Ternary complex dual-mode probe and acetylcholin esterase activity and enzyme inhibitor detection method thereof

Abstract

The invention provides a ternary complex dual-mode probe and an acetylcholin esterase activity and enzyme inhibitor detection method thereof. The ternary complex dual-mode probe is composed of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, a manganese dioxide nanoflower-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activated ssDNA (ssDNA) (MnO2 NFs-ssDNA) and internal reference fluorescence. When a target to be detected is acetylcholin esterase (AChE), hydrolysis of an acetylthiocholine substrate can be catalyzed, a thiocholine product can be generated, MnO2 NFs can be decomposed to release ssDNA, the trans-cleavage activity of Cas12a can be activated, fluorescence report nucleic acid can be cleaved, a fluorescence signal can be generated, and a more accurate ratio fluorescence signal can be output by taking internal reference fluorescence added into a system as an internal standard; the decomposition of the MnO2 NFs reduces the oxidase-like activity of the MnO2 NFs, and the colorimetric signal for catalyzing the color development of 3, 3 ', 5, 5'-tetramethyl benzidine is reduced; therefore, the higher the AChE activity is, the larger the yield of the thiocholine is, the more thorough the decomposition of the MnO2 NFs is, the stronger the fluorescence signal is, and the weaker the colorimetric signal is. The enzyme inhibitor organophosphorus pesticide can inhibit generation of thiocholine, and then an opposite experiment result appears.

Description

Technical Field

[0001] This invention relates to a ternary complex dual-mode probe and a method for detecting acetylcholinesterase activity and enzyme inhibitors, specifically to a method and application for detecting acetylcholinesterase activity and enzyme inhibitors based on CRISPR, manganese dioxide nanoflower-CRISPR activated ssDNA (MnO2NFs-ssDNA), and an internal control fluorescent probe. Background Technology

[0002] Acetylcholinesterase (AChE) is a key enzyme in nerve conduction. Organophosphorus pesticides and other neurotoxic agents exert their toxicity by irreversibly inhibiting AChE activity. Therefore, developing highly sensitive, selective, rapid, and reliable methods for detecting AChE activity and its inhibitors is of great significance in food safety, environmental monitoring, and biomedical fields.

[0003] Currently, fluorescence and colorimetric sensors based on nanomaterials have attracted widespread attention due to their simplicity and speed. Among them, manganese dioxide nanomaterials (such as nanosheets and nanoflowers) are often used as "switches" or signal converters in the construction of sensing platforms due to their good biocompatibility, tunable morphology, efficient fluorescence quenching ability, and peroxidase / oxidase-like activity. For example, existing technologies combine gold nanoclusters (AuNCs) with MnO2 nanosheets, utilizing the quenching of AuNCs fluorescence by MnO2 and its enzyme-like activity to construct a fluorescence-colorimetric dual-mode method for detecting organophosphorus pesticides. Other studies have used MnO2 nanosheets with copper-silver nanoclusters to construct ratiometric fluorescence sensors for detecting chlorpyrifos. However, these methods typically rely on a single signal output mode or simple fluorescence intensity changes, are easily affected by environmental and instrumental factors, and their specificity needs improvement.

[0004] On the other hand, CRISPR / Cas systems, especially proteins with "trans-cleavage" activity such as Cas12a and Cas13a, have been developed as a new generation of highly specific biosensors because they can non-specifically cleave surrounding reporter nucleic acids after recognizing specific nucleic acid sequences, producing a strong signal amplification effect. Recently, a study used MnO2 nanosheets as a carrier to adsorb single-stranded DNA (ssDNA) activators from the CRISPR / Cas12a system, constructing a fluorescent biosensor for detecting organophosphorus pesticides. The principle is that the AChE-catalyzed reaction product TCh reduces the MnO2 nanosheets, releasing ssDNA to activate Cas12a, which then cleaves the fluorescent reporter molecule to generate a signal. While this method utilizes the high specificity of CRISPR, the signal output is still limited to single-channel fluorescence, making multi-mode cross-validation impossible, and its reliability in complex real-world samples faces challenges.

[0005] Currently, there is a lack of integrated sensing probes that can simultaneously integrate the high specificity and signal amplification capabilities of the CRISPR system, ratiometric fluorescence signals, and the dual-mode (fluorescence / colorimetric) signal output and conversion capabilities of MnO2 nanomaterials. Existing technologies have shortcomings in terms of signal output redundancy, interference resistance, and on-site visualization detection. Summary of the Invention

[0006] Based on the deficiencies of existing technologies, the first objective of this invention is to develop a ternary complex dual-mode probe; the second objective of this invention is to achieve accurate and rapid detection of acetylcholinesterase; and the third objective of this invention is to achieve accurate and rapid detection of acetylcholinesterase inhibitors, such as organophosphorus pesticides.

[0007] The objective of this invention is achieved through the following technical solution: On the one hand, the present invention provides a ternary complex dual-mode probe, which consists of a CRISPR system, MnO2NFs-ssDNA and an internal control fluorescence; The CRISPR system includes Cas12a, gRNA, Cas Smart Buffer, and fluorescent reporter nucleic acid; The MnO2NFs in MnO2NFs-ssDNA are manganese dioxide nanoflowers synthesized by hydrothermal method; The ssDNA sequence in MnO2NFs-ssDNA is shown in SEQ ID NO.1; Among the internal control fluorescence candidates are AIE-FM fluorescent microspheres and BSA-Au NCs gold clusters; On the other hand, the present invention provides a ratiometric fluorescence detection method for acetylcholinesterase activity, which utilizes the aforementioned ternary complex dual-mode probe to detect acetylcholinesterase activity, and the steps are as follows: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 15 μL BSA-Au NCs gold cluster to a centrifuge tube and mix at room temperature for 30 min; (2) Add AChE solution and ATCh substrate to the centrifuge tubes above, and add water to make up to 250 μL. Mix well and react at 37°C for 1 h. (3) Then scan the fluorescence emission spectrum of 510-750nm with an ELISA reader, record the fluorescence intensity ratio, and obtain the relationship between the fluorescence ratio change value and the AChE concentration.

[0008] On the other hand, the present invention provides a colorimetric detection method for acetylcholinesterase activity, which utilizes the aforementioned ternary complex dual-mode probe to detect acetylcholinesterase activity, and the steps are as follows: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 2 μL AIE fluorescent microspheres to a centrifuge tube and mix at room temperature for 30 min; (2) Add AChE solution and ATCh substrate to the centrifuge tubes respectively, mix well, and react at 37°C for 1 h; (3) Then add 10 μL of 10 mM TMB solution, add water to make up to 250 μL, and react at 25℃ for 15 min; (4) Scan the ultraviolet absorption spectrum at 550-750 nm using an ELISA reader, record the absorbance value at 652 nm, and obtain A. 652 The relationship between AChE concentration and concentration.

[0009] On the other hand, the present invention provides a ratio fluorescence detection method for acetylcholinesterase activity inhibitors, which utilizes the aforementioned ternary complex dual-mode probe to detect acetylcholinesterase activity inhibitors, and the steps are as follows: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 15 μL BSA-Au NCs gold cluster to a centrifuge tube and mix at room temperature for 30 min; (2) Mix 10 μL of 6 U / mL AChE solution with 30 μL of inhibitor (organophosphorus pesticide, dichlorvos) solution of different concentrations at 37℃ for 1 h to obtain 40 μL of AChE-inhibitor solution of different concentrations. (3) Add AChE-inhibitor solution and 10 μL 10 mM ATCh to the centrifuge tube in (1), add water to make up to 250 μL, mix well, and react at 37℃ for 30 min. (4) Then scan the fluorescence emission spectrum of 510-750 nm with an ELISA reader, record the fluorescence intensity ratio, and obtain the relationship between the fluorescence ratio change value and the inhibitor concentration.

[0010] On the other hand, the present invention provides a colorimetric detection method for acetylcholinesterase activity inhibitors, which utilizes the aforementioned ternary complex dual-mode probe to detect acetylcholinesterase activity inhibitors, and the steps are as follows: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 2 μL AIE fluorescent microspheres, and mix at room temperature for 30 min; (2) Mix 10 μL of 6 U / mL AChE solution with 30 μL of inhibitor (organophosphorus pesticide, dichlorvos) solution of different concentrations at 37℃ for 1 h to obtain 40 μL of AChE-inhibitor solution of different concentrations. (3) Add AChE-inhibitor solution and 10 μL 10 mM ATCh to the centrifuge tube in (1), mix well, and react at 37℃ for 30 min; (4) Then add 10 μL of 10 mM TMB solution, add water to make up to 250 μL, and react at 25℃ for 15 min; (5) Scan the ultraviolet absorption spectrum at 550-750 nm using an ELISA reader, record the absorbance value at 652 nm, and obtain A. 652 The relationship between the concentration of the inhibitor and the concentration of the inhibitor.

[0011] On the other hand, the present invention provides an application of a ternary complex dual-mode probe in the detection of acetylcholinesterase and acetylcholinesterase activity inhibitors.

[0012] On the other hand, the present invention provides an application of a ratio fluorescence detection method in the detection of acetylcholinesterase and acetylcholinesterase activity inhibitors.

[0013] On the other hand, the present invention provides an application of a colorimetric detection method in the detection of acetylcholinesterase and acetylcholinesterase activity inhibitors.

[0014] The beneficial effects of this invention are: Dual-mode signal output with high reliability: For the first time, a CRISPR / Cas12a-mediated fluorescence signal and an enzyme-like catalytic colorimetric signal from MnO2NFs are integrated into a single probe. Both signals originate from the same triggering event, but their output modes are independent, allowing for mutual verification of results and effectively avoiding false positives or false negatives. This makes it particularly suitable for the analysis of complex matrix samples.

[0015] Introducing aggregation-induced emission (AIE) materials results in more stable signals: AIE fluorescent microspheres are used to replace traditional ACQ dyes or quantum dots. They exhibit strong aggregation-state luminescence and high photobleaching tolerance. As a fluorescent internal reference, they can provide more stable and reliable fluorescence signals and reduce background interference.

[0016] It has great potential for rapid on-site detection: the generation of colorimetric signals does not require complicated instruments. Semi-quantitative judgment can be made by observing color changes with the naked eye or by taking pictures and analyzing them with a smartphone, which greatly meets the needs of on-site, real-time and rapid screening.

[0017] The probe is integrated and easy to operate: ssDNA is preloaded onto MnO2NFs to form an "integrated" probe. When using it, it can be simply mixed with reaction buffer, Cas 12a protein, gRNA, fluorescent reporter nucleic acid, substrate, etc., which simplifies the operation steps. Attached Figure Description

[0018] Figure 1 Fluorescence characterization of a ternary complex dual-mode probe.

[0019] Figure 2 Enzyme-like activity characterization of a ternary complex dual-mode probe.

[0020] Figure 3 The detection principle of a dual-mode probe for ternary complexes.

[0021] Figure 4 Sensitivity of AChE detection in ratiometric fluorescence mode.

[0022] Figure 5 AChE detection sensitivity in colorimetric mode.

[0023] Figure 6 Sensitivity of the organophosphorus pesticide dichlorvos under ratio fluorescence mode.

[0024] Figure 7 Sensitivity of the organophosphorus pesticide dichlorvos in colorimetric mode. Detailed Implementation

[0025] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0026] Example 1: Synthesis and Construction of a Ternary Complex Dual-Mode Probe The probe constructed in this invention is a ternary complex, the core of which consists of a CRISPR system, MnO2NFs-ssDNA, and an internal control fluorescence.

[0027] Preparation of MnO2NFs: MnO2NFs were synthesized via a hydrothermal method. 0.05 g of KMnO4 was dissolved in 25 mL of distilled water and stirred at 800 rpm for 0.5 h. Then, 0.5 mL of oleic acid was added dropwise, and stirring continued for 48 h. The resulting mixture was transferred to a 50 mL centrifuge tube and centrifuged at 11000 rpm for 5 min to obtain a precipitate. The precipitate was washed five times each with distilled water and 75% ethanol to remove any remaining reactants. After washing, the precipitate was redispersed in 25 mL of distilled water to obtain MnO2NFs. This material exhibits oxidase-like activity and can catalyze the oxidation and color development of substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), generating a colorimetric signal.

[0028] Construction of Manganese Dioxide Nanoflower-CRISPR-Activated ssDNA (MnO2NFs-ssDNA): A single-stranded DNA (ssDNA, GCT TAG AGT ATA GTA GTT GAT CG, SEQ ID NO.1) that can be recognized by a specific gRNA was designed and synthesized as the "key" to activate the Cas12a protein. 50 μL of 0.2 μM ssDNA activator, 1000 μL of MnO2NFs, and 500 μL of 100 mM pH 7 Tris-HCl were mixed and reacted at room temperature with shaking at 200 rpm for 1 h. Unreacted reagents were removed by centrifugation at 12000 rpm for 5 min. After washing three times with Tris-HCl, the precipitate (MnO2NFs-ssDNA) was redispersed in 100 μL of Tris-HCl buffer for later use.

[0029] Synthesis of fluorescent microspheres (AIE-FM): 4 mL of 0.5 μmol / mL tannic acid (TA) solution and 4 mL of 0.5 μmol / mL ferric chloride solution were mixed and reacted at 500 rpm at room temperature for 10 min. Then, 2 mL of 1 mg / mL tetrahydrofuran solution of 2,3-bis(4'-(diphenylamino)-[1,1'-biphenyl]-4-yl)fumarin (TPAFN) was added dropwise, and the mixture was stirred continuously at 1000 rpm for 5 min. Next, 1 mL of 100 mM Tris-HCl buffer (pH 9) was added to adjust the pH. After gentle stirring and incubation at 500 rpm for 3 h, the mixture was centrifuged at 11000 rpm for 20 min at 4 °C. The supernatant was discarded, and the resulting precipitate was washed twice with ultrapure water to remove excess TA-Fe. 3+ After complexation, the precipitate was resuspended in 5 mL of ultrapure water and thoroughly mixed to obtain AIE-FM fluorescent microspheres. AIE-FM can be used as a stable fluorescent internal control.

[0030] Synthesis of gold nanoclusters (BSA-Au NCs): 5 mL of chloroauric acid (5-20 mM) was added to 5 mL of bovine serum albumin solution (20-50 mM), the pH was adjusted to alkaline, and the reaction was carried out at 37℃ for 10-24 h to obtain bovine serum albumin-coated BSA-Au NCs gold clusters. BSA-Au NCs can also be used as a stable fluorescent internal control.

[0031] Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid (FAM-TTTTTTT-BHQ, SEQ ID NO.2), 1 μL MnO2NFs-ssDNA, 2 μL AIE-FM fluorescent microspheres or 15 μL BSA-Au NCs gold clusters, and mix at room temperature for 30 min.

[0032] Example 2: Performance Characterization of a Ternary Complex Dual-Mode Probe To prepare the Cas12a-gRNA complex, mix 10 μL of Cas12a with 2.5 μL of gRNA (UAA UUU CUA CUA AGU GUA GAU AUC AAC UACUAU ACU CUA A, SEQ ID NO.3) and 25 μL of 10×Cas Smart Buffer at room temperature for 30 min. Take a series of centrifuge tubes and number them 1, 2, 3, 4, and 5. Add 2 μL of AIE-FM fluorescent microspheres to centrifuge tube 1; add 15 μL of BSA-Au NCs gold clusters to centrifuge tube 2; add 10 μL of 6 U / mL AChE, 10 μL of 10 mM ATCh, 1 μL of MnO2NFs-ssDNA, 7.5 μL of Cas12a-gRNA complex, and 0.5 μL of fluorescent reporter nucleic acid to centrifuge tube 3; add 10 μL of 6 U / mL AChE, 10 μL of 10 mM ATCh, 1 μL of MnO2NFs-ssDNA, 7.5 μL of Cas12a-gRNA complex, 0.5 μL of fluorescent reporter nucleic acid, and 2 μL of AIE-FM fluorescent microspheres to centrifuge tube 4; add 10 μL of 6 U / mL AChE, 10 μL of 10 mM ATCh, 1 μL of MnO2NFs-ssDNA, 7.5 μL of Cas12a-gRNA complex, 0.5 μL of fluorescent reporter nucleic acid, and 2 μL of AIE-FM fluorescent microspheres to centrifuge tube 5; add 10 μL of 6 U / mL AChE, 10 μL of 10 mM ATCh, 1 μL of MnO2NFs-ssDNA, and 7.5 μL of fluorescent reporter nucleic acid to centrifuge tube 5. μL of Cas12a-gRNA complex, 0.5 μL of fluorescent reporter nucleic acid, and 15 μL of BSA-Au NCs gold clusters were added to each tube to bring the total volume to 250 μL. The mixture was incubated at 37°C for 1 h, and then the fluorescence emission spectrum from 510 to 750 nm was scanned using a microplate reader. Results are as follows: Figure 1 As shown, tube 1, with its AIE-FM fluorescent microspheres, exhibits a distinct fluorescence emission peak at approximately 610 nm; tube 2, with its gold cluster, shows a distinct fluorescence emission peak at 674 nm; tube 3, with its CRISPR system, clearly activates Cas12a activity, cleaving the ssDNA sequence and releasing FAM fluorescence (524 nm); tube 4, with its ternary complex dual-mode probe using AIE-FM fluorescent microspheres as a stable fluorescent internal control, shows two fluorescence emission peaks at 524 nm and 610 nm; and tube 5, with its ternary complex dual-mode probe using BSA-Au NCs gold cluster as a stable fluorescent internal control, also shows two fluorescence emission peaks at 524 nm and 674 nm.

[0033] To verify the activity of MnO2NFs-like enzymes, a series of centrifuge tubes were taken and numbered 1, 2, 3, 4, 5, and 6. Add 10 μL of 10 mM TMB to centrifuge tube 1; 1 μL of MnO2NFs to centrifuge tube 2; 10 μL of 10 mM TMB and 1 μL of MnO2NFs to centrifuge tube 3; 10 μL of 10 mM TMB, 10 μL of 6 U / mL AChE and 1 μL of MnO2NFs to centrifuge tube 4; 10 μL of 10 mM TMB, 10 μL of 10 mM ATCh and 1 μL of MnO2NFs to centrifuge tube 5; 10 μL of 6 U / mL AChE and 10 μL of 10 mM ATCh to centrifuge tube 6; and 10 μL of 10 mM TMB, 10 μL of 6 U / mL AChE, 10 μL of 10 mM ATCh and 1 μL of MnO2NFs to centrifuge tube 7. Add water to each tube to bring the total volume to 250 mL. μL, reacted at 25℃ for 15 min. The UV absorption spectrum from 550-750 nm was scanned using a microplate reader. Results are as follows: Figure 2 As shown, MnO2NFs can catalyze the color change of TMB, and the presence of AChE and ATCh alone does not affect the enzyme activity of MnO2NFs. Only when AChE and ATCh coexist does AChE catalyze ATCh to produce the product thiocholine, which can reduce MnO2NFs to soluble Mn. 2+ Ions cause the nanoflower structure to decompose, thereby reducing its enzyme-like activity.

[0034] Example 3: Detection Principle of a Ternary Complex Dual-Mode Probe In the AChE activity assay, AChE in the system catalyzes an enzymatic reaction, hydrolyzing its substrate acetylthiocholine (ATCh) to produce thiocholine (TCh). TChh is a strong reducing agent that can reduce MnO2NFs to soluble Mn. 2+Ions cause the nanoflower structure to decompose. After decomposition, the ssDNA loaded on its surface is released into the solution. The free ssDNA binds to the Cas12a-gRNA complex, activating the trans-cleavage activity of Cas12a. The activated Cas12a indiscriminately cleaves the fluorescent reporter nucleic acid added to the system, causing the fluorophore FAM to separate from the quencher BHQ1, thereby generating a strong fluorescence recovery signal (“Turn-On”). At the same time, the internal control fluorescent AIE-FM or BSA-Au NCs in the system serve as reference fluorescent signals, further improving accuracy. The decomposition of MnO2NFs causes them to lose their oxidase-like activity, and they cannot catalyze the added colorimetric substrate (such as TMB) to produce a colorimetric reaction, and the solution color remains colorless or light blue. Therefore, the higher the AChE activity, the greater the TCh production, the more complete the decomposition of MnO2NFs, the stronger the fluorescence signal, and the weaker the colorimetric signal.

[0035] In the detection of AChE inhibitors (such as organophosphorus pesticides OPs), when OPs are present in the sample, they irreversibly bind to the active site of AChE, inhibiting its catalytic activity. Because AChE is inhibited, ATCh cannot be effectively hydrolyzed to generate TCh (or the amount generated is significantly reduced). Therefore, MnO2NFs cannot be effectively reduced and decomposed, and their structure remains intact. ssDNA cannot be released, the CRISPR / Cas12a system cannot be activated, the fluorescent reporter nucleic acid is not cleaved, and the fluorescence signal remains at a low level (“Turn-Off” mode). Simultaneously, the internal control fluorescent AIE-FM or BSA-Au NCs in the system serve as internal standards. Intact MnO2NFs retain their oxidase-like activity and can catalyze the oxidation of substrates such as TMB to generate blue products (oxidized TMB), producing a significant colorimetric signal. Therefore, the higher the concentration of OPs, the stronger the inhibition of AChE, the less TCh is generated, resulting in a weaker fluorescence signal and a stronger colorimetric signal.

[0036] The detection principle of this invention is based on a sophisticated cascade reaction, such as... Figure 3 As shown, dual-mode quantitative analysis of AChE activity or OPs concentration can be achieved by simultaneously or separately measuring ratio fluorescence intensity (the ratio of fluorescence intensity at a specific wavelength, such as 524 nm for FAM and 674 nm for BSA-Au NCs) and solution absorbance (at a specific wavelength, such as 652 nm for oxidized TMB). Changes in the colorimetric signal can be used for rapid on-site visualization and judgment, while the ratio fluorescence signal provides highly sensitive and accurate quantitative results. The two mutually verify each other, greatly improving the reliability and practicality of the detection.

[0037] Example 4: Ratio fluorescence mode for AChE activity detection Preparation of ternary complex dual-mode probe: Take a series of centrifuge tubes and add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas Smart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 15 μL LSA-Au NCs to them. Mix at room temperature for 30 min.

[0038] AChE detection: Add 10 μL of AChE solution of different concentrations (0.1, 0.2, 1, 3, 6 U / mL) and 10 μL of 10 mM ATCh to the centrifuge tubes respectively, add water to make up to 250 μL, mix well, and react at 37℃ for 1 h. Then scan the fluorescence emission spectrum from 510 to 750 nm using a microplate reader, and record the fluorescence intensity ratios I and I0. I and I0 are the ratios of the fluorescence intensity at 524 nm and 674 nm, respectively, for sample concentration and blank conditions (F0). 524 / F 674 The linear relationship between the fluorescence ratio change (I0-I) / I0 and the AChE concentration C was obtained: (I0-I) / I0 = -0.7805C + 0.2223. Figure 4 As shown in the figure. Using this linear relationship, AChE in unknown samples can be quantitatively analyzed, with a limit of detection (LOD) of 0.02 U / mL and a limit of quantitation (LOQ) of 0.06 U / mL.

[0039] Example 5: Colorimetric mode for AChE activity detection Preparation of ternary complex dual-mode probe: Take a series of centrifuge tubes and add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas Smart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 2 μL LAIE fluorescent microspheres to them. Mix at room temperature for 30 min.

[0040] AChE detection: Add 10 μL of AChE solution of different concentrations (0.1, 0.2, 1, 3, 6 U / mL) and 10 μL of 10 mM ATCh to the centrifuge tubes respectively, and react at 37℃ for 1 h. Then add 10 μL of 10 mM TMB solution, and add water to make up to 250 μL, and react at 25℃ for 15 min. Scan the UV absorption spectrum from 550-750 nm using a microplate reader, and record the absorbance value at 652 nm. 652 A was obtained 652 Linear relationship between AChE concentration C and A 652 = -0.0223C + 0.1827, as Figure 5As shown in the figure. Using this linear relationship, AChE in unknown samples can be quantitatively analyzed, with a limit of detection (LOD) of 0.04 U / mL and a limit of quantitation (LOQ) of 0.12 U / mL.

[0041] Example 6: Detection ratio fluorescence mode for inhibitory organophosphorus pesticides (dichlorvos) Preparation of ternary complex dual-mode probe: Take a series of centrifuge tubes and add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas Smart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 15 μL LSA-Au NCs gold cluster to each tube. Mix at room temperature for 30 min.

[0042] Dichlorvos detection: 10 μL of 6 U / mL AChE solution was mixed with 30 μL of dichlorvos solutions of different concentrations (0.5, 0.8, 3, 4, 5 mg / L) at 37℃ for 1 h to obtain 40 μL of AChE-dichlorvos solutions of different concentrations. 40 μL of the prepared AChE-dichlorvos solutions of different concentrations and 10 μL of 10 mMATCh were added to centrifuge tubes containing the ternary complex, and the volume was adjusted to 250 μL with water. After mixing, the mixture was reacted at 37℃ for 30 min. The fluorescence emission spectrum from 510 to 750 nm was then scanned using a microplate reader, and the fluorescence intensity ratios I and I0 were recorded. I and I0 are the ratios of the fluorescence intensity at 524 nm and 674 nm, respectively, for sample concentration and blank conditions, respectively (F0). 524 / F 674 The linear relationship between the fluorescence ratio change value (I0-I) / I0 and the dichlorvos concentration C was obtained: (I0-I) / I0 = 0.0908C + 0.2626. Figure 6 As shown in the figure. Using this linear relationship, dichlorvos in unknown samples can be quantitatively analyzed, with a limit of detection (LOD) of 0.03 mg / L and a limit of quantification (LOQ) of 0.09 mg / L.

[0043] Example 7: Colorimetric mode for detecting inhibitory organophosphorus pesticides (dichlorvos) Preparation of ternary complex dual-mode probe: Take a series of centrifuge tubes and add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas Smart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA and 2 μL LAIE fluorescent microspheres to them. Mix at room temperature for 30 min.

[0044] Dichlorvos (DDV) detection: 10 μL of 6 U / mL AChE solution was mixed with 30 μL of different concentrations of dichlorvos solution (0.8, 2, 3, 4, 5 mg / L) at 37℃ for 1 h to obtain 40 μL of AChE-DDV solutions of different concentrations. 40 μL of the prepared AChE-DDV solutions of different concentrations and 10 μL of 10 mMATCh were added to centrifuge tubes containing the ternary complex, mixed well, and reacted at 37℃ for 30 min. Then, 10 μL of 10 mM TMB solution was added, and the volume was adjusted to 250 μL with water, and reacted at 25℃ for 15 min. The UV absorption spectrum from 550 to 750 nm was scanned using a microplate reader, and the absorbance value A at 652 nm was recorded. 652 A was obtained 652 Linear relationship between A and dichlorvos concentration C 652 =0.0114C + 0.1307, as Figure 7 As shown in the figure. Using this linear relationship, dichlorvos in unknown samples can be quantitatively analyzed, with a limit of detection (LOD) of 0.09 mg / L and a limit of quantitation (LOQ) of 0.31 mg / L.

[0045] Example 8: Cross-validation of acetylcholinesterase activity detection results using a ternary composite probe Ratio fluorescence mode: Take two centrifuge tubes and add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas Smart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA, and 15 μL LSA-Au NCs gold clusters to each tube. Mix at room temperature for 30 min. Add 10 μL 0.5 U / mL AChE solution and 10 μL 10 mM ATCh to one centrifuge tube; add 10 μL distilled water and 10 μL 10 mM ATCh to the other centrifuge tube as a blank control. Add water to both centrifuge tubes to a final volume of 250 μL, mix well, and incubate at 37℃ for 1 h. Then, scan the fluorescence emission spectrum from 510 to 750 nm using a microplate reader and record the fluorescence intensity ratios I and I0. I and I0 are the ratios of the fluorescence intensity at 524 nm and 674 nm, respectively, for the sample concentration and blank control. 524 / F 674 ).

[0046] Colorimetric mode: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×Cas SmartBuffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2NFs-ssDNA, and 2 μL LAIE fluorescent microspheres to a centrifuge tube and mix at room temperature for 30 min. Then, add 10 μL 0.5 U / mL AChE solution and 10 μL 10 mM ATCh, and react at 37℃ for 1 h. Finally, add 10 μL 10 mM TMB solution and dilute to 250 μL with water, and react at 25℃ for 15 min. Scan the UV absorption spectrum from 550-750 nm using a microplate reader and record the absorbance value at 652 nm. 652 The calculated measured concentrations and recoveries are shown in the table below. The results show that both detection methods can accurately detect acetylcholinesterase concentration, with recoveries of 102.8% and 91.5% for ratio fluorescence and colorimetric detection modes, respectively. The results from the two detection modes can be cross-validated, improving the accuracy of the detection.

[0047] Table 1. Results of acetylcholinesterase activity assay using the ternary composite probe.

Claims

1. A ternary complex dual-mode probe, characterized in that, It consists of a CRISPR system, MnO2 NFs-ssDNA, and an internal control fluorescence. The CRISPR system includes Cas12a, gRNA, Cas Smart Buffer, and fluorescent reporter nucleic acid; The MnO2 NFs in the MnO2 NFs-ssDNA are manganese dioxide nanoflowers synthesized by hydrothermal method; The ssDNA sequence in the MnO2 NFs-ssDNA is shown in SEQ ID NO.1; The internal control fluorescent candidates include AIE-FM fluorescent microspheres and BSA-Au NCs gold clusters.

2. A ratiometric fluorescence detection method for acetylcholinesterase activity, characterized in that, The detection of acetylcholinesterase activity using the ternary complex dual-mode probe of claim 1 is carried out through the following steps: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2 NFs-ssDNA and 15 μL BSA-Au NCs gold cluster to a centrifuge tube and mix at room temperature for 30 min; (2) Add AChE solution and ATCh substrate to the centrifuge tubes above, and add water to make up to 250 μL. Mix well and react at 37°C for 1 h. (3) Then scan the fluorescence emission spectrum of 510-750nm with an ELISA reader, record the fluorescence intensity ratio, and obtain the relationship between the fluorescence ratio change value and the AChE concentration.

3. A colorimetric detection method for acetylcholinesterase activity, characterized in that, The detection of acetylcholinesterase activity using the ternary complex dual-mode probe of claim 1 is carried out through the following steps: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2 NFs-ssDNA and 2 μL AIE fluorescent microspheres to a centrifuge tube and mix at room temperature for 30 min; (2) Add AChE solution and ATCh substrate to the centrifuge tubes respectively, mix well, and react at 37°C for 1 h; (3) Then add 10 μL of 10 mM TMB solution, add water to make up to 250 μL, and react at 25℃ for 15 min; (4) Scan the ultraviolet absorption spectrum at 550-750 nm using an ELISA reader, record the absorbance value at 652 nm, and obtain A. 652 The relationship between AChE concentration and concentration.

4. A ratio fluorescence detection method for an acetylcholinesterase activity inhibitor, characterized in that, The detection of acetylcholinesterase activity inhibitors using the ternary complex dual-mode probe of claim 1 is carried out through the following steps: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2 NFs-ssDNA and 15 μL BSA-Au NCs gold cluster to a centrifuge tube and mix at room temperature for 30 min; (2) Mix 10 μL of 6 U / mL AChE solution with 30 μL of inhibitor solutions of different concentrations at 37℃ for 1 h to obtain 40 μL of AChE-inhibitor solutions of different concentrations; (3) Add AChE-inhibitor solution and 10 μL 10 mM ATCh to the centrifuge tube in (1), add water to make up to 250 μL, mix well, and react at 37℃ for 30 min. (4) Then scan the fluorescence emission spectrum of 510-750 nm with an ELISA reader, record the fluorescence intensity ratio, and obtain the relationship between the fluorescence ratio change value and the inhibitor concentration.

5. A colorimetric detection method for acetylcholinesterase activity inhibitors, characterized in that, The detection of acetylcholinesterase activity inhibitors using the ternary complex dual-mode probe of claim 1 is carried out through the following steps: (1) Preparation of the ternary complex dual-mode probe: Add 2 μL Cas12a, 0.5 μL gRNA, 5 μL 10×CasSmart Buffer, 0.5 μL fluorescent reporter nucleic acid, 1 μL MnO2 NFs-ssDNA and 2 μL AIE fluorescent microspheres, and mix at room temperature for 30 min; (2) Mix 10 μL of 6 U / mL AChE solution with 30 μL of inhibitor solutions of different concentrations at 37℃ for 1 h to obtain 40 μL of AChE-inhibitor solutions of different concentrations; (3) Add AChE-inhibitor solution and 10 μL 10 mM ATCh to the centrifuge tube in (1), mix well, and react at 37℃ for 30 min; (4) Then add 10 μL of 10 mM TMB solution, add water to make up to 250 μL, and react at 25℃ for 15 min; (5) Scan the ultraviolet absorption spectrum at 550-750 nm using an ELISA reader, record the absorbance value at 652 nm, and obtain A. 652 The relationship between the concentration of the inhibitor and the concentration of the inhibitor.

6. The application of the ternary complex dual-mode probe as described in claim 1 in the detection of acetylcholinesterase.

7. The application of the ternary complex dual-mode probe as described in claim 1 in acetylcholinesterase activity inhibitors.

8. The application of the ratio fluorescence detection method as described in claim 2 in the detection of acetylcholinesterase.

9. The application of the ratio fluorescence detection method as described in claim 4 in the detection of acetylcholinesterase activity inhibitors.

10. The application of the colorimetric detection method as described in claims 3 and 5 in the detection of acetylcholinesterase and acetylcholinesterase activity inhibitors.

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