Sensitive biological analysis method for aflatoxin AFB1 based on single-particle inductively coupled plasma mass spectrometry and CRISPR / Cas12a

Abstract

Nucleic acid amplification strategies are usually necessary biological detection means, but signal leakage caused by amplification often affects analysis results. Therefore, the invention provides an amplification-free aflatoxin B1 detection method based on the cleavage enzyme. Meanwhile, the single particle mode inductively coupled plasma mass spectrometry (SPICPMS) is a sensitive nanoparticle counting tool because a single particle can generate a signal which is obviously different from the background. When a cancerogen exists, the designed aptamer lock is specifically combined with a target object to release an activation chain for starting the trans-cleavage of the cleavage enzyme so as to cut the probe marked by the nano-particles, and the cleavage nano-particles are analyzed through a frequency mode of SpICPMS (Space Inductively Coupled Plasma Mass Spectrometry). According to the method disclosed by the invention, the picomole level detection limit is reached in an amplification-free manner. Meanwhile, the analysis on the standard substance is efficiently realized. The method disclosed by the invention is expected to become an effective tool for accurately detecting and evaluating small molecules of cancerogens.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and relates to the field of analytical chemistry sensing of single particle inductively coupled plasma mass spectrometry (Sp-ICPMS), and particularly relates to a sensitive amplification-free analysis method for a carcinogen aflatoxin AFB1 based on single particle inductively coupled plasma mass spectrometry assisted by the shearing enzyme CRISPR / Cas12a. Background Art

[0002] Aflatoxin produced by Aspergillus flavus is considered to be one of the most threatening risk molecules in the global food industry. In warm and humid environments, agricultural products such as corn, peanut oil, and wheat flour are easily contaminated. Among the classifications of aflatoxins, aflatoxin B1 (AFB1) has the greatest toxicity and poses the most serious threat to human health. High performance liquid chromatography (HPLC) is currently the efficient standard method for detecting toxic small molecules, but sometimes it also faces challenges such as difficult operation, complex sample processing, and time-consuming detection processes. The powerful functions of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated effector proteins (Cas) have promoted the development of CRISPR / Cas in a wide range of scientific fields. Features such as convenient operation procedures, outstanding biocompatibility, flexible programmability, and unique trans-cleavage have also been used to develop various advanced analytical strategies. Various signal amplification strategies, such as PCR, RPA, and EDC, etc., can also be combined with it, but inevitable signal leakage and interference from various proteases often have an adverse impact on detection. This paper proposes a single particle counting biosensing method for sensitive detection of AFB1 without amplification. Single particle inductively coupled plasma mass spectrometry (Sp-ICPMS) has the advantages of good resolution, high sensitive detection ability, and no mass spectrometry overlap. Magnetic beads (MBs) crosslinked with nucleic acids and noble metal nanoparticles (AuNPs) are used as probes (MB-DNA-AuNPs). In the presence of the target small molecule, AFB1 will specifically bind to the designed aptamer lock and release the DNA that can activate the shearing enzyme Cas12a-crRNA. This DNA will hybridize with the crRNA and activate the trans-cleavage of Cas12a. The gold nanoparticles on the probe MB-DNA-AuNPs will be sheared and released into the supernatant for downstream Sp-ICPMS counting analysis. This method has successfully achieved a detection limit (LODs) at the picomolar level. At the same time, through the inspection of certified reference materials (CRMs) and spiked recovery analysis, this method can provide effective analysis results. This method is expected to become an effective analytical tool for carcinogenic small molecules. Summary of the Invention

[0003] The object of the present invention is to provide an efficient CRISPR / Cas12a-based amplification-free aflatoxin B1 (AFB1) single nanoparticle counting bioassay method and its application in the detection and evaluation of the carcinogenic small molecule aflatoxin B1.

[0004] The principle of the present invention is as follows: This method is based on the principle of specific binding of aptamers to target small molecules. When the aptamer lock and AFB1 are mixed, two AFB1 aptamer strands will bind to AFB1 and release nucleic acids for activating Cas12a-crRNA. This nucleic acid can specifically hybridize with crRNA and release the trans-cleavage ability against single-stranded DNA (ssDNA). The probe MB-DNA-AuNPs mixed in the solution will be detected and release free gold nanoparticles. This cleavage induced by the target small molecule causes the gradual increase in the number of gold nanoparticles in the solution, thus resulting in the change in the number of gold nanoparticles in the solution. By counting and analyzing the gold nanoparticles in the AFB1 solution with different concentrations through the frequency pattern of Sp-ICPMS, this method achieves sensitive detection of AFB1. Description of the Drawings

[0005] The present invention adopts the principle of small molecule-induced aptamer strand release, and by virtue of the high sensitivity advantages of single nanoparticle inductively coupled plasma mass spectrometry and the cleavage enzyme CRISPR / Cas12a, realizes the amplification-free and highly sensitive detection and analysis of the carcinogenic small molecule AFB1.

[0006] Figure 1 It is the mechanism diagram of the analysis method of the present invention and the sequence display diagram designed;

[0007] Figure 2 It is the electron microscope characterization diagram of gold nanoparticle labeling and signal probe MB-DNA-AuNPs in the analysis method of the present invention;

[0008] Figure 3 It is the time-resolved data diagram of gold nanoparticles in the analysis of Sp-ICPMS and the linearity of the probe in the analysis method of the present invention;

[0009] Figure 4 It is the electrophoresis characterization diagram of the sequences used in the analysis method of the present invention and the aptamer lock synthesized for the target small molecule;

[0010] Figure 5 It is the linear diagram of the analysis of the target small molecule by the Sp-ICPMS frequency pattern in the analysis method of the present invention;

[0011] Figure 6 It is the specificity analysis diagram of the analysis of the target small molecule by the Sp-ICPMS frequency pattern in the analysis method of the present invention;

[0012] Figure 7 This is the application diagram of the Sp-ICPMS frequency pattern for standard substances and spike recovery in the analysis method of the present invention. Specific embodiments

[0013] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The water used in the following examples is all ultrapure water, which is processed by a Milli-Q ultrapure water purification system.

[0014] The present invention is carried out according to the following preferred specific steps.

[0015] A. Preparation of 42 nm AuNPs.

[0016] A1. Take 100 µL of 10% chloroauric acid (w / v) and add it to 60 mL of ultrapure water. Place a magnetic stir bar in a three-necked flask and mix well. Reflux and boil in a heating mantle and keep boiling for 20 min.

[0017] A2. Quickly add 1 mL of 1% sodium citrate reducing agent solution and keep refluxing for 30 min while stirring and boiling.

[0018] A3. Turn off the temperature control system of the heating mantle and keep the stir bar stirring continuously. Wait for the solution to cool slowly to room temperature, then transfer the solution to a black 100 mL storage bottle and make up to 100 mL.

[0019] B. Gold nanoparticle labeling (DNA-AuNPs).

[0020] B1. Take 1 mL of AuNPs and wash it three times with 0.01 M boric acid buffer solution (4 °C, 9600 rpm, 10 min / time), and adjust the pH to about 8.0 - 8.5.

[0021] B2. Add 1.32 nmol of a linker containing 75 T bases modified with a thiol (-SH) at the 5'-end and biotin at the 3'-end, and 0.1% Tween 20 respectively, vortex and mix well, and react at room temperature (25 °C) for 6 h.

[0022] B3. After the reaction, use the method of salt aging, add 2 M sodium chloride solution in 8 portions (20 min / time), and ultrasonically treat for 30 s to prevent aggregation of AuNPs. After completion, react overnight at room temperature in the dark.

[0023] B4. After the overnight reaction, centrifuge at 4 °C (9600 rpm) for 10 min, aspirate the supernatant to remove the excess linker, and redissolve it in Tris-HCl buffer solution (10 mM Tris-HCl, 100 mM NaCl, pH = 7.4).

[0024] C. Preparation of signal probe MB-DNA-AuNPs.

[0025] C1. Take 40 µL of streptavidin-modified magnetic microspheres (SA-MBs), wash them three times with washing buffer solution (10 mM Tris-HCl; 0.1% Tween-20; 0.01% PEG-4000; 50 mM NaCl), and redissolve them in 80 µL of the above buffer solution;

[0026] C2. Add 1 mL of 317 pM DNA-AuNPs to the above 80 µL of magnetic microspheres and react at room temperature (25 °C) for 2 h (1500 rpm);

[0027] C3. After the reaction, wash the above MB-DNA-AuNPs probe three times with magnetic force (the washing buffer solution is: 20 mM HEPES; 50 mM NaCl; 10 mM MgCl2; pH 7.4), redissolve it in the above HEPES buffer solution, and store the prepared probe at 4 °C.

[0028] D. Preparation of the aptamer lock for recognizing the target AFB1.

[0029] D1. Dissolve two aptamers (apt-1, apt-2) and the activator strand (initiator) in 20 mM HEPES buffer solution (50 mM NaCl; 10 mM MgCl2; pH 7.4);

[0030] Mix the above nucleic acids in a 200 µL PCR tube in a molar ratio of 2:1:2 (apt-1:initiator:apt-2), and perform an annealing reaction in a PCR instrument (program settings: react at 95 °C for 5 min, and then slowly cool to room temperature at 1 °C / min).

[0031] E. Preparation of Cas12a-crRNA mixed solution.

[0032] E1. In the DCEP-treated HEPES buffer, mix Cas12a and crRNA in a molar ratio of 1:4. The Cas12a-crRNA mixed solution can be incubated at room temperature with gentle shaking for 30 min.

[0033] F. Enzyme cleavage reaction.

[0034] F1. Add 15 µL of Cas12a-crRNA solution (concentration: 30 nM Cas12a, 120 nM crRNA), 20 µL of aptamer lock solution (concentration 5 nM), 15 µL of MB-DNA-AuNPs probe, and 50 µL of AFB1 with different concentrations (0 - 1000 pM) into a 200 µL PCR tube, and then perform the cleavage experiment by oscillating at 37 °C for 40 min;

[0035] F2. After the above reaction is completed, terminate the trans-cleavage reaction at 85 °C;

[0036] F3. Use the method of magnetic separation to collect the supernatant into a new 200 µL PCR tube;

[0037] F4. Take 20 µL of the above supernatant into a 2 mL centrifuge tube and dilute it with 2 mL of ultrapure water.

[0038] G. Sp-ICPMS parameter setting and cleaning.

[0039] G1. Insert the liquid suction pump tube of Sp-ICPMS into an aqueous solution containing 1% nitric acid;

[0040] G2. Set the frequency counting mode to collect signals from the solution, with a collection time of 20 s and a speed of 200 µs / time;

[0041] G3. Thoroughly clean with an aqueous solution containing 1% nitric acid for 5 min.

[0042] H. Sp-ICPMS counting analysis.

[0043] H1. Insert the liquid suction pump tube of Sp-ICPMS into the diluted sample solution;

[0044] H2. Collect single nanoparticle counting signals with the above signal collection parameters.

[0045] I. Preparation of certified reference material samples (CRMs).

[0046] I1. Weigh 2 g of CMRs (peanut oil containing AFB1 (22.0 ± 1.7 µg / kg) and wheat flour (175 ± 26 µg / kg)) in a 50 mL centrifuge tube respectively, and add 5 mL of methanol-water extraction solution (volume ratio = 7:3);

[0047] I2. After adding the extraction solution, perform strong oscillation and ultrasonic treatment to fully mix the mixed solution;

[0048] I3. After thorough mixing, centrifuge the sample (20 min, 4 °C, 16,000 rpm);

[0049] I4. Use a medical syringe and a 0.22 µm filter membrane to extract the supernatant of the sample into a new centrifuge tube;

[0050] I5. After diluting the sample sufficiently, the above single nanoparticle counting method can be used for analysis.

[0051] J. Spike recovery analysis.

[0052] J1. Add different amounts of AFB1 (0 - 1000 pM) to grain-related commodities (beer, peanut oil (without AFB1));

[0053] J2. Add the above methanol-water mixed extraction solution;

[0054] J3. After thorough shaking and mixing and sonication, dilute the sample sufficiently;

[0055] J4. Use a medical syringe and a 0.22 µm filter membrane to extract the supernatant of the sample into a new centrifuge tube and dilute it sufficiently;

[0056] J5. Use the above single nanoparticle counting method for analysis.

[0057] The following is a further description in conjunction with the accompanying drawings of the specification.

[0058] As Figure 1 shown, the present invention is a CRISPR / Cas12a-based amplification-free aflatoxin B1 (AFB1) single nanoparticle counting bioassay method.

[0059] Example 1. Characterization of gold nanoparticle probes.

[0060] To reveal the successful labeling of gold nanoparticle probes and the preparation of probe MB-DNA-AuNP in the Sp-ICPMS experiment, first, the performance of gold nanoparticles using DNA was characterized, and verification was carried out using ultraviolet-visible spectrophotometry and dynamic light scattering method respectively. As can be seen from the ultraviolet analysis results shown in Figure 2 (a), the gold nanoparticles labeled with DNA have an obvious red shift compared with the unlabeled bare gold nanoparticles. At the same time, from the particle size results shown by the dynamic light scattering in Figure 2 (b), it can also be seen that the linker sequence was successfully labeled on the surface of the gold nanoparticles. Subsequently, TEM characterization was carried out on the magnetic microspheres without attached gold nanoparticles and the probe MB-DNA-AuNP respectively. As shown in Figure 2It can be clearly observed from (c) and (d) that the labeling method adopted in this method can successfully connect gold nanoparticles to the surface of magnetic microspheres.

[0061] Example 2: Exploration of the performance of gold nanoparticle probes in single-particle analysis by Sp-ICPMS.

[0062] To reveal that the prepared gold nanoparticle probes can be detected by the single-particle analysis mode, the gold nanoparticles conjugated with linker were fully diluted and then analyzed using the Sp-ICPMS single-nanoparticle analysis mode. As can be seen from Figure 3 (a), (b), (c), and (d), the prepared gold nanoparticle probes can be sensitively detected by the Sp-ICPMS single-nanoparticle counting mode, and at the same time, good probe linearity (R2 = 0.99) is accompanied.

[0063] Example 3: Characterization of the aptamer lock prepared for specifically recognizing the target AFB1.

[0064] To demonstrate the successful preparation of the aptamer lock for specifically recognizing aflatoxin B1, electrophoresis analysis and characterization were performed on two aptamers, the shear enzyme activation chain, and the aptamer lock. As can be seen Figure 4 that the nucleic acid sequences designed in the present invention can successfully prepare a stable aptamer lock and be used for the recognition of aflatoxin B1. At the same time, the two aptamers do not affect each other in the absence of shear enzyme activation.

[0065] Example 4: Exploration of the linearity of the detection of the target AFB1 by this method.

[0066] The linearity of AFB1 detected by Sp-ICPMS single-nanoparticle counting analysis is shown in Figure 5 (a);

[0067] According to the concentration of AFB1 and the response signal of the Sp-ICPMS single-nanoparticle counting mode, the response trend of the single-particle frequency signal of different concentrations of AFB1 was established, and then logarithmic operation and linear processing were performed. A linear relationship of 1 - 1000 pM in the single-particle counting mode was obtained (the detection limit is 1.1 pM, R2 = 0.98). The Sp-ICPMS time-resolved data graph ( Figure 5 (c)-(d)) shows the results of real-time detection in the single-particle mode. This method can stably and successfully detect AFB1 and distinguish it from the background.

[0068] Example 5: Exploration of the specific recognition ability of this method for the target AFB1.

[0069] To reveal the specificity of this method for AFB1, the interference of common small molecules (ZEA, FB1, and OTA) on this analytical method was analyzed. 5 nM of ZEA, FB1, and OTA were added to the sample containing 500 pM AFB1, and the sample solutions containing AFB1 and the above interfering small molecules were detected in parallel separately in the counting mode of Sp-ICPMS. By Figure 6 (a), it can be seen that the interfering small molecules do not interfere with the detection of AFB1, indicating that this method can well distinguish the target AFB1 among different small molecules. Aflatoxins AFB2, AFG1, and AFG2, which are structurally similar to AFB1, were also used to test the specificity of this analytical method in the same steps. By Figure 6 From the results of (b), it can be seen that because of the structural similarity to AFB1, aflatoxins AFB2, AFG1, and AFG2 will cause certain signal interference. This phenomenon is consistent with the published results of the detection work for AFB1. Nevertheless, this analytical method can still distinguish the target AFB1 among AFB2, AFG1, and AFG2.

[0070] Example 6: Explore the detection of the actual AFB1-containing reference materials (CRMs) and the spike recovery analysis ability of this method.

[0071] 1. Sources of AFB1-containing reference materials (peanut oil and wheat flour) The peanut oil CRMs sample used in the application of the analytical method of the present invention is from the National Institute of Metrology, China (GBW10176, AFB1 content: 22.0 ± 1.7 μg / kg), and the wheat flour CRMs sample is from Beijing Micro-standards Technology Co., Ltd. (MCS-0021, AFB1 content: 175 ± 26 μg / kg). The AFB1 content in the CRMs sample is provided by the manufacturer's detection (HPLC detection method);

[0072] 2. Sources of cereal-related matrices used in spike recovery analysis The cereal-related matrix environments (beer and wheat flour) used in the spike recovery analysis of the analytical method of the present invention are all from the local farmers' market.

[0073] CRMs detection and spike recovery analysis results Using the above analytical steps, the samples were analyzed in the single nanoparticle mode of Sp-ICPMS after the reaction. The results of the analysis are shown in Figure 7 (a) and (b). Figure 7 (a) shows the comparison between the results obtained by this analytical method and the HPLC detection results of the provided CRMs. It can be seen that the final results of this analytical method are consistent with those of the standard method. Figure 7(b) shows the spike recovery analysis results of this analysis method. It can be seen that the established analysis method can obtain a recovery rate of 87.1 - 110. The above analysis results prove that the analysis method of the present invention has the ability to analyze and detect actual samples.

Claims

1. A sensitive bioanalysis method for aflatoxin AFB1 based on single-particle inductively coupled plasma mass spectrometry and CRISPR / Cas12a, characterized in that: The analysis method includes the sensitive detection of aflatoxin AFB1 using an aptamer-functionalized DNA sensor and Cas12a nuclease assistance based on the single nanoparticle mode of inductively coupled plasma mass spectrometry (SpICPMS); in the analysis method, the nucleic acid sequence includes a DNA aptamer sensor sequence for specifically recognizing AFB1 and an activation sequence for activating the nuclease Cas12a-crRNA; in the analysis method, the signal output probe consists of a single-stranded DNA with a long-chain T base, gold nanoparticles with a size of 42 nm, and magnetic microspheres. In the presence of the target small molecule, Cas12a-crRNA is activated and releases trans-cleavage, and the gold nanoparticles connected to the magnetic nanoparticles are cleaved and free in the supernatant. The gold nanoparticles free in the supernatant are counted and analyzed by the SpICPMS detection mode.

2. The aptamer-functionalized DNA sensor according to claim 1, wherein: By designing the AFB1 aptamer, two aptamer sequences can hybridize with the nuclease activation strand (apt-1, apt, 2, initiator) to form a stable ligand lock structure. In the presence of the target AFB1, the two aptamers bind to AFB1, and the nuclease activation strand is thus released for downstream detection; the aptamer sequences and the nuclease activation strand sequence are: aptamer 1: 5'-CACGTGTTGTCTCTCTGTGTCTCGTGTTGTGT-3', aptamer 2: 5'-GTTTCACGTGTTGTCTCTCTGTGTCTCGTG-3', and activation strand: 5'-CAACACGTGAAACACACAACACGAGAC-3'.

3. The magnetic microsphere probe based on gold nanoparticles connection according to claim 1, wherein: The linker sequence modified with a thiol group (-SH) at the 5' end and biotin at the 3' end is first modified on the surface of gold nanoparticles by forming a gold-sulfur bond (Au-S) through a salt aging method; after centrifugal washing (centrifugation conditions: 9600 rpm) to elute the excess linker sequence, it is redissolved in an equal volume of Tris-HCl buffer solution (10 mM Tris-HCl, 100 mM NaCl, pH = 7.5); subsequently, streptavidin-modified magnetic microspheres dissolved in Tris-HCl buffer solution are added and incubated at room temperature for 2 hours. A stable gold nanoparticle-modified magnetic microsphere probe can be obtained through the specific reaction of streptavidin and biotin; the linker sequence is: linker: 5'-SH-(T75)-biotin-3'.

4. The Sp-ICPMS detection method assisted by the shearing enzyme Cas12a according to claim 1, characterized in that: When the nuclease activation sequence (initiator) is released and hybridizes with the pre-incubated Cas12a-crRNA, trans-cleavage is released, and the gold nanoparticles connected to the magnetic microspheres are cleaved and free in the supernatant; the supernatant is collected, and after sufficient dilution, single nanoparticle counting analysis is performed in the single particle frequency mode of Sp-ICPMS. The measurement time is 20 s, and the scanning rate is 200 µs / time.

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