Liquid crystal sensor and aflatoxin detection method and application thereof

Abstract

The present invention belongs to the technical field of sensor detection, and specifically relates to a liquid crystal sensor and a method and application for detecting aflatoxin. The systematic combination of RCA reaction, magnetic beads and aptamers greatly improves the sensitivity and selectivity of the sensor. Due to the recognition of specific AFB1-aptamer, cDNA is released, triggering the cleavage and ligation of DNA by RNP. Therefore, since the RCA reaction is blocked, Myr is retained in the aqueous solution. When AFB1 is absent, Myr in the aqueous solution will be captured by the long-chain ssDNA generated by the RCA reaction, and thus a bright LC image can be observed. The designed portable device is inexpensive and convenient for data collection. Therefore, the CRISPR-RCA LC sensor can be used as a method for detecting food mycotoxins with high sensitivity, strong selectivity, convenient use and broad prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensor detection, and in particular relates to a liquid crystal sensor and a method and application of detecting aflatoxin thereof. Background Art

[0002] Liquid crystal (LC) is a material that has both the fluidity of a liquid and the anisotropy of a crystal. It has a high sensitivity to subtle changes in various interfaces. For example, surfactants have the ability to orient LC molecules at the water / LC interface, and changes in the LC image can be observed using polarizing microscopy (POM). Due to its superior properties, various LC-based sensors have been constructed to detect different targets, such as biomarkers and environmental pollutants. The detection principle is usually based on the rational coupling of the target with the surfactant concentration. High concentrations of surfactants usually cause LC to be vertically oriented at the water / LC interface, resulting in a dark LC image. In contrast, at low concentrations of surfactants, LC maintains a planar orientation and produces a bright LC image. By analyzing the changes in the LC image, qualitative and quantitative analysis of the target can be achieved. Although great progress has been made in the past few decades, there is still an urgent need to build high-performance LC-based sensors in the following aspects: 1) The sensitivity and selectivity need to be improved to improve the accuracy and reliability of detection; 2) The matrix effect needs to be reduced to avoid potential interference leading to inaccurate signals; 3) High portability and high-fidelity POMs are widely welcomed to reduce costs and be suitable for rapid on-site testing.

[0003] Aflatoxin (AF) is a type of mycotoxin that is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. Among them, aflatoxin B1 (AFB1) has the highest risk of mutagenicity, carcinogenesis, and teratogenesis. Currently, a variety of technologies have been invented to detect AFB1. Traditional detection methods generally require expensive instruments and complex sample preparation, such as high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Although the development of different rapid detection methods, such as immunoassays, electrochemical detection, and fluorescence detection, may overcome these problems, the development of innovative AFB1 detection methods with higher performance remains a priority. Summary of the invention

[0004] The purpose of the present invention is to provide a liquid crystal sensor and a method and application for detecting aflatoxin thereof, so as to overcome the shortcomings of the prior art. The system combination of rolling circle amplification (RCA) reaction, magnetic beads and aptamers greatly improves the sensitivity and selectivity of the sensor for aflatoxin detection.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a liquid crystal sensor, comprising an aptamer DNA, a complementary strand cDNA, a T4 DNA ligase, a Phi 29 DNA polymerase, a connecting DNA, a padlock DNA, a Cas12a-crRNA complex, and magnetic beads;

[0007] The aptamer DNA can specifically bind to the detection object, and the 3' end of the aptamer DNA is modified with biotin.

[0008] In some other embodiments, the magnetic beads are streptavidin-modified magnetic beads.

[0009] In some other embodiments, the nucleotide sequence of the aptamer DNA is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the complementary chain cDNA is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the connecting DNA is shown in SEQ ID NO.3;

[0012] The nucleotide sequence of the padlock DNA is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of crRNA in the Cas12a-crRNA complex is shown in SEQ ID NO.5.

[0014] In a second aspect, the present invention provides application of the liquid crystal sensor described in the first aspect in fungal toxin detection.

[0015] In some other embodiments, the mycotoxin is aflatoxin.

[0016] In a third aspect, the present invention provides a method for detecting aflatoxin, using the liquid crystal sensor described in the first aspect, comprising the following steps:

[0017] (1) Mixing the aptamer DNA with the complementary chain cDNA and annealing them to obtain dsDNA; mixing the dsDNA with magnetic beads and incubating them to obtain dsDNA-modified magnetic beads;

[0018] (2) Mixing the linked DNA with the magnetic beads, and incubating to obtain the linked DNA-modified magnetic beads;

[0019] (3) Mixing the aflatoxin to be tested with the dsDNA-modified magnetic beads to separate the supernatant containing cDNA; mixing crRNA and Cas12a to obtain a Cas12a-crRNA complex;

[0020] (4) The supernatant containing cDNA, the Cas12a-crRNA complex and the magnetic beads modified with linked DNA are mixed for the first incubation, the supernatant is removed and the padlock DNA is added, the magnetic beads are separated after the second incubation; the separated magnetic beads, T4 DNA ligase reaction buffer, T4 DNA ligase and water are mixed, the third incubation is carried out, the supernatant is removed and the mixture is mixed with Phi 29 DNA polymerase reaction buffer, water, deoxynucleotide mixed solution and Phi 29 DNA polymerase, the fourth incubation is carried out to perform RCA reaction, and magnetic beads containing RCA products are obtained;

[0021] (5) Mixing the magnetic beads containing the RCA product and myristoylcholine chloride (Myr), and incubating to prepare aflatoxin detection solution;

[0022] (6) Testing the aflatoxin test solution to obtain the concentration of the aflatoxin to be tested.

[0023] In some other embodiments, in step (1), the concentration of the aptamer DNA is 0.2-0.5 μM, the concentration of the complementary chain cDNA is 0.2-0.5 μM, and the concentration of the dsDNA is 10-12 nM;

[0024] The annealing temperature is 90-100°C and the time is 1-10 min;

[0025] In step (2), the concentration of the linked DNA is 25-250 nM; the concentration of the magnetic beads is 0.02-0.05 mg / mL;

[0026] In step (1) or step (2), the incubation temperature is room temperature and the incubation time is 20-30 min.

[0027] In some other embodiments, in step (3), the concentration of the aflatoxin to be detected is 0-20 ng / mL; the mixing temperature is room temperature, and the mixing time is 20-30 min;

[0028] The concentration of the crRNA is 0.1-0.2 μM, and the concentration of the Cas12a is 0.05-0.15 μM;

[0029] The solvent used for mixing the crRNA and Cas12a is a mixed solvent of NEBuffer and water.

[0030] In some other embodiments, in step (4), the temperature of the first incubation is 30-40°C and the time is 20-30 minutes;

[0031] The second incubation temperature is at room temperature and the time is 20-30 minutes;

[0032] The third incubation temperature is 10-20°C and the time is 50-70min;

[0033] The temperature of the fourth incubation is 30-40° C., and the time is 70-80 min.

[0034] In some other embodiments, in step (4), the concentration of the T4 DNA ligase is 2-5 U / μL;

[0035] The concentration of the padlock DNA is 50-70 nM;

[0036] The Phi 29 DNA polymerase is 25-150 mU / μL;

[0037] In step (5), the incubation temperature is room temperature and the incubation time is 10-15 min.

[0038] Beneficial effects of the present invention:

[0039] (1) This paper reports for the first time an advanced CRISPR-RCA LC sensor for the detection of mycotoxins in food, which has been successfully used to detect AFB1 in real samples.

[0040] (2) The systematic combination of CRISPR / Cas12a, RCA reaction, magnetic beads and aptamers in the present invention greatly improves the sensitivity and selectivity of the sensor.

[0041] (3) The portable device designed by the present invention is inexpensive and convenient for data collection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0043] Figure 1 Schematic diagram of a CRISPR-RCA LC sensor for detecting AFB1 in an embodiment of the present invention, wherein (a) is the addition of AFB1 to trigger the cleavage of the linked DNA, (b) is the initiation of the RCA reaction in the absence of AFB1, (c) is the response of LC to the presence and absence of AFB1, and (d) is an LC image obtained by a portable device;

[0044] Figure 2: This is a feasibility analysis of the CRISPR-RCA LC sensor in an embodiment of the present invention, wherein (a) is the influence of various conditions of the RCA reaction and the corresponding images of the LC produced under different conditions; (b) is the corresponding bright area coverage of the LC image in (a); (c) is the influence of various conditions of the CRISPR-RCA LC sensor and the corresponding LC images under different conditions; (d) is the corresponding bright area coverage of the LC image in (c); (e) is a TEM image of the RCA on the magnetic beads before (i) reaction and after (ii) reaction; (f) is an agarose gel electrophoresis image, and (g) is the fluorescence intensity of the CRISPR / Cas12 system at different concentrations of AFB1;

[0045] Figure 3 The figure is a result diagram of the optimization of experimental conditions in the embodiment of the present invention, wherein (a) is the concentration of the connected DNA, (b) is the concentration of Phi29 DNA polymerase, and (c) is the incubation time of the RCA product and Myr;

[0046] Figure 4 : The sensitivity and selectivity of the CRISPR-RCA LC sensor in the embodiment of the present invention, wherein (a) is the LC image of the CRISPR-RCA LC sensor in response to different concentrations of AFB1, (b) is the relationship between the bright area coverage and AFB1, (c) is the LC image of the CRISPR-RCA LC sensor to different fungal toxins, and (d) is the corresponding bright area coverage of the LC image in (c);

[0047] Figure 5 Detection of AFB1 in corn using the CRISPR-RCA LC sensor in an embodiment of the present invention, wherein (a) is the response image of the CRISPR-RCA LC sensor to different concentrations of AFB1 in corn, (b) is the bright area coverage corresponding to the LC image in (a), (c) is the layout of the portable LC imaging device, and (d) is the comparison of the portable LC imaging device and the laboratory POM in LC imaging. DETAILED DESCRIPTION

[0048] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein (Cas) (CRISPR / Cas) is an emerging method that is widely used in gene editing. The discovery of various Cas provides more possibilities for gene editing. Under the guidance of CRISPR RNA (crRNA), CRISPR / Cas12a has strong cis- and trans-cutting capabilities for single-stranded DNA and has been widely used in fields such as sensing.

[0049] Aptamers are short oligonucleotides that are often used as sensing elements and are highly specific for target recognition. They have unique advantages such as a wide range of target molecules, small molecular weight, chemical synthesis, small batch differences, and easy modification. In the detection of small molecules, aptamers are often used to enhance the ability of the CRISPR / Cas 12a system to achieve its application.

[0050] Rolling circle amplification (RCA) is an isothermal nucleic acid amplification technology that is achieved by polymerase-catalyzed rolling circle replication of circular DNA molecules. The RCA reaction can produce long-chain ssDNA containing tens of thousands of nucleotides, which is used to enhance the sensing signal. Therefore, with the assistance of the CRISPR / Cas12a system, aptamers and RCA reaction, the constructed LC-based sensor can significantly improve its sensing performance.

[0051] The present invention constructs a CRISPR-RCA LC sensor to detect aflatoxin B1 (AFB1) as shown in the schematic diagram of the principle Figure 1 As shown, (a) the addition of AFB1 triggers the cleavage of the linked DNA, (b) the initiation of the RCA reaction in the absence of AFB1, (c) the response of LC to the presence and absence of AFB1, and (d) the LC image acquired by the portable device.

[0052] from Figure 1 It can be seen that biotin-modified AFB1 hybridizes with complementary strand cDNA to form double-stranded DNA (dsDNA), and dsDNA binds to streptavidin-modified magnetic beads. In the presence of AFB1, it releases cDNA from dsDNA-modified magnetic beads, thereby activating the Cas12a-crRNA complex formed by Cas12a and crRNA, namely ribonucleoprotein (RNP). Then, the modified linker DNA on the magnetic beads is cut by the activated RNP. Since the cut linker DNA cannot form dsDNA with the padlock DNA, the RCA reaction cannot be initiated ( Figure 1 a in the figure). However, in the absence of AFB1, the cDNA in the dsDNA cannot be released to activate RNP. The ligated DNA on the magnetic beads binds to the padlock DNA and initiates the RCA reaction under the guidance of T4 DNA ligase and Phi29 DNA polymerase, thereby generating long ssDNA ( Figure 1 b). After the RCA reaction, the cationic surfactant Myr was added. Since the long-chain ssDNA captures Myr, the concentration of Myr in the aqueous solution decreases. Therefore, in the absence of AFB1, a bright image is observed. In contrast, in the presence of AFB1, long-chain ssDNA cannot be formed to capture Myr in the aqueous solution, so a dark image is observed ( Figure 1CRISPR-RCA LC sensor exponentially amplifies the signal generated by CRISPR / Cas12a through the RCA reaction, enhancing the response to AFB1 and greatly improving sensitivity. At the same time, the magnetic beads separate the RCA product from the complex matrix in the solution, thereby improving sensitivity and selectivity. It is worth noting that detection can be achieved using a homemade portable imaging device ( Figure 1 d) in.

[0053] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Specific conditions are not specified in the examples, and the conditions recommended by the manufacturer are followed. The components used without manufacturer's indication are all conventional products available commercially. The nucleotide sequences used in this example are shown in Table 1.

[0054] Table 1 Nucleotide sequence

[0055]

[0056] Example 1

[0057] 1. Experimental part

[0058] 1.1 Instruments and reagents

[0059] Main instruments: Polarized microscope (XPF-800C, Shanghai Tiansheng Instrument Co., Ltd., Shanghai, China)

[0060] Reagents: Myristoylcholine chloride (Myr) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., streptavidin-modified magnetic beads were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., DEPC water and tris(hydroxymethyl)aminomethane buffer (pH 8.0, 1 M Tris-HCl) were purchased from Beyotime Biotechnology Co., Ltd., phosphate buffered saline (PBS, pH 7.4, 1×) was purchased from Wuhan Saiweier Biotechnology Co., Ltd., EnGen Lba Cas12a, NEBuffer (10×), T4 DNA ligase (400 U / μL), T4 DNA ligase reaction buffer (10×), deoxynucleotide mixed solution (dNTPs, 10 mM), Phi29 polymerase (10 U / μL), and Phi 29 DNA polymerase reaction buffer (10×) were purchased from New England Biotechnology (Beijing) Co., Ltd., 4'-n-Pentyl-4-cyanobiphenyl (5CB) was purchased from Anage (Shanghai) Pharmaceutical Chemical Co., Ltd., TEM copper net was purchased from Shenzhen Puruisaisi Trading Co., Ltd., all mycotoxins used (aflatoxin B1, ochratoxin A, deoxynivalenol, zearalenone, patulin and citrinin) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., the nucleotides shown in Table 1 were provided by Sangon Biotechnology Co., Ltd., Shanghai, China, and the water used in the experiment was deionized water with a conductivity of 18.25 MΩ / cm.

[0061] 1.2 Pre-assembly of DNA-modified magnetic beads

[0062] The method for modifying magnetic beads with dsDNA is as follows: AFB1 aptamer (0.5 μM) and cDNA (0.5 μM) are mixed and annealed at 95 °C for 5 minutes to form dsDNA and then slowly cooled to room temperature. The magnetic beads (0.03 mg / mL) are washed three times with buffer solution II (Buffer II, composed of: 10 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, 0.05% Tween-20). Then, dsDNA (11.25 nM) is mixed with the magnetic beads and incubated at room temperature for 30 minutes to obtain dsDNA-modified magnetic beads. Then, the dsDNA-modified magnetic beads are washed three times with Buffer II to remove unbound dsDNA. Finally, they are dispersed in Buffer II and stored at 4 °C for subsequent use.

[0063] The method for modifying magnetic beads with ssDNA is as follows: First, the magnetic beads (0.04 mg / mL) were washed three times with buffer solution I (Buffer I, composed of: 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.05% Tween-20). Then, the ligated DNA (ssDNA, 40 nM) was mixed with the magnetic beads and incubated at room temperature for 20 minutes to obtain ssDNA-modified magnetic beads. Then, the ssDNA-modified magnetic beads were washed three times with Buffer I to remove the unbound ligated DNA. Finally, they were dispersed in Buffer I and placed at 4 °C for use.

[0064] 1.3 AFB1 detection method is as follows:

[0065] First, AFB1 was mixed with dsDNA-modified magnetic beads at room temperature for 30 minutes, and the supernatant was separated for use. At the same time, crRNA (0.15 μM), NEBuffer (1 μL, 10×), DEPC water (4.25 μL), and Cas12a (0.1 μM) were mixed at room temperature to form a Cas12a-crRNA complex, i.e., RNP. Then, the cDNA obtained from AFB1 recognition was mixed with RNP (5 μL), NEBuffer (6 μL, 10×), DEPC water (28 μL), and ssDNA-modified magnetic beads and incubated at 37°C for 30 minutes. Next, the ssDNA-modified magnetic beads were washed three times with Buffer I and then incubated with padlock DNA (60 nM) at room temperature for 30 minutes. Then, 2 μL 10× T4 DNA ligase reaction buffer, 0.2 μL T4 DNA ligase, and 17.8 μL water were incubated with the magnetic beads at 16°C for 60 minutes. The separated magnetic beads were then mixed with 10× Phi 29 DNA (4 μL) polymerase reaction buffer, water (30.5 μL), dNTPs (5 μL), and Phi 29 DNA polymerase (0.5 μL) at 30 °C for 80 min. After the RCA reaction, the magnetic beads were washed three times with PBS. The magnetic beads containing the RCA product were then mixed with Myr (40 μL, 15 μM) and left at room temperature for 10 min. Finally, the resulting sample was imaged with a polarizing microscope to determine the AFB1 concentration.

[0066] 1.4 Construction of a homemade portable imaging device

[0067] The homemade portable imaging device includes a magnifying glass, a compensator, a rotating platform, a light source, a polarizing filter, a column, a bracket and a smart phone. The bracket is composed of a rectangular plate and a circular plate, one end of the rectangular plate is vertically connected to the column, and the other end of the rectangular plate is horizontally connected to the circular plate. The column is cylindrical. The bracket includes a first bracket, a second bracket, a third bracket and a fourth bracket, and the first bracket, the second bracket, the third bracket and the fourth bracket are respectively arranged parallel to each other from the bottom to the top of the column. The polarizing filter includes a first polarizing filter and a second polarizing filter. The first bracket is provided with a light source and a first polarizing filter, and the light source is arranged below the first polarizing filter, and the first polarizing filter can be rotated at any angle; the second bracket is provided with a compensator and a rotating platform, and the compensator is arranged on the rotating platform, and the rotating platform is a cylindrical rotating platform, and the rotation angle can be adjusted arbitrarily; the third bracket is provided with a magnifying glass and a second polarizing filter, and the magnifying glass is arranged above the second polarizing filter, and the magnifying glass is used to magnify the optical image of LC, and the two polarizing filters can be rotated and arranged crosswise, and the cross angle can be adjusted according to actual needs, so as to adjust the brightness of the light; the fourth bracket is provided with a smart phone, and the smart phone is used to obtain the optical image of LC. All the brackets of the platform are printed using 3D printing technology.

[0068] The portable imaging device works as follows: When two polarizers are rotated to an orthogonal position, a dark field is obtained. The sample is placed on a platform consisting of a compensator and a rotating stage. A smartphone placed on the top bracket obtains an optical image of the LC.

[0069] 1.5 Detection of AFB1 in real samples

[0070] First, 1 g of corn was crushed into powder and added to a centrifuge tube. Then, 5 mL of methanol was added to the centrifuge tube, shaken for 10 minutes, and then sonicated for 10 minutes. Subsequently, the precipitate was removed by centrifugation at 8000 rpm for 10 minutes. Then, after filtering with a 0.22 μm filter, the supernatant was dried using nitrogen. Finally, the obtained product was re-dissolved using Buffer II, and 0-20 ng / mL of AFB1 was added to the above solution to prepare corn samples containing different concentrations of AFB1. Corn samples with different concentrations of AFB1 were mixed with dsDNA-modified magnetic beads at room temperature for 30 minutes, and then the supernatant was separated for use. The following steps are consistent with 1.3. Finally, the obtained samples were imaged using a homemade portable imaging device and a polarizing microscope to determine the AFB1 concentration.

[0071] 2. Results and Discussion

[0072] 2.1 Feasibility of CRISPR-RCA LC sensor

[0073] Figure 2The feasibility of CRISPR-RCA LC sensor in detecting AFB1 is demonstrated, where (a) shows the effects of various conditions of RCA reaction and the corresponding images of LC produced under different conditions; (b) shows the corresponding bright area coverage of the LC image in (a); (c) shows the effects of various conditions of CRISPR-RCA LC sensor and the corresponding LC images under different conditions; (d) shows the corresponding bright area coverage of the LC image in (c); (e) is the TEM image of RCA on magnetic beads before (i) and after (ii) reaction; (f) is the agarose gel electrophoresis image, and (g) is the fluorescence intensity of the CRISPR / Cas12 system at different concentrations of AFB1, with a scale bar of 500 nm, lane M: DNA Marker; lane 1: linked DNA; lane 2: padlock DNA; lane 3: RCA product.

[0074] First, the occurrence of the RCA reaction was verified. Various RCA reaction conditions were tested. It was found that the RCA reaction occurred only when the ligation DNA, padlock DNA, T4 DNA ligase, and Phi 29 DNA polymerase were all present, resulting in a bright image in the POM. Under other conditions, a dark image was obtained (a in Figure 2). Figure 2 b shows the corresponding bright area coverage of the LC image. When the cationic surfactant Myr is present, a self-assembled monolayer is formed, and the hydrophobic interaction between Myr and 5CB molecules causes the LC at the water / LC interface to be vertically arranged. Therefore, a dark image is observed. However, due to the electrostatic and hydrophobic interactions between ssDNA and Myr, the long-chain ssDNA produced by the RCA reaction can capture Myr from the aqueous solution, resulting in a bright image. Therefore, the occurrence of the RCA reaction can be demonstrated by the change from dark to bright. Next, the feasibility of the CRISPR-RCA LC sensor was also verified. Figure 2 As shown in c, when AFB1, dsDNA, RNP, and RCA are all present, a dark image is observed, but under other conditions, a bright image is observed. Figure 2 The corresponding bright area coverage is shown in d. This is because the introduction of AFB1 leads to the release of cDNA, which activates RNP to cut the connecting DNA. Since the RCA reaction is not initiated, long ssDNA cannot be generated and a dark image is observed. In contrast, under other conditions, the connecting DNA is intact and the RCA reaction can be initiated. Since Myr is captured by the generated long ssDNA, a bright image is obtained. Therefore, these results demonstrate the feasibility of using CRISPR-RCA LC sensor to detect AFB1.

[0075] In addition, transmission electron microscopy (TEM) and agarose gel electrophoresis were used to verify the occurrence of RCA reaction. Figure 2 Figure e shows that after the RCA reaction, a DNA shell is formed on the surface of the magnetic beads, which indicates the occurrence of the RCA reaction on the magnetic beads. In addition, according to the DNA Marker, the molecular weight of the ssDNA in lane 3 is much higher than 1.5 kbp (Figure 2f), proving that the RCA reaction produces long-chain ssDNA. In addition, different concentrations of AFB1 also have an effect on the fluorescence intensity of the CRISPR / Cas12 system (Figure 2g). This indicates that the added AFB1 activates the CRISPR / Cas12a system.

[0076] 2.1 Optimization of experimental conditions for CRISPR-RCA LC sensor

[0077] Figure 3 1 is a graph showing the optimization results of experimental conditions in an embodiment of the present invention, wherein (a) is the concentration of the ligated DNA, (b) is the concentration of the Phi29 DNA polymerase, and (c) is the incubation time of the RCA product and Myr.

[0078] First, the concentration of Myr was optimized. As the concentration of Myr increased, the coverage of the bright area became smaller and smaller. When the concentration of Myr was higher than 15 μM, the image of LC observed under POM became completely dark. Therefore, 15 μM Myr was selected. To determine the concentration of the ligated DNA, different concentrations of ligated DNA were tested ( Figure 3 When the concentration of ligated DNA exceeded 40 nM, bright images were observed. However, excess ligated DNA resulted in a decrease in the bright area coverage of the LC image. Therefore, 40 nM ligated DNA was selected. The optimal concentration of T4 DNA ligase was tested using different concentrations of T4 DNA ligase. An increase in the concentration of T4 DNA ligase resulted in an increase in the RCA product and bright area coverage. Therefore, 4 U / μL of T4 DNA ligase was selected for further experiments. Similarly, the optimal concentration of Phi 29 DNA polymerase was 125 mU / μL (b in Figure 3). In addition, the incubation time of RCA products with Myr was optimized. As the incubation time increased, the bright area coverage increased ( Figure 3 c). When the incubation time reaches 10 minutes, a completely bright image can be observed. Therefore, the optimal incubation time is 10 minutes.

[0079] 2.2 Performance of CRISPR-RCA LC sensor

[0080] The performance of the above strategy was evaluated using different concentrations (0-20 ng / mL) of AFB1. Figure 4Figure 2 shows the sensitivity and selectivity of the CRISPR-RCA LC sensor in an embodiment of the present invention, wherein (a) is the LC image of the CRISPR-RCA LC sensor in response to different concentrations of AFB1, (b) is the relationship between the bright area coverage and AFB1, (c) is the LC image of the CRISPR-RCA LC sensor to different fungal toxins (including aflatoxin B1, ochratoxin A, deoxynivalenol, zearalenone, patulin and citrinin), and (d) is the corresponding bright area coverage of the LC image in (c).

[0081] The results showed that as the concentration of AFB1 increased, the LC image changed from bright to dark (a in Figure 4). This is because the increase in the concentration of AFB1 led to a decrease in the RCA product, leaving Myr in the aqueous solution. Figure 4 Figure b shows the relationship between the bright area coverage and AFB1 concentration. In the range of 0.5-10 ng / mL, the bright area coverage was inversely linearly correlated with the AFB1 concentration. The correlation coefficient (R 2 ) was 0.992. In addition, the detection limit (LOD) of the CRISPR-RCA LC sensor was calculated to be approximately 0.312 ng / mL (3σ / slope).

[0082] The selectivity of the CRISPR-RCA LC sensor was evaluated using AFB1, ochratoxin A, deoxynivalenol, zearalenone, patulin, and citrinin. Notably, only the introduction of AFB1 resulted in a dark image, while the other mycotoxins produced bright images (Figure 4c). These results suggest that the RCA reaction can only be initiated when AFB1 is absent because its aptamer specifically recognizes AFB1. Figure 4 Panel d shows the bright area coverage corresponding to the above LC image. It is evident from these data that the CRISPR-RCA LC sensor is highly selective in detecting AFB1.

[0083] 2.3 Detection of AFB1 in corn using a portable device

[0084] A standard addition method was used to detect AFB1 in corn, and the application of the sensor in real samples was evaluated. Figure 5As shown, (a) is the response image of the CRISPR-RCA LC sensor to different concentrations of AFB1 in corn, (b) is the bright area coverage corresponding to the LC image in (a), (c) is the layout of the portable LC imaging device, and (d) is the comparison of the portable LC imaging device and the laboratory POM in LC imaging. I, II and III correspond to the portable device; IV, V and VI correspond to the laboratory POM, and I, II and III (IV, V and VI) correspond to AFB1 concentrations of 1, 10 and 20 ng / g in corn, respectively.

[0085] As the concentration of AFB1 increased, the LC images gradually turned darker, which indicated that the orientation of LCs changed from planar to vertical ( Figure 5 a). Figure 5 The b in the figure shows the coverage of the bright area corresponding to the LC image. Table 2 shows the statistical results, and the accuracy of the strategy was verified by LC-MS. The results showed that the recovery rate of the method was 84.13% - 93.88%, and the relative standard deviation was 7.03% - 14.65%. Therefore, the proposed detection method can meet the detection of AFB1 in actual samples with high reliability.

[0086] Table 2 Recovery of CRISPR-RCA LC sensor for AFB1 detection in corn

[0087]

[0088] The disadvantages of laboratory POM, such as high cost and inconvenient mobility, limit its potential application in rapid on-site detection. Therefore, we developed a portable and inexpensive LC imaging device. The layout of the optical device is shown in Figure 2. Figure 5 The LC image magnified by the lens can be observed with the naked eye or taken with a smartphone. AFB1 in corn was detected using the portable device. The image was consistent with the laboratory POM image (Fig. 5d). These results show that the portable device has high reliability and can be used for LC-based sensors in the field of food mycotoxin detection.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A liquid crystal sensor, characterized in that: Including myristoylcholine chloride, 4'-n-pentyl-4-cyanobiphenyl, aptamer DNA, complementary chain cDNA, T4 DNA ligase, Phi 29 DNA polymerase, ligated DNA modified magnetic beads, padlock DNA, Cas12a-crRNA complex and streptavidin modified magnetic beads; Wherein, the aptamer DNA can specifically bind to the detection object, and the 3' end of the aptamer DNA is modified with biotin; The nucleotide sequence of the aptamer DNA is shown in SEQ ID NO.1; The nucleotide sequence of the complementary chain cDNA is shown in SEQ ID NO.2; The nucleotide sequence of the connecting DNA is shown in SEQ ID NO.3; The nucleotide sequence of the padlock DNA is shown in SEQ ID NO.4; The nucleotide sequence of crRNA in the Cas12a-crRNA complex is shown in SEQ ID NO.

5.

2. Use of the liquid crystal sensor according to claim 1 in the detection of aflatoxin B1.

3. A method for detecting aflatoxin B1, characterized in that: The liquid crystal sensor according to claim 1 comprises the following steps: (1) Mixing the aptamer DNA with the complementary chain cDNA and annealing them to obtain dsDNA; mixing the dsDNA with magnetic beads and incubating them to obtain dsDNA-modified magnetic beads; (2) Mixing the linked DNA with the magnetic beads, and incubating to obtain the linked DNA-modified magnetic beads; (3) Mixing the aflatoxin B1 to be tested with the dsDNA-modified magnetic beads to separate the supernatant containing cDNA; mixing crRNA and Cas12a to obtain a Cas12a-crRNA complex; (4) The supernatant containing cDNA, the Cas12a-crRNA complex and the magnetic beads modified with linked DNA are mixed for the first incubation, the supernatant is removed and the padlock DNA is added, the magnetic beads are separated after the second incubation; the separated magnetic beads, T4 DNA ligase reaction buffer, T4 DNA ligase and water are mixed, the third incubation is carried out, the supernatant is removed and the mixture is mixed with Phi 29 DNA polymerase reaction buffer, water, deoxynucleotide mixed solution and Phi 29 DNA polymerase, the fourth incubation is carried out to perform RCA reaction, and magnetic beads containing RCA products are obtained; (5) Mixing the magnetic beads containing the RCA product and myristoylcholine chloride, and incubating them to prepare aflatoxin B1 detection solution; (6) The aflatoxin B1 test solution is tested. Through the hydrophobic interaction between myristoylcholine chloride and 4'-n-pentyl-4-cyanobiphenyl molecules, the liquid crystals on the water / liquid crystal interface are arranged differently to produce different light and dark images, thereby obtaining the concentration of the aflatoxin B1 to be tested.

4. The method for detecting aflatoxin B1 according to claim 3, characterized in that: In step (1), the concentration of the aptamer DNA is 0.2-0.5 μM, the concentration of the complementary chain cDNA is 0.2-0.5 μM, and the concentration of the dsDNA is 10-12 nM; The annealing temperature is 90-100°C and the time is 1-10 min; In step (2), the concentration of the linked DNA is 25-250 nM; the concentration of the magnetic beads is 0.02-0.05 mg / mL; In step (1) or step (2), the incubation temperature is room temperature and the incubation time is 20-30 min.

5. The method for detecting aflatoxin B1 according to claim 3, characterized in that: In step (3), the concentration of aflatoxin B1 to be tested is 0-20 ng / mL; the mixing temperature is room temperature, and the mixing time is 20-30 min; The concentration of the crRNA is 0.1-0.2 μM, and the concentration of the Cas12a is 0.05-0.15 μM; The solvent used for mixing the crRNA and Cas12a is a mixed solvent of NEBuffer and water.

6. The method for detecting aflatoxin B1 according to claim 3, characterized in that: In step (4), the first incubation temperature is 30-40°C and the time is 20-30 minutes; The second incubation temperature is at room temperature and the time is 20-30 minutes; The third incubation temperature is 10-20°C and the time is 50-70min; The temperature of the fourth incubation is 30-40° C., and the time is 70-80 min.

7. The method for detecting aflatoxin B1 according to claim 3, characterized in that: In step (4), the concentration of the T4 DNA ligase is 2-5 U / μL; The concentration of the padlock DNA is 50-70 nM; The Phi 29 DNA polymerase is 25-150 mU / μL; In step (5), the incubation temperature is room temperature and the incubation time is 10-15 min.

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