstream and downstream cascade amplification, realize the super-sensitive and high-specificity detection of the protein marker, it is a general protein marker detection platform, and has wide application prospect in the fields of medical diagnosis and food safety.

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

[0006] The third purpose of the present application is to provide an application method of the above-mentioned protein marker detection platform based on DNA nanoflower cascade CRISPR system in the protein marker detection, through the application method, the super-sensitive and high-specificity detection of the protein marker can be realized, and the detection limit can reach 500 fg / mL.

[0007] Technical principle of the present application

[0008] According to the protein marker, hundreds of nucleic acids on the DNA nanoflower can be recognized and activated by the Cas-crRNA complex and the Cas protein cis and trans cleavage activity, the DNF is cut in cis, and the reporter chain of the fluorescence quenching pair marked on the two ends is cut in trans, and the fluorescence signal is released.

[0009] Technical scheme of the present application

[0010] A protein marker detection platform based on DNA nanoflower cascade CRISPR system comprises an upstream module for amplifying protein marker input signal by using DNA nanoflower, and a downstream module for amplifying protein marker output signal by using CRISPR system.

[0011] The upstream module for amplifying protein marker input signal by using DNA nanoflower comprises an antibody pair, a single-stranded DNA, a coupling agent, a template strand and a primer strand for preparing DNA nanoflower with repeated amplification chain.

[0012] The antibody pair is a capture antibody and a detection antibody of the protein marker;

[0013] The sequence of the single-stranded DNA is SEQ ID NO. 1;

[0014] The sequence of the amplification chain on the DNA nanoflower is SEQ ID NO. 2;

[0015] The sequence of the primer strand is SEQ ID NO. 3;

[0016] The sequence of the template strand is SEQ ID NO. 4;

[0017] The coupling agent is sulfo-SMCC;

[0018] The downstream module for amplifying the output signal using the CRISPR system includes: crRNA, Cas protein, reporter strand with fluorescent and quenching groups at both ends;

[0019] The sequence of the crRNA is SEQ ID NO. 5;

[0020] The sequence of the reporter strand is TTTTTT;

[0021] Preferably, the upstream module for amplifying the input signal of the protein marker using the DNA nanoflower further includes T4 DNA ligase, EquiPhi29 DNA polymerase, dNTP, DTT, EquiPhi29 buffer used for preparation of the DNA nanoflower;

[0022] Preferably, in the downstream module for amplifying the output signal using the CRISPR system, the Cas protein is Cas12a or Cas14;

[0023] In the reporter strand with fluorescent and quenching groups at both ends, the fluorescent group is 5-carboxyfluorescein (FAM), cyanidin 3 (Cy3) or cyanidin 5 (Cy5), and the quenching group is black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2) or black hole quencher 3 (BHQ3).

[0024] A construction method of a protein marker detection platform based on a DNA nanoflower cascade CRISPR system, comprising the following steps:

[0025] (1) An upstream module for amplifying the input signal of the protein marker based on the DNA nanoflower, comprising the following construction steps:

[0026] ① According to the protein marker to be detected, select a specific antibody pair (capture antibody and detection antibody); wherein the capture antibody is used to coat the microplate to specifically capture and bind the protein marker; wherein the detection antibody is coupled with single-stranded DNA through a coupling agent to form an antibody-DNA conjugate (Ab-oligo);

[0027] The sequence of the single-stranded DNA is SEQ ID NO. 1;

[0028] The coupling agent is sulfo-SMCC;

[0029] The capture antibody coating concentration is 8 μg / mL, coated overnight at 4℃;

[0030] The molar concentration ratio of the detection antibody to the sulfo-SMCC coupling agent is 1:100, the reaction concentration is 6 μg / mL, the reaction is carried out at 4℃ for 30 min, and the molar concentration ratio to single-stranded DNA is 1:4, the reaction is carried out at 37℃ for 4h;

[0031] ②, design an amplification chain capable of hybridizing with single-stranded DNA and the corresponding primer chain and template chain. The template chain and the primer chain undergo rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase to generate DNA nanoflower with repeated amplification chain sequence, and the synthesized DNA nanoflower is used as an upstream module;

[0032] The amplification chain sequence on the DNA nanoflower is SEQ ID NO. 2;

[0033] The sequence of the primer chain is SEQ ID NO. 3;

[0034] The sequence of the template chain is SEQ ID NO. 4;

[0035] The reaction time of the rolling circle amplification is 2-6h, the reaction temperature is 37-42℃, and the working concentration of EquiPhi29 DNA polymerase is 10 U / μL;

[0036] After the rolling circle amplification is terminated, the reaction solution is centrifuged at 10000 rpm for 4 min, the lower sediment is reserved, resuspended in Milli-Q water, repeated 3 times to obtain DNA nanoflower, and the DNA nanoflower is dispersed in Milli-Q water and stored at 4℃ for standby;

[0037] The DNA nanoflower obtained above is in spherical flower shape, with a diameter of about 200-800nm, preferably 200-650nm;

[0038] ③, after the antibody of the antibody-DNA conjugate detects the target protein, the other end of the single-stranded DNA 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 the repeated amplification chain signal on the DNA nanoflower, realizing the upstream amplification of the input signal;

[0039] 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℃, and the reaction time is 10-30min;

[0040] (2) Constructing a downstream module for amplifying the output signal based on the CRISPR / Cas system, including the following construction steps:

[0041] ① Designing a crRNA hybridized with the DNA nanoflower sequence, pre-incubating the crRNA with the Cas protein to obtain a Cas-crRNA complex, and using the Cas-crRNA complex as the downstream module;

[0042] The sequence of the crRNA is SEQ ID NO. 5;

[0043] The Cas protein is Cas12a or Cas14;

[0044] The molar concentration ratio of the Cas protein to the crRNA is 1:1, the reaction is carried out at room temperature, and the reaction time is 10-30 min;

[0045] ② The thousands of repeated amplified chains on the DNA nanoflower can be recognized and activated by the Cas-crRNA complex, and the Cas protein has cis and trans cleavage activity, the DNA nanoflower is cut in cis, and the reporter chain modified with a fluorescence quenching group at both ends is cut in trans, releasing a fluorescence signal to amplify the output signal;

[0046] The sequence of the reporter chain is TTTTTT;

[0047] The fluorescence group is a fluorescence group 5-carboxyfluorescein (FAM), cyanine 3 (Cy3) or cyanine 5 (Cy5), and the quenching group is black hole quencher 1 (BHQ1), black hole quencher 2 (BHQ2) or black hole quencher 3 (BHQ3);

[0048] The Cas-crRNA complex is co-incubated with the DNA nanoflower for 30-60 min.

[0049] The above-mentioned protein marker detection platform based on the DNA nanoflower cascade CRISPR system is applied to the detection of protein markers, and the protein markers are neutrophil gelatinase lipocalin (NGAL), kidney injury molecule-1 (kim-1), interleukin-18 (IL-18) or cystatin C (CysC) and the like.

[0050] The beneficial technical effects of the present application

[0051] The protein marker detection platform based on the DNA nanoflower cascade CRISPR system can convert protein markers into DNA signals, and is hundreds or thousands of DNA signals, because the upstream module of the protein marker input signal amplification based on the DNA nanoflower is contained.

[0052] Further, the protein marker detection platform based on the DNA nanoflower cascade CRISPR system can specifically recognize the DNA sequence on the DNA nanoflower and activate the Cas protein activity, cut the DNA nanoflower in cis, and multiple inverted transverse cutting signal report chains, to realize downstream amplification of the output signal, because the output signal amplification downstream module based on the CRISPR / Cas system is contained.

[0053] Further, the protein marker detection platform based on the DNA nanoflower cascade CRISPR system is a universal detection platform, because the DNA nanoflower and the CRISPR system do not need to be changed, when used for detection of any protein marker, only specific antibody pairs for the protein marker need to be selected, and other DNA, crRNA sequences, construction conditions, etc. of the detection platform do not need to be changed, so that the detection of the protein marker can be realized.

[0054] The construction method of the protein marker detection platform based on the DNA nanoflower cascade CRISPR system is simple in construction process and convenient in operation.

[0055] Further, the protein marker detection platform based on the DNA nanoflower cascade CRISPR system in the application of protein marker detection can realize high sensitivity and rapid detection of protein markers, especially in the detection of the kidney injury biomarker neutrophil gelatinase lipid carrier protein (NGAL), and the sensitivity reaches 500 fg / mL. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 is a polyacrylamide gel electrophoresis characterization diagram of the DNA nanoflower.

[0057] Figure 2 is a scanning electron microscope and transmission electron microscope characterization diagram of the DNA nanoflower before and after being cut by the Cas12a-crRNA complex.

[0058] Figure 3 is a polyacrylamide gel electrophoresis characterization diagram of the detection antibody coupled single-stranded DNA.

[0059] Figure 4 is the absorbance characterization map of antibody pairing specificity verification.

[0060] Figure 5 is the fluorescence signal map generated by single-stranded DNA activated Cas12a.

[0061] Figure 6 is the fluorescence signal map generated by DNA nanoflower activated Cas12a.

[0062] Figure 7 is the fluorescence signal intensity change curve (a) of the DNF-CRISPR platform and the NGAL detection limit determination map (b) of the neutrophil gelatinase lipocalin (NGAL) marker within a certain concentration range. DETAILED DESCRIPTION

[0063] The application will be further described below by specific examples and in conjunction with the drawings. It should be understood that the following examples are only used to illustrate the application and are not used to limit the scope of the application. The experimental methods not specified in the following examples are selected according to the conventional methods and conditions, or according to the product instructions. Example 1

[0064] DNA and RNA sequence design for detection platform. The DNA sequence in the table can be replaced by other DNA sequences as long as the template strand and the primer can hybridize to perform subsequent amplification reaction.

[0065] For the neutrophil gelatinase lipocalin (NGAL: NO.D147611, purchased from Shenguo Bioengineering Co., Ltd.) in the kidney injury protein biomarker, a high-specificity antibody pair (capture antibody: 10222-MM03, purchased from Beijing Yiqioshenzhou Technology Co., Ltd.; detection antibody: D261026, purchased from Shenguo Bioengineering Co., Ltd.) was selected, and a single-stranded DNA sequence (SEQ ID NO. 1) coupled with the detection antibody was designed (the 5' end of the single-stranded DNA sequence was thiolated). According to the single-stranded DNA sequence, an amplification strand (SEQ ID NO. 2) hybridized with the single-stranded DNA, the corresponding primer strand (SEQ ID NO. 3), and the template strand (SEQ ID NO. 4) were designed (the 5' end of the template strand sequence was phosphorylated); and a crRNA (SEQ ID NO. 5) corresponding to the DNA nanoflower was designed according to the template strand through rolling circle amplification to generate a DNA nanoflower with repeated amplification strand fragments. In addition, a reporter strand with a terminal modification of a fluorescence and a quenching group was designed. The fluorescence group is Cy5, and the quenching group is BHQ2.

[0066] Table 1 DNA and RNA sequences involved in the examples and base sequence numbers

[0067] .

[0068] Example 2 Preparation and characterization of upstream module DNA nanoflower

[0069] Preparation of circular template: the reaction mixture for synthesizing circular DNA template consists of 0.5 μΜ 5' phosphorylated template strand, 0.5 μΜ primer strand and 1 × T4 ligation buffer. The reaction conditions are 95 ℃ heating for 2 min, gradually cooling from 95 ℃ to 4 ℃ at a rate of -1 ℃ min -1 , adding T4 DNA ligase at a concentration of 10 U μL -1 , incubating overnight at 4 ℃ to form a circular template (the sequence of the circular template is the same as that of the template strand); then terminating the reaction at 65 ℃ for 10 min.

[0070] Preparation of DNA nanoflower: mix the circular template (10 μL), EquiPhi29 DNA polymerase (1 μL, 10 U / μL), dNTPs (containing dATP, dCTP, dGTP, dTTP, all 100 mM, 2.5 μL), DTT (100 mM, 2 μL), EquiPhi29 DNA polymerase buffer (10 ×, 10 μL), Milli-Q water (67 μL) at 37 ℃ for 2 h of incubation, terminate the amplification reaction at 65 ℃ for 10 min; mechanically break the hydrogel product and centrifugally wash it with Milli-Q water for 3 times, disperse the lower precipitate in Milli-Q water, and store it at 4 ℃ for standby, finally forming the DNA nanoflower. Polyacrylamide gel electrophoresis is used to verify the synthesis of DNA nanoflower.

[0071] The experimental results are shown in Figure 1 . The circular template strand undergoes rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase, generating DNA nanoflower with repeated amplified strand sequences. The product DNA nanoflower is blocked in the gel hole due to its large molecular weight, and the migration rate is significantly reduced, proving the successful synthesis of DNA nanoflower. Example 3

[0072] Preparation and activity verification of downstream module Cas12a-crRNA complex

[0073] The Cas12a protein is purchased from New England Biolabs (NEB) Technology Co., Ltd. of the United States. Mix the crRNA and Cas12a protein at a molar concentration ratio of 1:1 in 1 × NEB buffer r2.1, and incubate at room temperature for 20 min to form a Cas12a-crRNA complex;

[0074] The pre-prepared DNA nanoflower was added to the Cas12a-crRNA complex solution (molar concentration ratio of 1:1), and the cis cleavage reaction was carried out at 37°C for 30 minutes. The morphology of the DNA nanoflower before and after cleavage was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0075] The experimental results are shown in Figs. 2a, 2b, 2c and 2d. Figure 2 As shown in Figs. 2a, 2b, 2c and 2d, the DNA nanoflower was co-incubated with the Cas12a-crRNA complex and was efficiently cleaved, proving that the DNA nanoflower before the reaction was in a spherical flower-like morphology with a diameter of about 200-650 nm; the Cas12a protein can be activated by the DNA nanoflower after co-incubation, and the DNA nanoflower is cleaved in cis. Example 4

[0076] Coupling and purification of detection antibody and single-stranded DNA

[0077] The detection antibody was reacted with Sulfo-SMCC in PBS at 4°C for 2h, wherein the molar concentration ratio of the detection antibody to Sulfo-SMCC was 1:100, and the excess sulfo-SMCC was removed by 3kd ultrafiltration.

[0078] The single-stranded DNA was added to the above reaction solution and co-incubated at 37°C for 4h, 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.

[0079] The experimental results are shown in Figs. 3a, 3b, 3c and 3d. Figure 3 As shown in Figs. 3a, 3b, 3c and 3d, the final product only expressed a single band and showed significantly reduced mobility, proving the successful synthesis and purification of the antibody-DNA conjugate. Example 5

[0080] Verification of matching effect of antibody pair

[0081] The capture antibody was coated on a microplate at 4°C overnight, and after coating was completed, each well was washed with washing solution (0.05% Tween + PBS); 5% bovine serum albumin (BSA) was used as blocking solution, and after incubation at 37°C, the washing solution was used for washing.

[0082] 100 ng ml -1 NGAL or buffer was added to each reaction well, and after sealing, incubation was carried out at 37°C; the liquid was discarded, and the microplate was spun dry. The detection antibody was added to the microplate, which was incubated at 37°C and then washed.

[0083] The horseradish peroxidase-labeled goat anti-rabbit secondary antibody was diluted according to the proportion, 100 μL was added to each well, color developing agent 3, 3', 5, 5'-tetramethylbenzidine (TMB) was added at 37°C in the dark, and the absorbance was measured at 450 nm by a multifunctional enzyme label instrument after termination.

[0084] The experimental results are shown in Figure 4 When the target protein NGAL is present, the enzyme color developing signal is significantly enhanced, which proves that the antibody exhibits strong specificity in recognizing and binding the protein marker (NGAL). Example 6

[0085] Effect of DNA nanoflower on CRISPR / Cas cleavage activity

[0086] The crRNA and the Cas12a protein were mixed in a 1:1 molar concentration ratio in a 1×NEB buffer r2.1 (purchased from New England Biolabs Technology Co., Ltd., USA) solution, and incubated at room temperature for 20 min to form a Cas12a-crRNA complex;

[0087] The same molar concentration of single-stranded DNA or the previously prepared DNA nanoflower was added to the Cas12a-crRNA complex solution (molar concentration ratio of 1:1) and incubated at room temperature for 60 min to activate the activity of the Cas12a-crRNA complex, trans-cleavage of the reporter strand, and release of the readable fluorescence signal. The signal was collected every 10 min, and the fluorescence intensity was measured by a fluorescence spectrophotometer (excitation wavelength of 630 nm and emission wavelength range of 650 nm-670 nm).

[0088] The experimental results are shown in Figure 5 and Figure 6 When the concentration of the DNA nanoflower is the same as the molar concentration of the single-stranded DNA, the fluorescence intensity signal generated by the DNA nanoflower activated Cas12a-crRNA complex is 20-32 times that of the single-stranded DNA, which is much higher than that of the single-stranded DNA, indicating that the DNA nanoflower has a cascade signal amplification effect with the CRISPR / Cas system. Example 7

[0089] Construction of DNF-CRISPR protein detection platform and determination of detection limit

[0090] 8 μg mL -1 NGAL capture antibody was coated on a microplate at 4°C overnight. After coating was completed, each well was washed 3 times with 300 μL of washing solution (0.05% Tween+PBS); 200 μL of 5% BSA was added to each well as blocking solution, and incubated at 37°C for 1 h. After incubation was completed, the wells were washed 3 times;

[0091] 100 uL of different concentrations of protein marker NGAL (concentrations are 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 respectively) were added to each reaction well, and after sealing, incubated at 37℃ for 30 min; the liquid was discarded and dried, then 100 μL of antibody-DNA conjugate was added to each well, and incubated at 37℃ for 30 min, after incubation, washed 3 times;

[0092] 100 μL of DNA nanoflower was added to each well, and the single-stranded DNA on the antibody-DNA conjugate hybridized with the DNA nanoflower at room temperature for 20-30 min;

[0093] 100 μL of Cas12a-crRNA complex and reporter chain were added to each well, and reacted at 37℃ in the dark for 40 min, and the fluorescence intensity was measured by multifunctional enzyme label instrument (excitation wavelength was 493 nm, and emission wavelength was 520 nm).

[0094] The experimental results are shown in Figure 7 As shown in the table, within a certain concentration range of protein marker NGAL (500 fg / mL~2.5 μg / mL), as the concentration increased, the fluorescence signal intensity output by the DNF-CRISPR platform increased; the detection limit of the protein marker NGAL was 500 fg / mL. The detection result further illustrates that the protein detection platform based on the DNA nanoflower cascade CRISPR system proposed in the present application can significantly improve the detection sensitivity of the protein marker, so as to realize how to sensitively and accurately detect the protein marker in the actual complex clinical sample.

[0095] The above is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. Any person skilled in the art can modify or change the above embodiment without departing from the spirit and scope of the present application. Therefore, any simple, equivalent changes and modifications made according to the content of the claims and description of the present application fall within the scope of the claims of the present application. The present application is not described in detail.

Claims

1. A DNA nanoflower-based cascade CRISPR system protein marker detection platform, characterized in that, The detection platform of the protein marker based on the DNA nanoflower cascade CRISPR system comprises an upstream module for amplifying the input signal of the protein marker by using the DNA nanoflower, and a downstream module for amplifying the output signal of the protein marker by using the CRISPR system; The upstream module for amplifying the input signal of the protein marker by using the DNA nanoflower comprises an antibody pair, a single-stranded DNA, a coupling agent, a template strand and a primer strand for preparing the DNA nanoflower with repeated amplification chains; The antibody pair is a capture antibody and a detection antibody of the protein marker; The sequence of the single-stranded DNA is SEQ ID NO. 1; The sequence of the amplification chain 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 module for amplifying the output signal by using the CRISPR system comprises a crRNA, a Cas protein, and a reporter strand with a fluorescent group and a quenching group at two ends; The sequence of the crRNA is SEQ ID NO. 5; The base sequence of the reporter strand is TTTTTT.

2. The DNA nanoflower-based cascade CRISPR system protein marker detection platform of claim 1, wherein, The upstream module for amplifying the input signal of the protein marker by using the DNA nanoflower further comprises T4 DNA ligase, EquiPhi29 DNA polymerase, dNTP, DTT and EquiPhi29 buffer used for preparing the DNA nanoflower.

3. The DNA nanoflower-based cascade CRISPR system protein marker detection platform of claim 1, wherein: The protein marker is neutrophil gelatinase lipocalin, kidney injury molecule-1, interleukin-18 or cystatin C in a kidney injury disease marker; the Cas protein of the CRISPR system is Cas12a or Cas14; in the reporter strand with a fluorescent group and a quenching group at two ends, the fluorescent group is 5-carboxyfluorescein, cyanidin 3 or cyanidin 5, and the quenching group is black hole quencher 1, black hole quencher 2 or black hole quencher 3.

4. The method for constructing a DNA nanoflower-based cascade CRISPR system protein marker detection platform according to claim 1, 2 or 3, characterized in that The construction steps include: (1) The upstream module for amplifying the input signal of the protein marker based on the DNA nanoflower comprises the following construction steps: ① According to the protein marker to be detected, an antibody pair specifically combined is selected; wherein the capture antibody is used for coating a microplate to specifically capture and combine the protein marker; wherein the detection antibody is coupled with a 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 An amplification chain capable of hybridizing with the single-stranded DNA is designed, as well as a corresponding primer strand and a template strand; the template strand and the primer strand are subjected to a rolling circle amplification reaction under the action of EquiPhi29 DNA polymerase to generate a DNA nanoflower with repeated amplification chain sequences, and the synthesized DNA nanoflower is used as the upstream module; The sequence of the amplification chain 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; ③The detection antibody in the antibody-DNA conjugate detects the protein marker, and the single-stranded DNA at the other end hybridizes with the DNA nanoflower, converting the biological signal of the protein marker into the repeated amplified chain signal on the DNA nanoflower, so as to realize the upstream amplification of the input signal; The hybridization sequence is the hybridization of SEQ ID NO. 1 and SEQ ID NO. 2; (2) The downstream module for amplifying the output signal is constructed based on the CRISPR / Cas system, including the following construction steps: ① The crRNA hybridized with the DNA nanoflower sequence is designed, and the Cas-crRNA complex is obtained by pre-incubation with the Cas protein and used as the downstream module; The sequence of the crRNA is SEQ ID NO. 5; The Cas protein is Cas12a or Cas14; ② The repeated amplified chain on the DNA nanoflower is recognized by the Cas-crRNA complex and activates the Cas protein cis and trans cleavage activity, and the DNA nanoflower is cut in cis, and the reporter chain modified with a fluorescence quenching group at both ends is cut in trans, releasing the fluorescence signal and amplifying the output signal.

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

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

7. The method for constructing a protein biomarker detection platform based on a DNA nanoflower cascade CRISPR system as described in claim 4, characterized in that, The DNA nanoflower in step (1) is in a spherical flower shape with a diameter of 200-800 nm.

8. The application of the DNA nanoflower-based cascade CRISPR system for detecting the protein marker in the protein marker detection of claim 1, 2 or 3.

9. Use of the DNA nanoflower-based cascade CRISPR system protein marker detection platform of claim 8 in protein marker detection, characterized in that, The obtained DNA nanoflower is used as the upstream module for amplifying the input signal, and the Cas-crRNA complex is used as the downstream module.

10. The application of the protein biomarker detection platform based on the DNA nanoflower cascade CRISPR system as described in claim 9 in protein biomarker detection, characterized in that, After the Cas-crRNA complex is activated by the DNA nanoflower corresponding to the protein marker, the reporter chain is cut in trans, and the detection of the protein marker is realized by using the cascade amplification principle.

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