Preparation and detection method of crisper-immune combined small molecule detection probe

Abstract

This invention specifically relates to the preparation of a CRISPR-immunoassay combined with small molecule detection probe, its related detection method, and its applications. The probe preparation mainly employs a chemical-biological conjugation method, using the bifunctional cross-linking agents 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide sodium salt (Sulfo-SMCC) and N-succinimide-S-acetylthioacetate (SATA) to covalently conjugate dsDNA modified with -NH2 at the 5' end and a monoclonal antibody to prepare a dsDNA-antibody probe. Secondly, this detection method uses the dsDNA-antibody probe as a bridge to combine the immune system and the CRISPR / Cas12a system. It utilizes the antibody's specific recognition ability for small molecules, and leverages RPA nucleic acid amplification for signal amplification and the cis-cleavage activity and / or trans-single-strand DNA cleavage activity of CRISPR / Cas12a to generate a fluorescent signal, achieving the conversion of the immune response signal into a high-intensity fluorescent signal, thus establishing a CRISPR-immunoassay combined with small molecule detection system. The detection method has advantages such as simple operation, high specificity, extremely high sensitivity, and stable signal.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for preparing a CRISPR-immunoassay combined with a small molecule detection probe and its related detection methods and applications. Background Technology

[0002] In recent years, the discovery and research of the CRISPR system has provided a novel method for the detection of nucleic acid targets, but the application of CRISPR systems to non-nucleic acid targets (such as small molecules) has been very limited. However, the detection of small molecule markers or pollutants is of great importance in fields such as medicine, environment, and food safety. β-agonists are a representative example of small molecule pollutants, often illegally used in animal husbandry, which can cause acute or chronic cumulative poisoning in humans and have long-term, incalculable impacts on the food chain and the environment. Despite the serious harm, instrumental analysis methods are currently very mature for detecting prohibited small molecule pollutants such as β-agonists. However, these methods suffer from drawbacks such as complex sample pretreatment, reliance on expensive instruments and specialized technicians; the instability of biosensor methods makes them difficult to apply in practice; immunoassay methods are advantageous due to their simplicity, short processing time, and good stability, but their main disadvantage is that their sensitivity depends heavily on the properties of the antibody itself, making them insufficient for detecting β-agonists, which are prohibited by law. By combining the specificity and controllability of immunoassays with the ultra-high sensitivity and signal stability of the CRISPR / Cas system, a CRISPR-immunoassay co-detection system can be established to compensate for the shortcomings of current immunoassay methods.

[0003] Currently, CRISPR detection of small molecules mainly utilizes aptamers, antibodies, and bacterial allosteric transcription factors as bridges to achieve nucleic acid signal conversion. For example, Chinese patent CN111812066A describes a detection method that uses bacterial allosteric transcription factors and the CRISPR / Cas12a system to convert small molecule signals into optical signals through the cis- and trans-cleavage activities of CRISPR / Cas12a. Compared to antibodies, screening suitable aptamers and bacterial allosteric transcription factors as non-nucleic acid target recognition elements is more difficult. Australian patent WO2023019290A1 proposes a CRISPR / Cas12a-driven immunosensor for detecting whole Cryptosporidium oocysts in water samples. However, research on the detection of small molecules using a CRISPR-immunoassay combined system is still lacking. Therefore, there is an urgent need for a new method for detecting small molecules that can integrate the CRISPR / Cas system with an immunoassay system, is simple to operate, highly specific, highly sensitive, and has the potential for rapid on-site detection. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a dsDNA-antibody probe, composition, small molecule detection kit, and related detection methods and applications that organically combine an immune reaction, an RPA isothermal amplification system, and a CRISPR / Cas12a system. The dsDNA-antibody probe acts as a bridge to combine the immune system and the CRISPR system. On the one hand, the antibody portion of the dsDNA-antibody probe specifically recognizes small molecules; on the other hand, the nucleic acid portion of the dsDNA-antibody probe, with the aid of the RPA amplification system and the CRISPR / Cas12a system, achieves signal amplification and signal output.

[0005] Therefore, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for synthesizing a dsDNA-antibody probe, wherein the dsDNA-antibody probe is covalently coupled to dsDNA and a monoclonal antibody through a chemical-biological conjugation method.

[0007] The dsDNA can be any type of dsDNA that can be amplified by RPA isothermally and recognized and activated by the CRISPR system.

[0008] Wherein, the dsDNA is dsDNA with a -NH2-modified 5' end;

[0009] The monoclonal antibody includes, but is not limited to, salbutamol monoclonal antibody, and may also be replaced by other small molecule monoclonal antibodies.

[0010] In this chemical-bioconjugation method, bifunctional cross-linking agents 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate sodium succinimide ester (Sulfo-SMCC) and N-succinimide-S-acetylthioacetate (SATA) are used to chemically react with dsDNA and antibodies to form covalent bonds, as shown in Example 1.

[0011] Secondly, the present invention also includes a method for constructing the dsDNA-antibody probe: dsDNA and a monoclonal antibody are covalently linked to obtain the dsDNA-antibody probe. The dsDNA-antibody probe is prepared by reacting dsDNA and antibody at a molar ratio of 1:1.

[0012] Thirdly, the present invention provides a kit for detecting small molecules, the kit comprising a recognition reagent, an amplification reagent, and a signal output reagent.

[0013] The recognition reagent comprises a dsDNA-antibody probe, wherein the dsDNA contains a -NH2 modified portion that is chemically and biologically conjugated with the antibody, a target sequence for the RPA isothermal amplification system, a recognition site PAM for the CRISPR / Cas12a system, and a sequence complementary to the crRNA portion.

[0014] The amplification reagent includes the RPA isothermal amplification system, comprising forward and reverse primers for target dsDNA, MgOAc, dNTPs, target dsDNA, 2×Reaction Buffer, 10×Basic E-mix, 20×Core ReactionMix, and / or water.

[0015] The 2×Reaction Buffer, 10×Basic E-mix, and 20×Core Reaction Mix are all reagents from TwistDx's DNA Isothermal Rapid Amplification Kit. The RPA forward and reverse primers were designed to target dsDNA.

[0016] The signal output reagent comprises the CRISPR / Cas12a system, which includes Cas12a enzyme, crRNA, single-stranded DNA probe (ssDNA-reporter), NEBuffer, RNase inhibitor and / or water.

[0017] The ssDNA-reporter is conjugated with a FAM reporter group and a BHQ quencher group at its two ends, respectively.

[0018] Fourthly, this invention provides a CRISPR-immunoassay combined with small molecule detection method, comprising the following steps:

[0019] S1. The coating agent is embedded in an ELISA plate, and dsDNA-antibody probe is added. The antibody binds specifically to the coating agent. After washing the plate, the dsDNA-antibody probe is immobilized on the ELISA plate.

[0020] S2. Add RPA reagent to the above system to perform exponential amplification of dsDNA;

[0021] S3. Add the CRISPR reagent to the above reaction system. The crRNA in CRISPR specifically recognizes the PAM site on the target dsDNA and performs cis-cleavage. At the same time, it activates the side-cutting activity of the CRISPR protein, which arbitrarily cuts the fluorescent reporter molecule to generate a fluorescent signal. The quantification or qualitative analysis of the analyte is achieved by using QuantStudio3 Real-time PCR or a portable UV-blue light gel excimer with a wavelength of 470nm.

[0022] Specifically, in step S1, the original coating concentration is 2.56 pg / mL, and the dsDNA-antibody probe dilution ratio is 1:16000, as shown in Example 2.

[0023] Specifically, the reaction conditions for the RPA amplification reaction in step S2 were a 10 μL reaction volume, which contained 0.5 μM forward and reverse primers, 2.0 mM dNTPs, and 22.4 mM MgOAc. The entire amplification process was completed by heating at 37°C for 25 min, as shown in Experiment 4 of the series.

[0024] Specifically, in step S3, the reaction conditions of the CRISPR system were as follows: 10 μL reaction volume, containing 125 nM Cas12a protein, 50 nM crRNA, 1000 nM ssDNA-reporter, and a reaction time of 10 min (see Experiment 2).

[0025] The present invention has the following beneficial effects:

[0026] This invention combines enzyme-linked immunosorbent assay (ELISA), RPA amplification technology, and CRISPR detection technology. It uses a synthesized dsDNA-antibody probe as a bridge to integrate the immune system and the CRISPR / Cas12a system. Utilizing the antibody's specific recognition ability for small molecules, RPA nucleic acid amplification amplifies the signal, and CRISPR / Cas12a cleavage activity generates a fluorescent signal, thus converting the immune signal into a fluorescent signal. This establishes a CRISPR-immunoassay combined with small molecule detection system. The detection method exhibits high specificity and sensitivity.

[0027] This invention introduces a dsDNA-antibody probe, which is formed by covalently coupling dsDNA and a monoclonal antibody. On one hand, the antibody portion of the dsDNA-antibody probe specifically recognizes small molecules; on the other hand, the nucleic acid portion of the dsDNA-antibody probe is used to amplify and output signals via RPA and CRISPR / Cas12a systems. This probe can simultaneously perform immune and nucleic acid signal transduction functions, simplifying operation.

[0028] The CRISPR-immunoassay combined with small molecule detection method described in this invention only requires selecting the appropriate antibody according to different small molecules and synthesizing dsDNA-antibody probes. Other components do not need to be changed, which can realize the detection of different small molecules and has strong versatility. Attached Figure Description

[0029] Figure 1 CRISPR-immunoassay combined with small molecule detection system detection protocol;

[0030] Figure 2: Target dsDNA sequence design diagram;

[0031] Figure 3 The coupling principle of dsDNA-antibody probe;

[0032] Figure 4 Figure 1 shows the purification and characterization results of the dsDNA-antibody probe. Figure A is the chromatogram of the dsDNA-antibody probe purified by molecular sieve chromatography, Figure B is the agarose nucleic acid electrophoresis result, and Figure C is the SDS-PAGE result.

[0033] Figure 5 Standard curves of antibody versus dsDNA-antibody probe;

[0034] Figure 6 Optimized fluorescence curves for the CRISPR / Cas12a detection system corresponding to dsDNA;

[0035] Figure 7 Fluorescence values ​​at 10 min after optimizing the CRISPR / Cas12a detection system conditions corresponding to the target dsDNA, respectively, correspond to the optimization of Cas12a concentration, crRNA concentration, and ssDNA-reporter concentration.

[0036] Figure 8 The sensitivity of the CRISPR / Cas12a detection system to the target dsDNA;

[0037] Figure 9 Figure 1 shows the optimization results of the RPA amplification system conditions corresponding to the target dsDNA. Figure 2 shows the primer concentration optimization, Figure 3 shows the dNTPs concentration optimization, Figure 4 shows the MgOAc concentration optimization, and Figure 5 shows the amplification time optimization.

[0038] Figure 10 Figure 1 shows the results of optimizing the concentration of the coating agent and the dsDNA-antibody probe. Figure 2 shows the optimized concentration of the coating agent, and Figure 3 shows the optimized concentration of the dsDNA-antibody probe.

[0039] Figure 11 Standard curve of CRISPR-immunoassay combined with small molecule detection system;

[0040] Figure 12 Standard curve of SAL in pig urine samples. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0042] pass Figure 1 The principle of the CRISPR-immunoassay combined with small molecule detection method of this invention is briefly described as follows: The coating antigen is immobilized on an ELISA plate, and dsDNA-antibody probes are added and incubated with the analyte. The analyte and the coating antigen compete for the antibody. Then, excess probes and drugs are washed away. Next, the CRISPR / Cas12a detection system is directly added to the wells of the ELISA plate. More analyte will bring stronger competition, resulting in fewer dsDNA-antibody probes bound to the coating antigen, fewer Cas12a activations, and lower signal values. A standard curve showing a negative correlation between analyte concentration and fluorescence intensity is established.

[0043] Based on the above principles, design is required for dsDNA. Since the specific recognition of Cas12a is affected by steric hindrance, the design of the target dsDNA needs to consider whether the sequence characteristics of the target dsDNA will affect the specific binding of Cas12a to the target dsDNA. Therefore, the target dsDNA sequence consists of four parts (e.g., Figure 2 As shown): (1) A linker arm that connects to the antibody, with a length of 5-10 bp; (2) A sequence containing the PAM site "TTTV" and a target sequence complementary to crRNA; (3) Providing sufficient binding space for the Cas12a enzyme / crRNA complex; (4) An RPA primer recognition sequence. The linker arm serves to increase the distance between the antibody and the CRISPR / Cas12a recognition site, preventing Cas12a from failing to recognize the target DNA due to steric hindrance. Simultaneously, it provides sufficient distance between the dsDNA and the antibody, preventing the target dsDNA in the dsDNA-antibody probe from blocking the antibody's specific binding site.

[0044] The following examples all use salbutamol (SAL), a β-agonist, as the detection target to establish a CRISPR-immunoassay combined with small molecule detection method. The antibody is a salbutamol monoclonal antibody, and the dsDNA is a 219 bp double-stranded DNA sequence containing the PAM site "TTTV" and conforming to the above four-part composition. The dsDNA-antibody probe synthesis method described in this invention is applicable to any monoclonal antibody, not just salbutamol monoclonal antibody.

[0045] Example 1. Synthesis of dsDNA-antibody probe

[0046] The principle of dsDNA antibody probe preparation is as follows: Figure 2As shown, the dsDNA modified with -NH2 at the 5' end first forms a covalent amide bond with the n-hydroxysuccinimide (NHS) ester at one end of the bifunctional cross-linking agent 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide sodium salt (Sulfo-SMCC). The NHS ester at the N-succinimide-S-acetylsthioacetate (SATA) end reacts with the -NH2 on the lysine residue of the antibody. Subsequently, hydroxylamine deprotects the thiol group, exposing it. The maleimide group at the other end of Sulfo-SMCC reacts with the exposed thiol group at pH 6.5-7.5 to form a stable thioether bond. The specific operation steps are as follows:

[0047] Considering the potential losses from subsequent purification and concentration, the initial amount of antibody conjugation should be at least 1 mg. Here, we take 9 mg of antibody as an example. The amount of each reagent can be increased or decreased proportionally according to actual needs.

[0048] (1) Dialyzing 9 mg of antibody (60 nmol) in phosphate buffered saline (PBS) solution at pH 7.4 at 4°C for 48 hours can replace the primary amine groups that may be present in the buffer solution for storing the antibody, preventing non-specific reactions in the subsequent coupling process, and adjust the pH of the antibody background solution to 7.4, which is conducive to the reaction between the antibody and SATA.

[0049] (2) SATA modified antibody: Take 10 times the molar volume of antibody prepared SATA solution (600 nmol) and add it to the dialyzed antibody. Mix well and react at room temperature for 30 min to obtain SATA modified antibody. The SATA modified antibody is concentrated and impurities are removed by ultrafiltration tube to remove excess SATA reagent. The ultrafiltration tube is centrifuged at 6000 r / min and 4℃. During the concentration and impurity removal process, the ultrafiltration tube needs to be removed every 10 minutes of centrifugation. Use a pipette to spray the liquid onto the membrane on both sides and repeat the blowing and aspiration several times to prevent the protein from precipitating due to excessive concentration. Continue until the solution is reduced to 200 μL. Finally, use PBS to make up the liquid volume to 900 μL.

[0050] (3) Sulfo-SMCC modified target dsDNA: Take 10 times the molar amount of DNA prepared Sulfo-SMCC solution (600 nmol) and add it to the prepared 60 nmol target dsDNA. Mix well and react at room temperature for 30 min to obtain SATA modified dsDNA. The SATA modified dsDNA is concentrated and impurities are removed according to step (2). Finally, the solution volume is supplemented to 900 μL with PBS and then temporarily stored in -20℃ environment.

[0051] (4) Weigh 1.74 g hydroxylamine hydrochloride and 0.365 g disodium EDTA and dissolve them in 40 mL PBS solution. Adjust the pH of the solution to 7.4 with sodium hydroxide solution to prepare hydroxylamine hydrochloride solution. Take 100 μL of the prepared hydroxylamine hydrochloride solution and add it to the concentrated and purified antibody. Mix well and react at room temperature for 2 hours.

[0052] (5) Add the Sulfo-SMCC modified target dsDNA stored in step (3). When adding, ensure that the molar ratio of target dsDNA to antibody is 1:1, mix well, and react at room temperature for 1 hour.

[0053] (6) Concentrate using an ultrafiltration tube, and remove excess reaction reagents during the process. The operation is the same as step (2) for concentration and impurity removal. After the solution is reduced to 250 μL, take out the liquid and add PBS solution to make up to 1 mL to obtain the initial product of dsDNA-antibody probe.

[0054] (7) The primary product of dsDNA-antibody probe was separated and purified by molecular sieve to obtain purified dsDNA-antibody probe.

[0055] During the conjugation process, unconjugated target dsDNA and antibodies remain. To prevent interference with subsequent quantitative systems, purification is necessary. The purification method utilizes a one-step molecular sieve chromatography approach, based on the difference in molecular weight between the dsDNA-antibody probe and the target dsDNA. Molecular sieve chromatography employs a Superdex 200 pre-packed column, separating the dsDNA-antibody and the unconjugated target dsDNA based on the difference in molecular weight.

[0056] The molecular weight of monoclonal antibodies is generally around 150 kDa. In this example, the dsDNA used is 219 bp, with a molecular weight of approximately 130 kDa. The molecular weight of the dsDNA-antibody probe (280 kDa) differs significantly from that of the uncoupled target dsDNA and antibody, thus molecular sieves can be used for separation. The first peak to elute is the dsDNA-antibody probe, followed by the uncoupled target dsDNA and antibody. Since the molecular weight difference between the latter two is only 20 kDa, the difference is small, and they elute together within the same retention time. The molecular sieve chromatography purification results of the dsDNA-antibody probe are shown below. Figure 4 As shown in Figure A.

[0057] The results of nucleic acid electrophoresis and SDS-PAGE identification of the purified dsDNA-antibody probe are as follows: Figure 4 As shown in Figures B and C, because the antibody in the dsDNA-antibody probe cannot move on the protein gel due to the dsDNA binding, the probe will be positioned higher than the antibody. Figure 4In Figures B and C, lanes 4 and 5 correspond to the dsDNA-antibody probe and the mixture of uncoupled antibody and target dsDNA, respectively. The probe band is single, indicating that a pure dsDNA-antibody probe has been obtained.

[0058] Series Experiment 1. Performance Comparison of dsDNA-Antibody Probe Before and After Conjugation

[0059] The activity of the unconjugated antibody and the purified dsDNA-antibody probe was identified using ic-ELISA. A suitable antigen-antibody concentration combination was determined using checkerboard titration, and a standard curve was established. The specific steps are as follows:

[0060] (1) Plate coating: Dilute the salbutamol (SAL) coating agent to 1 μg / mL with coating buffer, add 100 μL / well to the well of the microplate, and incubate in a 37°C water bath for 12 hours.

[0061] (2) Blocking: Take out the microplate, wash it twice with an automatic plate washer and pat it dry. Add 120 μL of 6% skim milk powder blocking solution and block for 3 hours. Shake off the blocking solution in the plate and dry at 37°C for 1 hour. The 6% skim milk powder blocking solution is prepared by dissolving 6g of skim milk powder in 100mL of phosphate Tween buffer solution (PBST).

[0062] (3) Sample addition: In the checkerboard titration method, the antibody with an initial concentration of 1 μg / mL was first diluted with PBST to 7 concentrations (0.5 μg / mL, 0.25 μg / mL, 0.13 μg / mL, 62.5 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL); 50 μL of PBST was added to the odd-numbered wells of the sealed ELISA plate, and 50 μL of salbutamol diluted with PBST (1 μg / mL) was added to the even-numbered wells. Then, 50 μL of antibody diluted with the same factor was added to each row, and the last row was set as a blank control (NTC) of 50 μL of PBST; during the standard curve establishment process, the salbutamol drug with an initial concentration of 10 μg / mL was first diluted with PBST to 7 concentrations (1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.0 ... (0.001 ng / mL, 0.001 ng / mL); Add 50 μL of the drug at the same dilution to each row of the sealed ELISA plate, and set the last row as a blank of 50 μL PBST. Then add 0.25 μg / mL antibody to each well, incubate at 37℃ for 40 min, wash the plate 5 times, and pat dry.

[0063] (4) Add secondary antibody: Dilute HRP-labeled goat anti-mouse antibody 5000 times with PBST, add 100 μL to each well, incubate at 37℃ for 30 min, wash the plate 5 times, and pat dry;

[0064] (5) Color development: Add 100 μL of color development solution to each well and incubate at 37℃ for 10 min;

[0065] (6) Termination: Add 50 μL of stop solution to each well to terminate the reaction, and read the absorbance value of each well at 450 nm wavelength in a multi-functional microplate reader.

[0066] The results are as follows Figure 4 As shown, SAL antibody IC 50 The concentration was 1.49 ng / mL, and the LOD was 0.18 ng / mL; the dsDNA-antibody probe IC50 was 1.49 ng / mL. 50 The concentration was 1.83 ng / mL, and the LOD was 0.2 ng / mL. The IC50 of the dsDNA-antibody probe was... 50 The LOD value did not change significantly compared to before conjugation, and the antibody performance was basically unaffected. Therefore, the dsDNA-antibody probe can be used to establish a CRISPR-immunoassay combined with small molecule detection method.

[0067] Experiment 2. Optimization of CRISPR / Cas12a detection system conditions

[0068] Before establishing the CRISPR-immunoassay combined with small molecule detection system, the corresponding CRISPR / Cas12a detection system was optimized for the target dsDNA. Based on the prepared target dsDNA and corresponding crRNA, an initial system was first used: 50 nM Cas12a, 150 nM crRNA, 200 nM ssDNA-reporter (FAM-TTTTTT-BHQ), 100 nM DNA, 2 μL NEBuffer 2.1, and sterile ultrapure water was added to a final volume of 20 μL.

[0069] First, the reaction time in the CRISPR / Cas12a detection system was optimized. The configured CRISPR / Cas12a detection system was incubated at 37°C using a QuantStudio3 Real-time PCR instrument, and fluorescence signals were continuously acquired. The results are as follows: Figure 5 As shown, the fluorescence essentially reached saturation after 10 minutes.

[0070] In the CRISPR / Cas12a system, Cas12a only exhibits trans-cleavage activity when it forms a ternary complex with Cas12a, crRNA, and target dsDNA, thus non-specifically cleaving the ssDNA-reporter probe and releasing a fluorescent signal. Therefore, the concentrations of Cas12a, crRNA, and ssDNA-reporter are the main factors affecting detection performance. Based on a reaction time of 10 min, the concentrations of Cas12a protein, crRNA, and ssDNA-reporter were further optimized. The optimized CRISPR / Cas12a detection system was as follows: Cas12a protein concentration 125 nM, crRNA concentration 50 nM, ssDNA-reporter concentration 1000 nM, 100 nM DNA, 2 μL NEBuffer 2.1, and sterile ultrapure water added to a final volume of 20 μL, as shown in Table 1. Figure 6 As shown.

[0071] Table 1: Optimization parameters of the CRISPR / Cas12a detection system

[0072]

[0073] Considering practical cost reasons, the CRISPR system was subsequently scaled down to a 10μL system, which did not affect the detection results.

[0074] Experiment 3. Performance of the CRISPR / Cas12a detection system

[0075] The established CRISPR / Cas12a detection system was tested for its detection efficacy by adding different concentrations of target dsDNA. The target dsDNA concentration was diluted to 10⁻⁶. 1 ng / μL, 10 0 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 The minimum recognition concentration of target dsDNA was tested using an optimized CRISPR / Cas12a detection system (obtained from Experiment 2 of the series) at ng / μL. A blank control was set up in each group, with the same system as the experimental group, using sterile ultrapure water instead of DNA template. Each experiment was performed in triplicate. The recognition performance of target dsDNA was compared to use as a screening criterion for universal dsDNA-antibody probes, and also as a reference for the template DNA concentration added during RPA amplification.

[0076] The results are as follows Figure 7 As shown, when the target dsDNA added to the system is 10... 1At ng / μL, the CRISPR / Cas12a detection system produced visible green fluorescence. Statistical analysis of the fluorescence values ​​with the blank control group revealed a significant difference, indicating good recognition performance.

[0077] The template amount for subsequent DNA is 10. 1 ng / μL.

[0078] Experiment 4. Optimization of RPA amplification system conditions

[0079] First, following the RPA amplification kit instructions, an initial RPA reaction system was prepared: 0.5 μM forward and reverse primers, 14 mM MgOAc, 0.8 mM dNTPs, and 10 μM MgOAc. 1 The following reagents were added: ng / μL DNA template, 5 μL 2×Reaction Buffer, 1 μL 10×Basic E-mix, and 0.5 μL 20×Core Reaction Mix, with sterile ultrapure water added to a final volume of 10 μL. This study optimized the primer concentration, dNTP concentration, MgOAc concentration, and reaction time in the RPA reaction system. The optimized RPA reaction system consisted of 0.5 μM forward and reverse primers, 2 mM dNTPs, 22.4 mM MgOAc, and 10 ng / μL reaction time. 1 Add ng / μL DNA template, 5μL 2×Reaction Buffer, 1μL 10×Basic E-mix, and 0.5μL 20×Core Reaction Mix, then add sterile ultrapure water to a final volume of 10μL. The reaction time is 25 min, as shown in Table 2. Figure 8 As shown.

[0080] Table 2 Optimization parameters for RPA amplification system

[0081]

[0082] The optimized RPA reaction system was obtained by adding the optimized CRISPR / Cas12a detection system after the RPA reaction was completed, incubating it in a QuantStudio3 Real-time PCR instrument at 37°C for 10 min, and then collecting fluorescence signals. The results were then observed visually using a portable blue light gel cutter at a wavelength of 470 nm.

[0083] Example 2. Establishment of a CRISPR-immunoassay combined with small molecule detection method

[0084] This method is based on the principle of CRISPR-immunoassay combined with small molecule detection and was established after analysis and optimization of multiple sets of experiments in this case.

[0085] Because this method involves RPA amplification, false positives are highly likely. The reason for this is likely that the open reaction site of the enzyme-labeled wells is susceptible to aerosol contamination during RPA amplification, leading to false positives. Therefore, the reaction site was transferred from the open enzyme-labeled wells to the closed EP tubes (tests showed that EP tubes can also perform immune reactions). Therefore, the specific implementation steps of the CRISPR-immunoassay combined small molecule detection system constructed in this invention are as follows:

[0086] It should be noted that this detection method is divided into three stages, which are used to determine the working concentration of antigen and antibody, establish a standard curve, and test real samples, respectively. Each stage is implemented in steps 1) to 7), the difference being that in the first stage, step 3) is implemented in step A, in the second stage, step 3) is implemented in step B, and in the third stage, step 3) is implemented in step C.

[0087] 1) Coating: Weigh 1.59g Na2CO3 and 2.93g NaHCO3 and dissolve them in 1L of tertiary water to prepare a coating buffer. Dilute the SAL coating to 2.56 pg / mL with this coating buffer (the original coating concentration was obtained from series 5). Add 10 μL / tube to the EP tube and incubate in a constant temperature water bath at 37℃ for 3 hours.

[0088] 2) Blocking: After removing the EP tube, manually shake off the coating solution inside the tube; add 200 μL of phosphate Tween buffer solution (PBST) to the EP tube, then manually shake off the solution inside the tube again, and use the PBST to hand wash the EP tube twice; add 20 μL of 6% skim milk powder blocking solution (prepared by dissolving 6 g of skim milk powder in 100 mL of PBST), incubate in a 37℃ constant temperature water bath for 1 hour, manually shake off the blocking solution inside the EP tube, and dry at 37℃ for 1 hour;

[0089] 3) Adding reagents: Steps A, B, and C in step (3) are respectively implemented in the three stages of the detection method experiment, as follows:

[0090] A. Dilute the 43.2 mg / mL dsDNA antibody probe to seven concentrations (1000, 2000, 4000, 8000, 16000, 32000, and 64000 times) using PBST. The initial concentration of the dsDNA antibody probe was measured to be 43.2 mg / mL using a micro spectrophotometer. Since the dsDNA antibody probe contains dsDNA, it will interfere with the accurate measurement of its absorbance at 280 nm. Therefore, this reference value was used. Add 50 μL of PBST and 50 μL of diluted dsDNA antibody probe to a sealed EP tube. Set a blank control (NTC) of 100 μL of PBST in the last row.

[0091] B. Dilute the initial concentration of 6 ng / mL of salbutamol with PBST to seven concentrations (1 ng / mL, 0.17 ng / mL, 0.027 ng / mL, 0.0046 ng / mL, 0.7 pg / mL, 0.13 pg / mL, and 0.021 pg / mL). Add 50 μL of the serially diluted salbutamol and 50 μL of dsDNA-antibody probe diluted 1:16000 (obtained from series 5) to a sealed EP tube. Set up a blank control in the last row with 50 μL of PBST and 50 μL of dsDNA-antibody probe.

[0092] C. Using pig urine as the real sample, the pig urine sample was first diluted with 5 times the volume of PBST. This dilution factor can basically eliminate the matrix interference caused by the pig urine sample (obtained from series 7). Then, 50 μL of diluted pig urine sample was added to the sealed EP tube, followed by 50 μL of dsDNA-antibody probe diluted 16000 times (obtained from series 5).

[0093] 4) After adding the reagents, incubate at 37°C for 40 min, and manually shake off the solution in the tube; add 200 μL of sterile ultrapure water to the EP tube, and then manually shake off the liquid in the tube. Wash the EP tube repeatedly with sterile ultrapure water five times; then wash the EP tube with sterile ultrapure water to thoroughly remove unbound probes and inorganic salts in the tube, reducing the impact on the subsequent RPA and CRISPR systems.

[0094] 5) RPA amplification: Add 10 μL of the optimal RPA reaction system, specifically with 0.5 μM each of forward and reverse primers, 2 mM dNTPs, 22.4 mM MgOAc, 5 μL 2 × Reaction Buffer, 1 μL 10 × Basic E-mix and 0.5 μL 20 × Core Reaction Mix, and add sterile ultrapure water to 10 μL (obtained from series 4). Incubate at 37℃ for 25 minutes.

[0095] 6) CRISPR / Cas12a detection system: Add 10 μL of the optimal CRISPR / Cas12a detection system, specifically Cas12a protein concentration 125 nM, crRNA concentration 50 nM, ssDNA-reporter concentration 1000 nM, 2 μL NEBuffer 2.1, and add sterile ultrapure water to 20 μL (obtained from series 2). Incubate at 37℃ for 10 minutes, then inactivate at 98℃ for 2 minutes, and then collect fluorescence signals using the QuantStudio3 Real-time PCR instrument.

[0096] 7) The acquired fluorescence signals were processed using Origin 2021 software. The optimal antigen concentration (i.e., working antigen concentration) was determined to be 2.56 pg / mL, and the dsDNA-antibody probe dilution ratio (i.e., working antibody concentration) was determined to be 1:16000. The IC50 standard curve of the CRISPR-immunoassay combined with small molecule detection system was determined. 10 It was 0.64 pg / mL, IC50 50 The concentration was 11.08 pg / mL, with a detection range of 1.83~66.97 pg / mL; and the detection of pig urine samples.

[0097] The CRISPR-immunoassay combined with small molecule detection method described in this technical solution integrates the specificity of immunoassay, the convenience of RPA isothermal amplification, and the ultra-high sensitivity and signal stability of the CRISPR / Cas system. Compared with the original antibody-based ic-ELISA method, this method demonstrates superior performance. 10 It has increased by 280 times, IC 50 The sensitivity was significantly improved by 134 times. Furthermore, this method is universally applicable; simply replacing the salbutamol monoclonal antibody with a corresponding small molecule monoclonal antibody can achieve highly sensitive detection of different small molecules, overcoming the shortcomings of existing immunoassay methods in detecting legally prohibited drugs.

[0098] Experiment 5. Results of Optimization of Coating Agent and dsDNA-Antibody Probe Concentration

[0099] Following the method in Example 2, to determine the working concentration of the antigen and antibody, single-factor optimization was performed on the concentrations of the coating antigen and the dsDNA-antibody probe (the initial concentrations were 2.2 mg / mL and 43.2 mg / mL, respectively; however, the latter, due to the presence of dsDNA, would interfere with the accurate measurement of its absorbance at 280 nm, so this concentration was only used as a reference, and subsequent experimental results were described using dilution ratios). The coating antigen concentrations were 0.16 pg / mL, 0.64 pg / mL, 2.56 pg / mL, 10.24 pg / mL, and 40.96 pg / mL, and the dsDNA-antibody probe dilutions were 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, to find the optimal combination of coating antigen / probe concentrations. The optimization results are as follows: Figure 9 As shown.

[0100] The results show that the fluorescence intensity decreases as the concentrations of the coating antigen and dsDNA-antibody probe decrease. Since lower concentrations of both can promote a competitive reaction and thus exhibit better performance, a coating antigen / probe concentration combination with lower signal intensity should be chosen. The results also show that with the antibody concentration unchanged (dilution ratio 1:1000), a significant decrease in fluorescence intensity occurs when the coating antigen concentration decreases from 2.56 pg / mL to 0.64 pg / mL. Based on the fluorescence intensity produced under the latter condition, it is determined that it will not provide a sufficient signal range for establishing a standard curve for the combined system. Furthermore, to prevent excessively low signal intensity from causing significant errors in the subsequent establishment of the combined system, a coating antigen concentration (i.e., working antigen concentration) of 2.56 pg / mL was chosen. The concentration of the dsDNA-antibody probe was optimized using the original coating concentration. When the dilution ratio of the dsDNA-antibody probe was increased from 1:16000 to 1:32000, the fluorescence intensity decreased significantly and remained basically unchanged thereafter. Based on the selection criteria of the above analysis, the dilution ratio of the dsDNA-antibody probe (i.e. the working concentration of the antibody) is 1:16000.

[0101] Experiment 6. Establishment of Standard Curve for CRISPR-Immunotherapy Combined with Small Molecule Detection System

[0102] Based on Experiment 5, the optimal antigen-antibody working concentrations were selected as 2.56 pg / mL for the original coating and 1:16000 for the dsDNA-antibody probe dilution. A standard curve for the CRISPR-immunoassay combined with small molecule detection system was established according to the method in Example 2.

[0103] Data processing and standard curve plotting: The B / B0 value was plotted on the ordinate and the drug concentrations on the abscissa, where B is the average absorbance of the three parallel wells and B0 is the average blank absorbance. The standard curve of ic-ELISA was obtained by fitting the data using Origin 2021 software.

[0104] The standard curve of the CRISPR-immunoassay combined with small molecule detection system is as follows: Figure 10 As shown, the IC10 was 0.64 pg / mL, the IC50 was 11.08 pg / mL, and the detection range was 1.83~66.97 pg / mL.

[0105] Experiment 7. Detection of porcine urine samples using a CRISPR-immunoassay combined with small molecule detection system.

[0106] Before testing actual pig urine samples, it is necessary to investigate the matrix effect of the samples. Adding an appropriate amount of PBST solution to eliminate the matrix effect can improve the accuracy of the system. The pretreated pig urine samples were diluted 2, 5, and 10 times with PBST solution, and these were used to dilute the SAL standard. Standard curves were established according to the operating procedures of the combined system and compared with the standard curves established by diluting the SAL standard with PBST.

[0107] The results are as follows Figure 10 As shown, when the pig urine sample was diluted 5 times, the fitted curve basically overlapped with the standard curve obtained by using PBST as the diluent, and the IC50 value (11.36 pg / mL) was the closest, indicating that this dilution factor can basically eliminate the matrix interference caused by the pig urine sample.

[0108] To evaluate the practical performance of the method, after testing the detection capability of the instrument, salbutamol standards (200, 1000, 5000 pg / mL) were added to pig urine samples. These spiked samples were then diluted 20-fold with pig urine samples (SAL concentrations of 10, 50, 250 pg / mL). The spiked samples were measured using the established CRISPR-immunoassay system, and LC-MS / MS was used to confirm the spiked samples. The accuracy and precision of the system were evaluated using the sample spiked recovery rate and the coefficient of variation (difference between parallel groups) as standards. Three parallel experiments were set up for each group. The recovery rate was calculated using formula (1), and the coefficient of variation was calculated using formula (2).

[0109] Recovery rate (%) = (Measured value - Blank value) * 100% / Added value (1)

[0110] Coefficient of variation (%) = Standard deviation * 100% / Mean (2)

[0111] Table 3. SAL addition and recovery experiment in pig urine samples (n=3)

[0112]

[0113] The results (as shown in Table 3) indicate that the recovery rate of SAL by the combined detection system established in this invention ranges from 90.9% to 134.0%, with a coefficient of variation ranging from 1.1% to 7.8%. These results are basically consistent with those obtained by the LC-MS / MS method (recovery rate of 103.2% to 118.4%, coefficient of variation of 1.7% to 10.2%). These results demonstrate that the CRISPR-immunoassay combined small molecule detection system established in this invention has high accuracy and precision, and can be used for the detection of SAL residues in actual pig urine.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A dsDNA-antibody probe, comprising an antibody and a target dsDNA, characterized in that: The antibody is any monoclonal antibody, and the dsDNA is a target dsDNA with a length of 100-300 bp containing the PAM site "TTTV". The two are synthesized into a dsDNA-antibody probe by maleimide-thiol coupling method. The target dsDNA sequence consists of four parts, including: a linker arm sequence of 5-10 bp in length that is linked to the antibody, a sequence containing the PAM site "TTTV" and a target sequence complementary to crRNA, a sequence that provides sufficient binding space for the Cas12a enzyme / crRNA complex, and an RPA primer recognition sequence. The nucleotide sequence of the target dsDNA is shown in SEQ ID No.

1.

2. The method for preparing the dsDNA-antibody probe according to claim 1, characterized in that, Includes the following steps: (1) 60 nmol of antibody was dialyzed in phosphate buffered saline (PBS) solution at pH 7.4 at 4°C for 48 hours to obtain the dialyzed antibody; (2) N-succinimide-S-acetylsiothioacetate (SATA) modified antibody: Add 600 nmol SATA solution to the dialyzed antibody, mix well, react at room temperature for 30 minutes to obtain SATA modified antibody, then transfer the SATA modified antibody to an ultrafiltration tube, and repeatedly centrifuge the ultrafiltration tube to concentrate and remove impurities from the SATA modified antibody until the solution is reduced to 200 μL, and then use phosphate buffer solution to make up the liquid volume to 900 μL; (3) Modification of target dsDNA with bifunctional cross-linking agent 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid sulfonyl succinimide sodium salt (Sulfo-SMCC): Add 600 nmol of prepared Sulfo-SMCC solution to 60 nmol of prepared target dsDNA, mix well, react at room temperature for 30 minutes to obtain Sulfo-SMCC modified dsDNA, transfer to ultrafiltration tube, centrifuge the ultrafiltration tube repeatedly, concentrate and remove impurities from Sulfo-SMCC modified dsDNA, make up the solution volume to 900 μL with phosphate buffer solution, and then temporarily store it at -20℃. (4) Add 100 μL of hydroxylamine hydrochloride solution to the concentrated and purified antibody, mix well, and react at room temperature for 2 hours; (5) Add the Sulfo-SMCC modified target dsDNA stored in step (3), wherein the molar ratio of Sulfo-SMCC modified target dsDNA to SATA modified antibody is 1:1, mix well, and react at room temperature for 1 hour. (6) The solution was concentrated and purified by centrifugation and ultrafiltration until 250 μL remained. The liquid was then removed and the solution was replenished to 1 mL with PBS to obtain the initial product of dsDNA-antibody probe. (7) The primary product of dsDNA-antibody probe was separated and purified by molecular sieve to obtain purified dsDNA-antibody probe.

3. The method for preparing the dsDNA-antibody probe according to claim 2, characterized in that, The concentration and impurity removal process in steps (2), (3) and (6) includes: centrifuging the ultrafiltration tube at 6000 r / min and 4℃, removing the ultrafiltration tube every 10 minutes of centrifugation, and using a pipette to spray the liquid onto both membranes, repeating the blowing and suction 3-5 times.

4. The method for preparing the dsDNA-antibody probe according to claim 3, characterized in that, The hydroxylamine hydrochloride solution comprises the following preparation steps: a) Weigh 1.74 g of hydroxylamine hydrochloride and 0.365 g of disodium ethylenediaminetetraacetic acid (EDTA) and add them to 40 mL of PBS solution. Mix well to obtain a mixed solution. b) Add sodium hydroxide solution to the mixed solution to adjust the pH of the mixed solution to 7.4, and obtain hydroxylamine hydrochloride solution.

5. The method for preparing the dsDNA-antibody probe according to claim 4, characterized in that, The antibody is a salbutamol monoclonal antibody.

6. A CRISPR-immunoassay combined with small molecule detection method, characterized in that, The detection method consists of three stages, each including steps 1)-7), which correspond to steps A, B, and C in step 3), respectively. Specifically: 1) Coating: Dilute the salbutamol coating agent to 2.56 pg / mL with coating buffer, add 10 μL / tube to EP tubes, and incubate in a 37℃ water bath for 3 hours; 2) Blocking: After removing the EP tube, manually shake off the coating solution inside the tube; add 200 μL of phosphate Tween buffer solution (PBST) to the EP tube, manually shake off the solution inside the tube, and wash the EP tube twice with PBST; then add 20 μL of 6% skim milk powder blocking solution to the EP tube, place the EP tube in a 37°C constant temperature water bath for 1 hour, manually shake off the blocking solution inside the EP tube, and dry at 37°C for 1 hour; 3) Add reagents: A. Dilute the dsDNA-antibody probe with PBST. Add 50 μL of PBST and 50 μL of diluted dsDNA-antibody probe to a sealed EP tube. Set a blank control (NTC) of 100 μL PBST in the last row. B. Dilute the initial concentration of salbutamol (6 ng / mL) with PBST. Add 50 μL of the salbutamol drug diluted by a factor of 1 and 50 μL of the dsDNA-antibody probe diluted at a ratio of 1:16000 to the sealed EP tube. Set a blank control in the last row with 50 μL of PBST and 50 μL of dsDNA-antibody probe. C. Dilute the pig urine sample with 5 times the volume of PBST, then add 50 μL of the diluted pig urine sample to the sealed EP tube, followed by 50 μL of dsDNA-antibody probe diluted 16000 times. 4) After adding the reagent, incubate the EP tube at 37°C for 40 minutes, manually shake off the solution in the tube, add 200 μL of sterile ultrapure water to the EP tube and then manually shake off the liquid in the tube. Wash the EP tube repeatedly with sterile ultrapure water 5 times. 5) RPA amplification: Add 10 μL of RPA reaction system, add sterile ultrapure water to 10 μL, and incubate at 37℃ for 25 min; 6) CRISPR / Cas12a detection system: Add 10 μL of RISPR / Cas12a detection system, add sterile ultrapure water to 20 μL, incubate at 37℃ for 10 minutes, then react at 98℃ for 2 minutes to inactivate, and then use the instrument to collect fluorescence signals; 7) Use software to process the collected fluorescence signals to obtain detection data.

7. The CRISPR-immunoassay combined with small molecule detection method according to claim 6, characterized in that, The RPA reaction system described in step 5) consists of 0.5 μM each of forward and reverse primers, 2 mM of dNTPs, 22.4 mM of MgOAc, 5 μL of 2×Reaction Buffer, 1 μL of 10×Basic E-mix, and 0.5 μL of 20×Core Reaction Mix.

8. The CRISPR-immunoassay combined with small molecule detection method according to claim 7, characterized in that, The CRISPR / Cas12a detection system described in step 6) specifically consists of Cas12a protein concentration of 125 nM, crRNA concentration of 50 nM, ssDNA-reporter concentration of 1000 nM, and 2 μL NEBuffer 2.

1.

9. The CRISPR-immunoassay combined with small molecule detection method according to claim 8, characterized in that, In step 3), step A, the dsDNA-antibody probe is diluted to seven concentrations: 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times. In step B, the salbutamol drug is diluted to seven concentrations: 1 ng / mL, 0.17 ng / mL, 0.027 ng / mL, 0.0046 ng / mL, 0.7 pg / mL, 0.13 pg / mL, and 0.021 pg / mL.

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