Method for detecting homogeneous protein, and detection platform

Abstract

Provided are a method for detecting a homogeneous protein, and a detection platform. The method comprises: constructing a target protein recognition element by using a small-molecule-modified primer; the protein recognition element recognizing a target protein; recognizing, by using a CRISPR / Cas12a system, a product from target protein recognition, and generating a detectable fluorescence signal. In the provided detection method and platform, the inhibitory effect resulting from specific binding to the target protein is introduced into the RCA amplification process, thereby affecting the signal output of CRISPR / Cas12a; and the method achieves a detection sensitivity below the picomolar level for both SA and Anti-dig, with a detection efficiency approximately 1000 times that of previously reported methods. The platform has great potential in the aspect of detecting real samples (such as human serum), and also shows excellent specificity during competitive experiments on free small molecules. In addition, by changing the recognition element, the present invention can be suitable for homogeneous analysis of different protein types, thereby achieving universality and providing a new direction for quantifying a protein biomarker at an ultra-low concentration in clinical practice.

Description

A homogeneous protein detection method and detection platform Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a homogeneous protein detection method and detection platform. Background Art

[0002] The healthcare sector is moving towards personalized, patient-centered healthcare. The demand for detecting protein biomarkers is growing across various fields. Heterogeneous immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays, combine antibody-specific recognition with specific signals generated by enzyme catalysis and have become the mainstay of clinical bioanalysis. However, heterogeneous analytical reactions typically require time-consuming surface fixation, multi-step incubation, and washing processes, as well as specialized personnel and expensive instrumentation, resulting in high costs and frequent false positives. Using simple and effective techniques for quantitative analysis in complex biological matrices remains a significant challenge.

[0003] Homogeneous immunoassays require only a single step, eliminating the need for washing steps. This offers significant advantages, significantly improving reaction kinetics and enabling portable testing. This approach is widely adopted in clinical diagnostics outside the laboratory. Homogeneous immunoassays often use aptamers or DNA-modified proteins as recognition elements, converting analyte-specific binding reactions into conformational changes in functional DNA to generate a signal output. The clustered regularly interspaced short palindromic repeat (CRISPR) and associated protein (Cas) system has demonstrated its ability to serve as an effective signal amplification tool for biosensors due to its portability and high cost-effectiveness. Tang et al. reported a CRISPR-based homogeneous assay that converts the detection of novel coronavirus antibodies into targeting recombinase polymerase amplification (RPA) products, further activating the trans-cleavage activity of Cas12a and generating a fluorescent signal. The development of various homogeneous assay technologies has improved detection speed and sensitivity, and has potential value in life science and clinical research. Technical issues

[0004] In view of this, it is necessary to provide a homogeneous protein detection method and detection platform that can detect in complex samples with high sensitivity and strong specificity to address the defects of the existing technology. Technical Solutions

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] One of the purposes of this application is to provide a homogeneous protein detection method, comprising the following steps:

[0007] Construct target protein recognition elements using small molecule modified primers;

[0008] The protein recognition element recognizes the target protein;

[0009] The CRISPR / Cas12a system is used to recognize the target protein recognition product and generate a detectable fluorescent signal.

[0010] In some embodiments, in the step of constructing the target protein recognition element with a small molecule modified primer, the small molecule modified primer includes a biotin-modified primer or a digoxigenin-modified primer, and the target protein includes SA or anti-Dig.

[0011] In some embodiments,

[0012] When the target protein is SA, the method specifically includes the following steps:

[0013] SA is mixed with a biotin-modified primer and incubated at 25-37° C. for 15-60 minutes to obtain a reaction mixture, the reaction mixture is added to a padlock solution, and the mixture is incubated at 25-37° C. for 1-4 hours to obtain a reaction solution, the reaction solution is mixed with a T4 DNA ligase system, and the mixture is incubated at 15-37° C. for 15-60 minutes to form a circular structure product to obtain a target protein recognition element;

[0014] When the target protein is anti-Dig, the method specifically includes the following steps:

[0015] Mixing anti-Dig with a digoxigenin-modified primer and incubating at 25-37° C. for 0.5-2 hours to obtain a reaction solution, adding the reaction mixture to a padlock solution and incubating at 25-37° C. for 1-4 hours to obtain a reaction solution, mixing the reaction solution with a T4 DNA ligase system, and incubating at 15-37° C. for 15-60 minutes to form a circular structure product to obtain a target protein recognition element;

[0016] The T4 DNA ligase system is a mixed solution of T4 DNA ligase and StickTogether™ DNA ligase buffer.

[0017] In some embodiments, the step of the protein recognition element recognizing the target protein specifically includes the following steps:

[0018] The protein recognition element is added to the phi29 reaction system and incubated for 2-6 hours to complete RCA and produce RCA products.

[0019] In some embodiments, the step of using the CRISPR / Cas12a system to identify the target protein recognition product and generate a detectable fluorescent signal specifically includes the following steps:

[0020] The RCA product was added to the CRISPR / Cas12a reaction system and incubated at 25-37°C for 0.5-1 hour to produce a reaction solution; the reaction solution was excited at a wavelength of 480 nm and the fluorescence spectrum was recorded in the range of 500-600 nm;

[0021] The CRISPR / Cas12a reaction system includes a mixture of NEBuffer r 2.1, Cas12a, FQ reporter gene, crRNA and HO.

[0022] In some embodiments, the following steps are also included:

[0023] The selectivity of the above-mentioned homogeneous protein detection method was evaluated using interfering proteins, including any one of bovine serum albumin, novel coronavirus N protein, novel coronavirus S protein and interleukin 6. The concentration of the interfering protein was set to 10 times the concentration of SA or anti-Dig, respectively.

[0024] The second purpose of this application is to provide a detection platform for a homogeneous protein detection method, comprising:

[0025] Target recognition element: construct target protein recognition element using small molecule modified primers;

[0026] Signal transduction element: The protein recognition element recognizes the target protein;

[0027] Signal output element: Uses the CRISPR / Cas12a system to recognize the target protein and generate a detectable fluorescent signal. Beneficial effects

[0028] This application adopts the above technical solution, and its beneficial effects are as follows:

[0029] The homogeneous protein detection method and platform provided herein utilize small molecule-modified primers to construct a target protein recognition element; this protein recognition element recognizes the target protein; the CRISPR / Cas12a system then recognizes the target protein recognition product and generates a detectable fluorescent signal. This detection method and platform utilizes the inhibitory effect of specific binding of the target protein to the RCA amplification process, thereby affecting the CRISPR / Cas12a signal output. This method achieves sub-picomolar sensitivity for both SA and Anti-dig, approximately 1000 times higher than previously reported. This platform has significant potential for detecting real samples (such as human serum) and demonstrates excellent specificity in competitive experiments with free small molecules. Furthermore, by varying the recognition element, it can be adapted for homogeneous analysis of different protein types, achieving versatility and providing a new approach for quantifying ultra-low-concentration protein biomarkers in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] FIG1 is a schematic diagram showing the design principles of the protein homogeneous detection method and platform provided in the embodiments of the present application.

[0032] FIG2 is a flowchart of the steps of the homogeneous protein detection method provided in an embodiment of the present application.

[0033] FIG3 is a schematic diagram of the structure of the homogeneous protein detection platform provided in an embodiment of the present application.

[0034] Figure 4 is a schematic diagram of the verification and optimization of protein-induced inhibition in the RCA-CRISPR / Cas12a system provided in the examples of the present application.

[0035] FIG5 is a schematic diagram showing platform parameter optimization according to an embodiment of the present application.

[0036] FIG6 is a schematic diagram of SA detection using a homogeneous immunoassay platform provided in an embodiment of the present application.

[0037] FIG7 is a schematic diagram of Anti-dig detection using a homogeneous immune platform provided in this embodiment.

[0038] FIG8 is a schematic diagram of the specificity of the competitive inhibition verification platform using free small molecules provided in this example. Modes for Carrying Out the Invention

[0039] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0040] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on this application.

[0041] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0042] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0043] This application provides a method and highly sensitive platform for protein detection in homogeneous solutions, in which specific recognition of the analyte hinders the detection effect of the rolling circle amplification (RCA)-assisted CRISPR / Cas12a system. As shown in the schematic diagram 1, (A) shows the RCA-CRISPR / Cas12a system using small molecule-modified DNA as primers. (B) shows the analyte-induced hindering effect within the RCA-CRISPR / Cas12a system. The small molecule-modified primer hybridizes with the padlock strand to form circular DNA, which is then amplified by RCA under the action of phi29 polymerase. The resulting single-stranded DNA (ssDNA) product activates the trans-cleavage activity of Cas12a, generating a fluorescent signal. Streptavidin (SA) or anti-digoxigenin (Anti-Dig) antibody is specifically detected using biotin- or digoxigenin-modified DNA primers, respectively. The detection limits of SA and Anti-Dig are 0.08 pmol / L and 0.2 pmol / L, respectively. The combination of RCA and CRISPR / Cas12a increases the detection sensitivity by 1000-fold compared to traditional homogeneous immunoassays.

[0044] This application uses small molecule modified DNA as a recognition element, which can specifically bind to the target protein. Due to the steric effect induced by the target protein, the hybridization between the primer and the padlock chain is hindered, which hinders RCA amplification and activation of Cas12a, resulting in a decrease in the fluorescent signal generated by the FQ-DNA reporter. The application is described in detail below with reference to the examples.

[0045] Please refer to Figure 2, which is a flowchart of the steps of the homogeneous protein detection method provided in an embodiment of the present application, including the following steps S110 to S130. The specific structure of each component and its connection relationship are described in detail below.

[0046] Step S110: constructing a target protein recognition element using a small molecule modified primer.

[0047] In this embodiment, in the step of constructing the target protein recognition element with a small molecule modified primer, the small molecule modified primer includes a biotin-modified primer or a digoxigenin-modified primer, and the target protein includes SA or anti-Dig.

[0048] In this embodiment, when the target protein is SA, the following steps are specifically included:

[0049] SA and a biotin-modified primer were mixed and incubated at 37°C for 30 minutes to obtain a reaction mixture, the reaction mixture was added to a padlock solution, and the mixture was incubated at 37°C for 3 hours to obtain a reaction solution, the reaction solution was mixed with a T4 DNA ligase system, and the mixture was incubated at 25°C for 30 minutes to form a circular structure product to obtain a target protein recognition element;

[0050] When the target protein is anti-Dig, the method specifically includes the following steps:

[0051] Mixing anti-Dig with a digoxigenin-modified primer and incubating at 37°C for 1 hour to obtain a reaction solution, adding the reaction mixture to a padlock solution and incubating at 37°C for 3 hours to obtain a reaction solution, mixing the reaction solution with a T4 DNA ligase system, and incubating at 25°C for 30 minutes to form a circular structure product to obtain a target protein recognition element;

[0052] The T4 DNA ligase system is a mixed solution of T4 DNA ligase and StickTogether™ DNA ligase buffer.

[0053] Step S120: The protein recognition element recognizes the target protein.

[0054] Specifically, the protein recognition element was added to the phi29 reaction system and incubated for 4 hours to complete RCA and produce RCA products.

[0055] Step S130: Use the CRISPR / Cas12a system to identify the target protein recognition product and generate a detectable fluorescent signal.

[0056] In this example, the RCA product was added to the CRISPR / Cas12a reaction system and incubated at 37 ° C for 1 hour to produce a reaction solution; the reaction solution was excited at a wavelength of 480 nm, and the fluorescence spectrum was recorded in the range of 500-600 nm; the CRISPR / Cas12a reaction system included a mixture of NEBuffer r 2.1, Cas12a, FQ reporter gene, crRNA and HO.

[0057] Furthermore, the homogeneous protein detection method provided in the above embodiment of the present application further includes the following steps:

[0058] The selectivity of the system was evaluated using bovine serum albumin (BSA), 2019-nCoV N protein, 2019-nCoV S protein, and interleukin-6 (IL-6). The concentration of the interfering proteins was set at 10-fold that of SA (13 pmol / L) or anti-Dig (26 pmol / L), respectively.

[0059] Please refer to FIG3 , which is a schematic diagram of the structure of the detection platform of the homogeneous protein detection method provided in the embodiment of the present application, including:

[0060] Target recognition element 110: A target protein recognition element is constructed using a small molecule modified primer.

[0061] In this embodiment, in the step of constructing the target protein recognition element with a small molecule modified primer, the small molecule modified primer includes a biotin-modified primer or a digoxigenin-modified primer, and the target protein includes SA or anti-Dig.

[0062] In this embodiment, when the target protein is SA, the following steps are specifically included:

[0063] SA and a biotin-modified primer were mixed and incubated at 37°C for 30 minutes to obtain a reaction mixture, the reaction mixture was added to a padlock solution, and the mixture was incubated at 37°C for 3 hours to obtain a reaction solution, the reaction solution was mixed with a T4 DNA ligase system, and the mixture was incubated at 25°C for 30 minutes to form a circular structure product to obtain a target protein recognition element;

[0064] When the target protein is anti-Dig, the method specifically includes the following steps:

[0065] Mixing anti-Dig with a digoxigenin-modified primer and incubating at 37°C for 1 hour to obtain a reaction solution, adding the reaction mixture to a padlock solution and incubating at 37°C for 3 hours to obtain a reaction solution, mixing the reaction solution with a T4 DNA ligase system, and incubating at 25°C for 30 minutes to form a circular structure product to obtain a target protein recognition element;

[0066] The T4 DNA ligase system is a mixed solution of T4 DNA ligase and StickTogether™ DNA ligase buffer.

[0067] Signal transduction element 120: The protein recognition element recognizes the target protein.

[0068] Specifically, the protein recognition element was added to the phi29 reaction system and incubated for 4 hours to complete RCA and produce RCA products.

[0069] Signal output element 130: uses the CRISPR / Cas12a system to recognize the target protein recognition product and generate a detectable fluorescent signal.

[0070] Specifically, the RCA product was added to the CRISPR / Cas12a reaction system and incubated at 37°C for 1 hour to produce a reaction solution; the reaction solution was excited at a wavelength of 480 nm, and the fluorescence spectrum was recorded in the range of 500-600 nm; the CRISPR / Cas12a reaction system included a mixture of NEBuffer r 2.1, Cas12a, FQ reporter gene, crRNA and HO.

[0071] The detection method and platform provided in this application introduce the blocking effect caused by the specific binding of the target protein into the RCA amplification process, thereby affecting the signal output of CRISPR / Cas12a. This method has achieved a detection sensitivity of less than picomolar for both SA and Anti-dig, and the detection effect is about 1000 times that of previous reports. This platform has great potential in detecting real samples (such as human serum) and has also demonstrated excellent specificity in competitive experiments with free small molecules. In addition, by changing the recognition element, it can be applied to homogeneous analysis of different protein types, achieving versatility and providing a new direction for quantifying ultra-low concentration protein biomarkers in clinical practice.

[0072] The technical solution of the present application is described in detail below with reference to specific embodiments.

[0073] Example

[0074] Chemicals and reagents

[0075] LbCas12a (cf p1), T4 DNA ligase, and phi29 DNA polymerase (Bacillus subtilis) were purchased from New England BioLabs, Inc. All oligonucleotides shown in Table S1 were synthesized and purified by Sangon Biotech (Shanghai, China). Streptavidin and anti-Dig polyclonal antibody (sheep) were purchased from Sigma-Aldrich (Shanghai, China). The novel coronavirus spike protein and nucleocapsid protein were purchased from Sino Biological (Beijing, China). Bovine serum albumin (BSA) was purchased from Solarbio (Beijing, China). All buffer solutions used in the experiments were prepared with DEPC water. Detection was performed using a HITACHI 7100 fluorescence spectrometer. PCR and polyacrylamide gel electrophoresis (PAGE) images were analyzed using a Tanon 1600 Series Multi-Purpose Gel Image Analysis System (Shanghai Tianneng Technology Co., Ltd.). Hybridization, amplification, and CRISPR processes were performed in a ProFlex 3×32-well PCR system (Thermo Fisher Technology Co., Ltd.).

[0076] Table S1. Oligonucleotide sequences

[0077] Primer5'→3'ModificationcrRNAGGGUAAUUUCUACUAAGUAGUAUGGUCAGAACUCACCUGUU-PrimerCACAGCTGAGGATAGGACAT-B-7-PCACAGCTGAGGATAGGACATBiotin, 7thB-13-PCACAGCTGAGGATAGGACATBiotin, 13thB-20-PCACAGCTGAGGATAGGACATBiotin, 20thD-7-PCACAGCTGAGGATAGGACATDigoxin, 7thD-13-PCACAGCTGAGGATAGGACATDigoxin, 13thD-20-PCACAGCTGAGGATAGGACATDigoxin, 20thPadlockCTCAGCTGTGTAACAACATGAAGATTGTAGGTCAGAACTCACCTGTTAGAAACTGTGAAGATCGCTTATTATGTCCTATC-FQ-DNAFAM-TTTATT-BHQ1-

[0078] The specific implementation process is as follows:

[0079] Mix 10 μL of 1 nmol / L target protein (including SA or anti-Dig) with 10 μL of 0.2 nmol / L small molecule-modified primers and incubate at 37°C. For SA, incubate with biotin-modified primers for 30 minutes. For anti-Dig, incubate with digoxigenin-modified primers for 1 hour. Subsequently, add the reaction mixture to 10 μL of 100 nmol / L padlock solution and incubate at 37°C for 3 hours. Subsequently, take 8 μL of the above reaction solution and mix with T4 DNA ligase system (0.5 μL T4 DNA ligase, 10 μL StickTogether™ DNA Ligase Buffer) and incubate at 25°C for 30 minutes to form a ring structure, which is the target protein recognition element. The resulting product is subjected to PAGE to verify the recognition of the target protein.

[0080] The protein recognition element was added to the phi29 reaction system and incubated for 4 hours to complete RCA and produce RCA products.

[0081] Add 20 μL of RCA product to an 80 μL CRISPR / Cas12a reaction system (10 μL NEBuffer r 2.1, 5 μL Cas12a (800 nM), 0.08 μL FQ reporter gene (4000 nM), 0.5 μL crRNA (1600 nM), and 63.4 μL HO) and incubate at 37°C for 1 hour. Excite at 480 nm, and record fluorescence spectra in the 500-600 nm range.

[0082] The selectivity of the system was evaluated using bovine serum albumin (BSA), 2019-nCoV N protein, 2019-nCoV S protein, and interleukin-6 (IL-6). The concentration of the interfering proteins was set at 10-fold that of SA (13 pmol / L) or anti-Dig (26 pmol / L), respectively.

[0083] The target protein (SA or anti-Dig, 26 pmol / L) was mixed with various concentrations of biotin (0, 0.065, 0.26, 2.6, 6.5, 13, and 26 pmol / L) or digoxigenin (0, 0.13, 13, 26, 130, 260, and 650 pmol / L) at 37°C for 30 minutes. Subsequently, 10 μL of small-molecule-modified primers (0.2 nmol / L) was added and incubated at 37°C. Subsequent hybridization, T4 DNA ligation, and RCA-assisted CRISPR / Cas12a procedures were similar to those described above.

[0084] Results and discussion

[0085] Construction of a homogeneous detection platform

[0086] The homogeneous detection platform consists of three main components: a target recognition element, a signal transduction element, and a signal output element. This example uses SA and Anti-Dig as typical models for analysis. Small molecule-modified DNA primers serve as target recognition elements, specifically binding to the target protein.

[0087] Please refer to Figure 4 for the validation and optimization of protein-induced inhibition in the RCA-CRISPR / Cas12a system provided in this example. (A) Electrophoresis analysis demonstrates the validation of SA recognition and the RCA-CRISPR / Cas12a system. Lane 1: Biotin-modified primer (B-20-P); Lane 2: Padlock strand; Lane 3: Hybridization between the biotin-modified primer and padlock strand; Lane 4: Effect of SA recognition on hybridization between the biotin-modified primer and padlock strand A; Lane 5: RCA amplification product generated using primer B-20-P and padlock strand as template; Lane 6: Effect of SA recognition on the RCA reaction system; Lane 7: RCA product cleaved by CRISPR / Cas12a; Lane 8: Effect of SA recognition on CRISPR / Cas12a cleavage of the RCA product. (B) Effect of the biotin / digoxigenin modification position on the output signal. The control is an unmodified primer. B-7-P / D-7-P indicates biotin / digoxigenin modification at the 7th base of the primer; B-13-P / D-13-P indicates biotin / digoxigenin modification at the 13th base of the primer; and B-20-P / D-20-P indicates biotin / digoxigenin modification at the 20th base of the primer. (C) Shows the effect of SA and biotin recognition at different sites on the output signal. (D) Shows the effect of Anti-Dig and digoxigenin recognition at different sites on the output signal.

[0088] As shown in Figure 4 (A), clear bands indicate the presence of a free biotin-modified primer (lane 1), free padlock strand (lane 2), and successful hybridization between the biotin-modified primer and padlock strand (lane 3). The presence of SA creates steric hindrance, inhibiting hybridization between the primer and padlock strand, confirming specific recognition of the target protein (lane 4). The signal transduction pathway underlying the RCA reaction converts specific binding of the target protein into a protein-induced RCA barrier. The padlock strand serves as a template for primer extension to complete RCA. Binding of the small molecule on the primer to the target protein hinders its hybridization with the padlock strand, thereby inhibiting nucleic acid amplification during the signal transduction process. Using the padlock strand as a template, the biotin-modified primer is extended using phi29 polymerase (lane 5). The introduction of SA reduces the number of RCA amplification products (lane 6). This demonstrates that specific recognition of the target protein is successfully converted into nucleic acid amplification through the RCA amplification process. As a signal output element, the CRISPR / Cas12a system recognizes the RCA product, generating a detectable fluorescent signal. The bands observed in lane 7 indicate that Cas12a / crRNA effectively recognizes the RCA product and cleaves it into short DNA strands, confirming the feasibility of the RCA-CRISPR / Cas12a system. Upon SA recognition, Cas12a's trans-cleavage activity is inhibited (lane 8), further demonstrating the protein-induced inhibitory effect in the RCA-CRISPR / Cas12a system.

[0089] Platform parameter optimization

[0090] Different parameters of the detection platform were optimized, including the reaction time between the primer and the padlock strand, the T4 ligase concentration, the extension time of the phi29 DNA polymerase, the trans-cleavage time of the Cas12a / crRNA system, and the position of the small molecule modification on the primer, all of which were aimed at improving the performance of homogeneous protein detection.

[0091] As shown in Figure 5, it is a schematic diagram of platform parameter optimization, where (A) represents the hybridization time between the small molecule modified primer and the padlock chain; (B) represents the concentration of T4 DNA ligase; (C) represents the extension time of phi29 DNA polymerase; and (D) represents the shearing time of the CRISPR / Cas12a system.

[0092] The fluorescence signal reached its maximum when the reaction time between the primer and padlock strand was 3 hours, the T4 ligase concentration was 100 U, the phi29 DNA polymerase extension time was 4 hours, and the CRISPR / Cas12a system reaction time was 1 hour. Furthermore, the position of the small molecule modification on the primer is crucial, as the steric hindrance caused by its interaction with the target protein directly affects the primer-padlock hybridization efficiency. As shown in Figure B, biotin or digoxigenin modification at bases 7, 13, and 20 of the primer had negligible effects on the RCA process. Furthermore, as shown in Figures C and D, when the small molecule modification at base 20 was specifically recognized by the corresponding target proteins (SA and anti-Dig), the fluorescence signal changed significantly, providing a basis for further experimental research.

[0093] Detection performance of SA and anti-digoxin

[0094] Please refer to Figure 6 for a schematic diagram of SA detection using a homogeneous immunoassay platform. (A) shows the fluorescence spectra generated by different SA concentrations (0, 0.13, 0.65, 1.3, 2.6, 7.8, 13, 19.5, and 26 pmol / L). (B) shows the linear relationship between SA concentration and fluorescence signal change. (C) shows the fluorescence signals generated by SA (13 pmol / L) and different nonspecific targets (130 pmol / L). (D) shows a comparison of the fluorescence signals generated by SA in serum and buffer.

[0095] Under optimal experimental conditions, the fluorescence signal decreased significantly as the SA concentration increased from 0 to 26 pmol / L (Figure A). The fluorescence change (ΔF) exhibited a strong linear relationship with SA concentration within the range of 0.13 to 13 pmol / L (Figure B), with a detection limit of SA as low as 0.08 pmol / L. To explore the selectivity of the detection platform for target proteins, we used BSA, SARS-CoV-2 N protein, SARS-CoV-2 S protein, and IL-6 as non-target controls. As shown in Figure C, SA significantly decreased the fluorescence signal, while other interfering proteins had no significant effect on the fluorescence signal even at concentrations 10-fold higher than SA. To evaluate the platform's applicability in complex samples, we tested SA in human serum. Figure D shows that SA effectively reduced the fluorescence signal, with similar changes in fluorescence signal observed in buffer and 10-fold diluted serum, demonstrating the suitability of this method for protein detection in complex samples.

[0096] Please refer to Figure 7, which shows Anti-dig detection using a homogeneous immunoassay platform. (A) shows the fluorescence spectra generated by Anti-dig at different concentrations (0, 0.65, 1.3, 7.8, 10.5, 13, and 26 pmol / L). (B) shows the linear relationship between Anti-dig concentration and fluorescence intensity. (C) shows the comparison of the fluorescence signal generated by Anti-dig (26 pmol / L) and a nonspecific target (260 pmol / L). (D) shows the fluorescence signal change generated by Anti-dig in serum and buffer.

[0097] Figures 7A and 7B show that when the anti-dig concentration increases from 0 to 26 pmol / L, the fluorescence intensity gradually decreases, and the change in fluorescence signal (ΔF) shows a significant linear relationship with the anti-dig concentration within the range of 0.65 to 26 pmol / L. The detection limit of anti-dig is 0.2 pmol / L. Figures 7B and 7C show the specificity of the platform in detecting anti-dig and its stable detection effect in actual samples. As listed in Table S2, the sensitivity of the detection platform in this study for detecting SA and anti-dig is approximately 10% higher than that previously reported. 3 times.

[0098] Please refer to Figure 8, which demonstrates the specificity of the platform using competitive inhibition with free small molecules. (A) shows the fluorescence signal generated by the reaction of SA (26 pmol / L) with free biotin at various concentrations (0, 0.065, 0.26, 2.6, 6.5, 13, and 26 pmol / L). The blank panel represents the absence of free SA and biotin. (B) shows the fluorescence signal generated by the reaction of Anti-Dig (26 pmol / L) with free digoxigenin at various concentrations (0, 0.13, 13, 26, 130, 260, and 650 pmol / L). The blank panel represents the absence of free Anti-Dig and digoxigenin.

[0099] When biotin or digoxin is present, it can competitively bind to the corresponding target protein, hindering the recognition of the target protein by the small molecule-modified primer, thereby interfering with the output fluorescence signal. As shown in Figure 8, as the concentration of free biotin or digoxin increases, the effect of the target protein on reducing fluorescence intensity decreases. This phenomenon once again demonstrates the remarkable specificity of this platform for detecting target proteins.

[0100] The protein homogeneous detection platform provided in the above embodiments of the present application introduces the hindering effect caused by the specific binding of the target protein into the RCA amplification process, thereby affecting the signal output of CRISPR / Cas12a. This method has a detection sensitivity of less than picomolar for both SA and Anti-dig, and the detection effect is about 1000 times that reported previously. This platform has great potential in detecting real samples (such as human serum), and also shows excellent specificity in competitive experiments of free small molecules. In addition, by changing the recognition element, it can be applied to homogeneous analysis of different protein types, achieving versatility and providing a new direction for quantifying ultra-low concentration protein biomarkers in clinical practice.

[0101] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A homogeneous protein detection method, characterized in that: The steps include: The target protein recognition element is constructed by using small molecule modified primers; The protein recognition element recognizes the target protein; The CRISPR / Cas12a system is used to recognize the target protein recognition product and generate a detectable fluorescent signal.

2. The homogeneous protein detection method according to claim 1, characterized in that: In the step of constructing a target protein recognition element with a small molecule modified primer, the small molecule modified primer includes a biotin modified primer or a digoxigenin modified primer, and the target protein includes SA or anti-Dig.

3. The homogeneous protein detection method according to claim 2, characterized in that: When the target protein is SA, the method specifically comprises the following steps: Mixing SA with a biotin-modified primer and incubating at 25-37° C. for 15-60 minutes to obtain a reaction mixture, adding the reaction mixture to a padlock solution and incubating at 25-37° C. for 1-4 hours to obtain a reaction solution, mixing the reaction solution with a T4 DNA ligase system, and incubating at 15-37° C. for 15-60 minutes to form a ring structure product to obtain a target protein recognition element; When the target protein is anti-Dig, the method specifically comprises the following steps: Mixing anti-Dig with a digoxigenin-modified primer and incubating at 25-37° C. for 0.5-2 hours to obtain a reaction solution, adding the reaction mixture to a padlock solution and incubating at 25-37° C. for 1-4 hours to obtain a reaction solution, mixing the reaction solution with a T4 DNA ligase system, and incubating at 15-37° C. for 15-60 minutes to form a ring structure product to obtain a target protein recognition element; The T4 DNA ligase system is a mixed solution of T4 DNA ligase and StickTogether™ DNA ligase buffer.

4. The homogeneous protein detection method according to claim 3, characterized in that: The step of the protein recognition element recognizing the target protein specifically includes the following steps: The protein recognition element is added to the phi29 reaction system and incubated for 2-6 hours to complete RCA and produce RCA products.

5. The homogeneous protein detection method according to claim 4, characterized in that: The step of using the CRISPR / Cas12a system to identify the target protein recognition product and generate a detectable fluorescent signal specifically includes the following steps: The RCA product was added to the CRISPR / Cas12a reaction system and incubated at 25-37°C for 0.5-1 hour to produce a reaction solution; the reaction solution was excited at a wavelength of 480 nm and the fluorescence spectrum was recorded in the range of 500-600 nm; The CRISPR / Cas12a reaction system includes a mixture of NEBuffer r 2.1, Cas12a, FQ reporter gene, crRNA and H2O.

6. The homogeneous protein detection method according to claim 5, characterized in that: The following steps are also included: The protein selectivity of the above-mentioned homogeneous protein detection method was evaluated using interfering proteins, including any one of bovine serum albumin, new coronavirus N protein, new coronavirus S protein and interleukin 6, and the concentration of the interfering protein was set to 10 times the concentration of SA or anti-Dig, respectively.

7. A detection platform for a homogeneous protein detection method according to any one of claims 1 to 6, characterized in that: include: Target recognition element: construct target protein recognition element with small molecule modified primers; Signal transduction element: the protein recognition element recognizes the target protein; Signal output element: The CRISPR / Cas12a system is used to recognize the target protein recognition product and generate a detectable fluorescent signal.

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