Sensor for detecting various mycotoxins and application of sensor in test strip

Abstract

The invention relates to a sensor for detecting various mycotoxins and application of the sensor in a test strip. The sensor disclosed by the invention comprises an Au (at) Ir (at) SH-PolyA-cDNA (complementary deoxyribonucleic acid) nanoprobe, a fungaltoxin aptamer, a complementary chain of the fungaltoxin aptamer and a developing solution, the mycotoxins are aflatoxin M1 (AFM1), aflatoxin B1 (AFB1) and ochratoxin A (OTA). The test strip for simultaneously detecting multiple mycotoxins comprises the sensor; the test strip comprises a plurality of detection channels. The AFM1, the AFB1 and the OTA are detected at the same time through the change of the colorimetric signal of the detection line. The nano-enzyme probe and the target in the solution compete with each other to form an aptamer. On the premise that the target exists, the signal probe which is not combined with the aptamer is captured by the detection line and forms a blue signal under the catalysis of the color developing agent. The method can realize rapid detection of three mycotoxins, and has high specificity and sensitivity. According to the invention, the problem of single target identification of a traditional paper-based sensor is solved.

Description

Technical Field

[0001] The present invention relates to the field of food safety biotechnology, and in particular to a sensor for detecting multiple mycotoxins and its application in a test strip, in particular to a lateral flow test strip for simultaneously detecting multiple mycotoxins by using nanozymes to amplify signals. Background Art

[0002] Mycotoxin contamination is widely present in food, among which agricultural and sideline products are the most seriously polluted. As a natural contaminant, it may occur in various links of agriculture and agricultural product processing, such as plant growth, harvesting, storage and agricultural product processing. Mycotoxins can easily enter the food chain and the environment with contaminated food, thereby endangering the ecosystem and human health. In 2020, Eskola et al. confirmed that the incidence of mycotoxins is as high as 60%-80%. In order to avoid the circulation of mycotoxins in food, it is crucial to develop sensitive and efficient methods to monitor mycotoxins in food. Officially approved typical detection methods, such as high performance liquid chromatography and liquid chromatography-mass spectrometry, have the characteristics of high accuracy and sensitivity. However, these methods are time-consuming and rely on expensive large-scale equipment, which cannot meet the needs of on-site detection. At present, the specific recognition of targets by aptamers has greatly promoted the development of lateral flow chromatography (LFA).

[0003] Since many toxin-producing fungi have similar growth environments and the same ability to produce toxins, agricultural products are often contaminated by multiple mycotoxins at the same time. The detection of a single pollutant is no longer applicable to situations where multiple pollutants exist. Therefore, it is particularly important to develop high-throughput multi-target detection. Currently, simultaneous detection based on multiple targets has been reported. Jin et al. used UCNPs with red, green, and blue fluorescent signals as detection probes to achieve simultaneous detection of multiple targets on multiple detection lines. Cheng et al. designed a three-channel LFA that can detect three pesticides at the same time. However, there are no reports on the detection of multiple mycotoxins by LFA based on aptamers, and lateral flow immunochromatography (LFIA) with antibodies as recognition molecules is still the mainstream.

[0004] Gold nanoparticles (AuNPs) are common signal tags due to their unique optical properties. However, their application in ultra-low detection limits is limited by their inherent plasmon activity. Therefore, the catalytic reaction of nanozymes becomes the key to improving sensitivity. The peroxide-like catalytic activity of nanozymes enables them to catalyze chromogenic substrates to produce strong color signals. Compared with the traditional colorimetric signal of AuNPs, the intensity of the catalytic color signal is increased by several orders of magnitude. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a fast and effective multi-channel paper-based sensor, which is used to simultaneously detect aflatoxin M1 (AFM1), aflatoxin B1 (AFB1) and ochratoxin A (OTA) by detecting changes in the colorimetric signal of the line. The nanozyme probe competes with the target in the solution for the aptamer. Under the premise of the presence of the target, the signal probe that is not bound to the aptamer is captured by the detection line and forms a blue signal after catalysis by the color developer. The sensor is designed based on the "Turnon" mode, and the target content is proportional to the intensity of the color of the detection line, which is in line with human intuitive feeling. This method can realize the rapid detection of three mycotoxins with high specificity and sensitivity. This invention solves the problem of single target recognition of traditional paper-based sensors.

[0006] The first object of the present invention is to provide a sensor for detecting multiple fungal toxins, the sensor comprising Au@Ir@SH-PolyA-cDNA nanoprobe, a fungal toxin aptamer, a complementary chain of the fungal toxin aptamer, and a color developing solution; the fungal toxins are AFM1, AFB1 and OTA.

[0007] In one embodiment of the present invention, the aptamer nucleic acid sequence of AFM1 is 5'-ACTGCTAGAGATTTTCCACAT-3' (SEQ ID No 17);

[0008] The aptamer nucleic acid sequence of OTA is 5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′ (SEQ ID No 14);

[0009] The aptamer nucleic acid sequence of AFB1 is 5′-GGGCACGTGTTGTCTCTCTGTGCCTCGTGCCC-3′ (SEQ ID No 15).

[0010] In one embodiment of the present invention, the aptamer complementary strand of AFM1 is one or more of M1-1, M1-2, M1-3, M1-2-6, M1-2-8, and M1-2-9; the aptamer complementary strand of OTA is one or more of O-1, O-2, O-3, O-3-10, O-3-14, and O-3-16; the aptamer complementary strand of AFB1 is 5'-GGCACAGAGAGA-3' (SEQ ID No 16);

[0011] The nucleic acid sequences of M1-1, M1-2, M1-3, M1-2-6, M1-2-8, and M1-2-9 are TAGCAGT, AAATCTC, ATGTGGA, AATCTC, AAATCTCT, and AAAATCTCT, respectively;

[0012] The nucleic acid sequences of O-1, O-2, O-3, O-3-10, O-3-14 and O-3-16 are shown in SEQ ID No. 1 to SEQ ID NO. 6.

[0013] In one embodiment of the present invention, the Au@Ir@SH-PolyA-cDNA nanoprobe is prepared by a "low pH" method:

[0014] Au@IrNPs were mixed with SH-PolyA-cDNA, and sodium citrate buffer was added for the first incubation. Then HEPES buffer was added several times to adjust the pH of the solution to neutral, and the second incubation was performed, followed by centrifugation.

[0015] In one embodiment of the present invention, the concentration of the sodium citrate buffer is 0.5 mM, pH 3.0;

[0016] The first incubation temperature was 25°C, the first incubation time was 3 h, and the rotation speed was 400 rpm;

[0017] The concentration of the HEPES buffer was 0.5 mM, pH 7.6; the second incubation temperature was 4° C., and the second incubation time was 6 h.

[0018] The second object of the present invention is to provide a test strip for simultaneously detecting multiple mycotoxins, wherein the test strip comprises the above-mentioned sensor; and the test strip contains multiple detection channels.

[0019] In one embodiment of the present invention, the plurality of detection channels is at least 3.

[0020] In one embodiment of the present invention, the test strip includes a bottom plate, a sample pad, and a plurality of binding pads, a nitrocellulose membrane, and a water absorbent pad connected to the sample pad in sequence, wherein the plurality of binding pads are radially distributed outward with the sample pad as the center; and the nitrocellulose membrane is provided with a detection line and a control line.

[0021] In one embodiment of the present invention, Au@Ir@SH-Poly A-cDNA nanoprobe is dropped on the conjugate pad.

[0022] In one embodiment of the present invention, the control line is provided with SA-Bio-DNA C The detection line is provided with SA-Bio-DNA T .

[0023] In one embodiment of the present invention, the distance between the detection line and the control line is 5 mm.

[0024] In one embodiment of the present invention, the test strip is a multi-channel test strip that can detect multiple mycotoxins simultaneously, thereby avoiding cross-reactions and interferences between different targets with similar molecular structures.

[0025] In the present invention, by introducing gold iridium nanozymes (Au@IrNPs) as signal tags, an aptamer-based lateral flow chromatography technology (Apt-LFA) for the simultaneous detection of aflatoxin M1 (AFM1), aflatoxin B1 (AFB1) and ochratoxin A (OTA) was designed. In order to avoid cross-reaction and interference between different targets with similar molecular structures, a three-channel test strip was designed. Each channel can detect a mycotoxin. In addition, the complementary sequences of AFM1 and OTA were optimized using fluorescent dyes to obtain optimal competition. After a series of studies, the effectiveness and reliability of the Apt-LFA were confirmed.

[0026] The present invention utilizes the interaction between streptavidin and biotin to bind biotinylated DNA T and DNA C (Bio-DNA T / C ) The detection line (T line) and control line (C line) pre-fixed on the NC membrane. DNA T A sequence containing an aptamer that can bind to the complementary sequence (cDNA) attached to the signal probe. DNA C It is a PolyT sequence containing 18 bases, which can easily capture the signal probe. Three toxin Au@Ir@SH-PolyA-cDNA nanoprobes are pre-sprayed on the binding pad. Subsequently, the aptamer is mixed and incubated with the sample solution, and the incubation solution is loaded onto a unified sample pad. When three target targets (the fungal toxins to be detected by the present invention) are contained, the three targets bind to their respective aptamers. At this time, the nanoprobes that are not bound to the aptamer are captured by the nucleic acid sequence fixed on the detection line. By loading the color developing solution, the catalytic effect of Au@IrNPs makes the detection line present a strong color signal. When the target is not present, the aptamer binds to the complementary sequence on the signal probe, thereby hindering the binding to the nucleic acid sequence on the detection line. Therefore, the target content in the sample solution is always positively correlated with the blue signal of the detection line, and the "Turn on" mode is more in line with human intuitive feeling. However, regardless of whether the target is present, the PolyA sequence on the signal probe always binds to the PolyT sequence on the control line, which is used to verify the effectiveness of the detection.

[0027] The above technical solution of the present invention has the following advantages compared with the prior art:

[0028] 1. The present invention screened the best complementary sequence of AFM1 and OTA aptamers, and the change in fluorescence intensity before and after adding the target is the best reflection. Through screening, the best complementary sequence maintains appropriate competition with the target to ensure the effectiveness of the sensor.

[0029] 2. The present invention synthesized gold-iridium nanozymes and successfully loaded nucleic acid sequences on the surface. The evaluation of the reaction kinetics and catalytic performance of the nanozymes demonstrated the feasibility of signal amplification of the detection results.

[0030] 3. The present invention is based on the "Turn on" mode, and the signal intensity of the detection result is proportional to the target concentration. Compared with the previous situation that the higher the target concentration, the lower the signal intensity, it is more in line with human intuitive feeling.

[0031] 4. Compared with conventional paper-based sensors, the present invention can achieve multiple detection of three targets, providing a reasonable idea for the simultaneous detection of multiple mycotoxins, and the strategy is simpler and more effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 This is a schematic diagram of the principle of the three-channel paper-based sensor in the present invention for detecting AFM1, AFB1 and OTA;

[0034] Figure 2 This is the assembly schematic diagram of the three-channel test strip;

[0035] Figure 3 The relevant data for the synthesis and functionalization of gold-iridium nanozymes are provided; (A) Flow chart of the synthesis and functionalization of Au@IrNPs; (B) TEM image of 40nmAuNPs (inset: SEM image); (C) Particle size distribution of 40nmAuNPs; (D) UV-visible absorption spectra of 40nmAuNPs and Au@IrNPs; (E) TEM image of Au@IrNPs (inset: SEM image); (F) Particle size distribution of Au@Ir NPs; (G) Absorbance in the supernatant after incubation of Au@IrNPs with different amounts of nucleic acid chains;

[0036] Figure 4Evaluation of the catalytic performance of gold-iridium nanozymes; (A) Reaction relationship between different concentrations of TMB (0mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM) and V; (B) Double reciprocal graph generated by (A); (C) The corresponding absorbance at 450nm and 653nm before and after Au@IrNPs catalyzed TMB (material concentration: 1.7, 2.6, 3.4, 5.7, 14.3, 28.7, 43, 57, 115, 229, 344, 459, 574, 717, 860, 2000, 5000, 8000, 10000, 20000fM);

[0037] Figure 5 Optimization diagram of complementary sequences of AFM1 and OTA; (A) Fluorescence intensity at different aptamer concentrations; (B) Fluorescence intensity at different target concentrations; (C) Difference in fluorescence intensity before and after adding AFM1 (the complementary sequences shown in the figure are M1-1, M1-2, and M1-3, see the table for details); (D) Difference in fluorescence intensity before and after adding OTA (the complementary sequences shown in the figure are O-1, O-2, and O-3, see the table for details); (E) Difference in fluorescence intensity before and after adding AFM1 (the number of bases in M1-2-6, M1-2, M1-2-8, and M1-2-9 are 6, 7, 8, and 9, respectively); (F) Difference in fluorescence intensity before and after adding OTA (the number of bases in O-3-10, O-3, O-3-14, and O-3-16 are 10, 12, 14, and 16, respectively); (G) Schematic diagram of complementary sequences;

[0038] Figure 6 The original data of fluorescence intensity before and after adding AFM1 and OTA;

[0039] Figure 7 This is the optimization diagram of the colorimetric solution composition; (A) Signal intensity of different TMB concentrations; (B) Signal intensity of different H2O2 concentrations;

[0040] Figure 8 The optimized graphs are for aptamer concentration and signal probe loading volume on the conjugate pad; signal intensities of aptamers at different concentrations, including AFM1 (A), AFB1 (B), and OTA (C); signal intensities of signal probes at different loading volumes on the conjugate pad, including AFM1 (D), AFB1 (E), and OTA (F);

[0041] Fig. 9 To characterize the stability of three-channel test strips during storage time;

[0042] Fig.10 This is the optimization diagram of the catalytic color development time of the three-channel test strip;

[0043] Fig.11Analysis of the sensitivity and specificity of the three-channel test strip; signal intensity of targets at different concentrations, including AFM1 (A), AFB1 (C) and OTA (E); calibration curves of signal intensity and logarithm of target concentration, including AFM1 (B), AFB1 (D) and OTA (F); (G) test results of test strips at different target concentrations; (H) signal intensity of different types of fungal toxins. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0045] In the following examples, the primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.:

[0046] The nucleotide sequence of the nanozyme probe of AFM1 is: 5′-AAAATCTCTAAAAAAAAAAAAAAAAAA-SH-3′ (the nanozyme probe of AFM1 is Au@Ir@SH-PolyA-cDNA1, SEQ ID No 7);

[0047] The nucleotide sequence of the nanozyme probe of AFB1 is: 5′-GGCACAGAGAGAAAAAAAAAAAAAAAAAAA-SH-3′ (the nanozyme probe of AFB1 is Au@Ir@SH-PolyA-cDNA2, SEQ ID No 8);

[0048] The nucleotide sequence of the nanozyme probe of OTA is: 5'-TGTCCGATGCTCAAAAAAAAAAAAAAAAAA-SH-3' (the nanozyme probe of OTA is Au@Ir@SH-PolyA-cDNA3, SEQ ID No 9);

[0049] The nucleotide sequence of the detection line of the nitrocellulose membrane of the AFM1 detection channel is: 5′-AGAGATTTTTAAA-Bioth-3′ (SEQ ID No 10);

[0050] The nucleotide sequence of the detection line of the nitrocellulose membrane of the AFB1 detection channel is: 5′-TCTCTCTGTGCCAAA-Bioth-3′ (SEQ ID No 11);

[0051] The nucleotide sequence of the detection line of the nitrocellulose membrane of the OTA detection channel is: 5′-GAGCATCGGACAAAA-Bioth-3′ (SEQ ID No 12);

[0052] The nucleotide sequence of the control line is: 5′-TTTTTTTTTTTTTTTTTT-Bioth-3′ (SEQ ID No 13);

[0053] The standard stock solutions of aflatoxin M1 (Aflatoxin M1, AFM1), aflatoxin B1 (Aflatoxin B1, AFB1), ochratoxin A (OchratoxinA, OTA), and zearalenone (ZEN) used in the present invention were purchased from Qingdao Prebon Biotechnology Co., Ltd.; the SH-Poly A-cDNA and biotinylated DNA used in the present invention were T (Bio-DNA T ), biotinylated DNA C (Bio-DNA C ) was purchased from Shenggong Bioengineering Co., Ltd.

[0054] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad used in the present invention are all conventional raw materials in the art.

[0055] Further, preferred are a sample pad (fusion3), a nitrocellulose membrane (CN140), an absorbent pad (H5072), a PVC base plate (DB-6), and a conjugate pad (8965).

[0056] Table 1 Nucleotide sequences of complementary chains

[0057]

[0058] Example 1: Synthesis and functionalization of gold iridium nanoparticles (Au@IrNPs)

[0059] (1) Synthesis of Au@IrNPs: 1.7 mL of AuNPs solution with a particle size of 15 nm was added to a round-bottom flask and diluted to 20 mL with ultrapure water and stirred at room temperature (25°C, 500 rpm). 10 mL of HAuCl4 aqueous solution (0.037%, w / v) and 10 mL of a mixed solution containing sodium citrate (0.03%, w / v) and L-ascorbic acid (0.06%, w / v) were injected into the mixture at a rate of 12 mL / h. After the two solutions were completely added, they were placed in an oil bath and heated and stirred (300 rpm, 120°C, 30 min). Synthesized 40 nm AuNPs were cooled to room temperature and stored at 4°C for later use. 12 mL of 40 nm AuNPs were placed in a beaker and stirred at room temperature (25°C, 500 rpm). Then, 4 mL of Na3IrCl6·xH2O aqueous solution (1.25 mg / mL) and 4 mL of cold NaBH4 aqueous solution (2.25 mg / mL) were injected using a syringe pump at injection rates of 1 mL / h and 12 mL / h, respectively. After complete injection, the reaction lasted for 2 h. The mixture was centrifuged (8000 rpm, 10 min, 25 ° C) to collect the precipitate, which was washed twice with ultrapure water to obtain Au@IrNPs. Finally, the precipitate was dispersed in 6 mL of sodium citrate buffer (0.01%, w / v) and stored for later use.

[0060] (2) Functionalization of Au@IrNPs: The SH-PolyA-cDNA of the three toxins was activated before use: SH-PolyA-cDNA (100 μM) was mixed with tri(2-carboxyethyl)phosphine solution (10 mM) and vortexed thoroughly, incubated at 4°C for 2 h, and the SS bond was reduced to -HS to obtain three activated SH-PolyA-cDNAs. 6 μL of activated SH-PolyA-cDNA was added to 500 μL Au@IrNPs obtained in step (1), mixed evenly, and then 2 μL of sodium citrate buffer (0.5 M, pH = 3) was added. The resulting mixture was incubated at room temperature for 3 h. Subsequently, 12 μL of HEPES buffer (0.5 M, pH = 7.6) was added to the reaction solution in portions. The resulting mixed reaction solution was incubated at 4°C for another 6 h. The incubated mixture was centrifuged at 8000 rpm for 10 min to remove excess DNA. The precipitate was washed twice with ultrapure water and resuspended in 50 μL of resuspension solution (a mixture of 5wt% sucrose, 0.5wt% Tween-20, 5wt% BSA and 0.05wt% NaN3) and stored at 4°C for later use. Three nanozyme probes were obtained: the nanozyme probe of AFM1 was Au@Ir@SH-PolyA-cDNA1, the nanozyme probe of AFB1 was Au@Ir@SH-PolyA-cDNA2, and the nanozyme probe of OTA was Au@Ir@SH-PolyA-cDNA3.

[0061] Characterization of related materials: The transmission electron microscopy image of 40 nm AuNPs in step (1) is as follows: Figure 3 As shown in B, the overall spherical shape is relatively smooth. In the presence of the reducing agent NaBH4, Ir grows on the surface of the gold seed. A closer look reveals that the overall spherical shape of the gold seed is still retained, and the surface of the Au@IrNPs is rough, which means that Ir grows on the seed surface in an island or Franck-van der Merwe pattern ( Figure 3 In the UV-visible absorption spectrum, 40 nm AuNPs have a typical absorption peak around 525 nm. When their surface is coated with Ir, the main LSPR peak of Au@IrNPs blue-shifts and becomes broader ( Figure 3 The mass ratio of Au:Ir in Au@IrNPs was determined to be 12:1 by inductively coupled plasma mass spectrometry (ICP-MS).

[0062] The concentration of AuNPs was calculated based on the absorbance: the ratio of the absorbance of AuNPs at the surface plasmon resonance peak (Aspr) to the absorbance at 450nm (A450) is related to the particle size (Formula 1-1). Compared with the standard table, the particle size of the gold seed is about 40nm, which is consistent with the observation in TEM. The concentration of 40nm AuNPs was calculated according to Formula 1-2, where ε450 is the molar extinction coefficient when λ=450nm. The concentration of Au@IrNPs calculated by absorbance is 8.6×10 -11 M.

[0063]

[0064]

[0065] SH-PolyA-cDNA solution has a typical absorption peak at around 260nm. Figure 3 As shown in G, when the amount of SH-PolyA-cDNA added increased from 1 μL to 15 μL, the absorbance of the supernatant also increased accordingly, indicating that the surface of Au@IrNPs was successfully modified. At the same time, the optimal addition amount was determined to be 6 μL.

[0066] Example 2: Evaluation of the catalytic performance of Au@IrNPs

[0067] In this experiment, TMB and H2O2 were used as colorimetric solutions and reacted in a fixed volume of NaAc buffer (pH 4.0, 0.2 M, 200 μL). In order to study the reaction kinetics of the material, a fixed concentration of Au@IrNPs (1.7×10 -12The catalytic performance was studied in a fixed concentration of H2O2 (1M) and a fixed concentration of TMB (0.5mM) to explore the absorbance changes of Au@IrNPs at different concentrations. The apparent kinetic parameters were calculated according to the Michaelis-Menten equation, as follows:

[0068]

[0069] Where [S] is the substrate concentration, V is the initial velocity, V max is the maximum reaction rate, K m is the Michaelis constant.

[0070] The present invention uses the catalytic reaction of TMB and H2O2 to evaluate the peroxidase catalytic activity of Au@IrNPs. Under the catalytic reaction, a blue product (oxidized TMB) is generated. In order to obtain kinetic parameters, including the Michaelis-Menten constant (K m ) and the maximum reaction rate (V max ), by plotting the relationship between the initial reaction rate and the TMB concentration, the Michaelis-Menten curve was obtained, such as Figure 4 At the same time, the curve is converted into a double reciprocal graph (such as Figure 4 (B) When TMB is used as substrate, the K m Value and V max The values ​​were 2.5mmol / L and 9.78×10 -8 Ms -1 . This indicates that the peroxidase activity of Au@IrNPs is good. In addition, the peroxidase activity of nanomaterials at different concentrations was compared. In the absence of a catalytic reaction, by recording the absorbance at 450nm, the minimum detection concentration of Au@IrNPs was 10000fM. In the presence of TMB and H2O2, the absorbance at 653nm was recorded, and the minimum detection concentration of the material was 2.6fM. By comparison, it was found that the material achieved a certain degree of signal amplification, and the LOD value of Au@IrNPs decreased by nearly 4000 times compared with before catalysis.

[0071] Example 3: Selection of the best complementary sequence of AFM1 and OTA aptamers

[0072] The uniformly mixed AFM1, OTA aptamer and aptamer complementary chain (50 μL) (wherein the aptamer complementary chain of AFM1 is M1-1, M1-2, M1-3, M1-2-6, M1-2-8, M1-2-9, and the aptamer complementary chain of OTA is O-1, O-2, O-3, O-3-10, O-3-14, O-3-16) were heated at 95°C for 5 min and cooled to room temperature. 100 μL SYBR greenⅠ (1×) was added to the above mixture and reacted at 25°C, 400 rpm for 30 min. Then, a certain concentration of target was evenly mixed with the above solution and incubated for another 30 min. After the reaction, the fluorescence intensity of the solution was measured using F-7000. The aptamer nucleic acid sequence of AFM1 is 5'-ACTGCTAGAGATTTTCCACAT-3'; the aptamer nucleic acid sequence of OTA is 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'.

[0073] In order to obtain the best complementary sequence of AFM1 and OTA, the experiment was carried out at a fixed concentration of SGI solution (1×) and a fixed volume of the final system (200 μL). First, the fluorescence intensity of different aptamer concentrations (0.1, 0.3, 0.5, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0 μM) and different target concentrations (0, 0.8, 1.0, 2.0, 4.0 μg / mL) was explored. Considering the cost factor and fluorescence intensity, the optimal aptamer concentration and target concentration were finally determined to be 3.0 μM and 1.0 μg / mL, respectively. Figure 5 On this basis, it is necessary to determine the optimal complementary position and number of bases in the complementary sequence, such as Figure 5 C and D in the figure. First, the competitiveness of the left, middle and right parts of the aptamer complementary sequence with the target was explored in the experiment. After optimization, the complementary positions of AFM1 and OTA were finally selected as M1-2 and O-3, respectively. In addition, the optimization of the number of bases was achieved by adding or reducing some bases in the sequence of the best complementary position. Figure 5 In E and F, the sequence with the largest decrease in fluorescence intensity before and after the addition of the target is the best complementary sequence. After optimization, the complementary sequences of AFM1 and OTA were finally selected as M1-2-9 and O-3.

[0074] Example 4: Preparation of three-channel test strips

[0075] Three-channel test strips according to Figure 2 Methods Cut and assemble on the bottom plate. First, prepare the test line and control line solutions. The specific process is as follows: In 50 μL Bio-DNA T1 (5'-AGAGATTTTAAA-Bioth-3'), Bio-DNAT2 (sequence: 5'-TCTCTCTGTGCCAAA-Bioth-3'), Bio-DNA T3 (5'-GAGCATCGGACAAAA-Bioth-3'), Bio-DNA C 15 μL SA (1 mg / mL) was added to each solution of 5′-TTTTTTTTTTTTTTTTTT-Bioth-3′ to obtain a mixture, and the mixture was incubated at 4°C for 2 h, and then centrifuged (6000 rpm, 20 min, 4°C) using an ultrafiltration tube (30 Kda, Millipore) to remove excess Bio-DNA. T / C . Under the same conditions, the PBS buffer was used for washing twice. The inner tube was centrifuged inverted (1500 rpm, 8 min, 4°C) to collect the liquid and adjust it to the original volume with PBS buffer to obtain SA-Bio-DNA T1 、SA-Bio-DNA T2 、SA-Bio-DNA T3 、SA-Bio-DNA C .

[0076] Next, the prepared SA-Bio-DNA T1 、SA-Bio-DNA T2 、SA-Bio-DNA T3 、SA-Bio-DNA C Spray on the nitrocellulose membrane to form a test line (T line) and a control line (C line), and the distance between the two lines is about 5mm. The sprayed nitrocellulose membrane is placed at 37℃ to dry for 2h. Figure 2 Installed on the bottom plate, the components overlap each other by 2mm to ensure good liquid transfer, and the test strip is obtained. The strip is cut into 4mm pieces and stored in a dry and dark environment for future use.

[0077] Example 5: Optimization of the detection process and detection parameters of the three-channel test strip

[0078] Three Au@Ir@SH-Poly A-cDNA nanoprobes (AFM1 nanozyme probe is Au@Ir@SH-PolyA-cDNA1, AFB1 nanozyme probe is Au@Ir@SH-PolyA-cDNA2, OTA nanozyme probe is Au@Ir@SH-PolyA-cDNA3) were loaded onto the binding pads. Different concentrations of aptamers were mixed with different concentrations of target standard solutions and incubated at room temperature for 30 minutes. The running buffer solution (2×SSC) was adjusted to 180 μL and loaded onto the sample pad. After 10 minutes, 0.5 μL of colorimetric solution was added to the detection line of the test strip, and the detection results were recorded after catalysis for 20 seconds.

[0079] A series of detection conditions of the present invention are optimized, and the concentration of the fixed target (toxin to be detected) is 100 ng / mL. First, the components of the color developing solution are optimized, such as Figure 7 As shown. The concentration of H2O2 was fixed at 2M, and the catalytic effect of the same gradient concentration of TMB (0.7, 0.9, 1.1, 1.3, 1.5, 1.7mM) was explored. Considering the convenience of the test strips, the TMB concentration of the three targets was uniformly determined to be 1.5mM. In addition, the concentration of TMB was fixed at 1.5mM, and the catalytic performance of different concentrations of H2O2 (0.5, 1.0, 1.5, 2.0M) was evaluated. As a result, the concentration of H2O2 was selected to be 1.0M. Secondly, the optimal concentration of the aptamer and the loading volume of the signal probe on the binding pad were optimized (see Figure 8 ). In order to obtain an appropriate color development effect, the detection results of blank samples and spiked samples of the test strips under different aptamer concentrations (5, 10, 15, and 20 μM) were compared. Through intuitive observation of grayscale, the optimal aptamer concentrations of the three targets were all 20 μM. The design of the present invention realizes the simultaneous detection of multiple toxins in three channels. Since the strips are separated from each other and do not interfere with each other, selecting the optimal nanozyme signal probe volume for each target is both convenient and effective. Here, the signal response of nanozyme signal probes with different volumes (0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 μL) was studied. The results showed that the optimal nanozyme signal probe loading volumes of AFM1, AFB1, and OTA were 0.4 μL, 0.1 μL, and 0.5 μL, respectively. Finally, the optimal detection parameters were obtained through single-factor experimental analysis: (1) The test strips have excellent storage stability; (2) The optimal reading time after the color development solution is added is 20 s, as shown in the results. Fig. 9 , 10 .

[0080] Example 6: Analysis of sensitivity and specificity of three-channel test strips

[0081] The concentrations of the three targets were fixed at 25, 50, 100, 250, and 500 ng / mL for testing. Fig.11As shown in Figure G, it can be clearly observed that as the target concentration increases, the color intensity of the detection line also gradually increases, indicating that the target concentration is proportional to the signal intensity of the detection line. In order to quantify the detection results, the gray value obtained by Image J software was explored. Fig.11 As shown in B, D, and F, the standard curve constructed by the logarithm of the detection line signal intensity and the target concentration shows a good linear relationship. After statistical analysis, the detection limits of AFM1, AFB1, and OTA were 0.39 ng / mL, 0.36 ng / mL, and 0.82 ng / mL, respectively. In order to test the specificity of the test strip based on gold iridium nanozyme, four fungal toxins were tested at a concentration of 100 ng / mL, including AFB1, AFM1, OTA, and ZEN. Fig.11 As shown in H, when AFB1, AFM1, and OTA are present alone, the detection line detects a signal response. On the contrary, when ZEN is detected, the detection line does not show a signal response. Therefore, a high signal response can only be observed when the target is present.

[0082] Example 7: Actual sample spike recovery

[0083] Peanuts and corn were purchased from supermarkets for spike recovery test. The standard was added to 2.5 g of ground sample and then ultrasonically extracted in 10 mL of methanol / water (8:2 v / v) for 30 min. The mixture was centrifuged at 6000 rpm for 10 min and the supernatant was collected.

[0084] The results are shown in Table 1. The recoveries of AFM1, AFB1 and OTA in peanut samples were distributed in 93.33%-97.01%, 95.72%-102.67% and 106.88%-109.33%, and all relative standard deviations (RSD) were less than 3%. The results show that the detector has high reliability and accuracy for real sample detection.

[0085] Table 1 Recovery test results of peanut samples (n=3)

[0086]

[0087] Note: ND means not detected; NC means not calculated; RSD = (SD / mean) × 100%, each data represents the average of three replicates.

[0088] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A sensor for detecting multiple mycotoxins, characterized in that: The sensor comprises Au@Ir@SH-PolyA-cDNA nanoprobe, mycotoxin aptamer, complementary chain of mycotoxin aptamer and color developing solution; the mycotoxins are AFM1, AFB1 and OTA.

2. A sensor for detecting multiple mycotoxins according to claim 1, characterized in that: The aptamer nucleic acid sequence of AFM1 is 5′-ACTGCTAGAGATTTTCCACAT-3′; The aptamer nucleic acid sequence of OTA is 5′-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3′; The aptamer nucleic acid sequence of AFB1 is 5′-GGGCACGTGTTGTCTCTCTGTGCCTCGTGCCC-3′.

3. A sensor for detecting multiple mycotoxins according to claim 1, characterized in that: The aptamer complementary chain of AFM1 is one or more of M1-1, M1-2, M1-3, M1-2-6, M1-2-8, and M1-2-9; the aptamer complementary chain of OTA is one or more of O-1, O-2, O-3, O-3-10, O-3-14, and O-3-16; the aptamer complementary chain of AFB1 is 5'-GGCACAGAGAGA-3'; The nucleic acid sequences of M1-1, M1-2, M1-3, M1-2-6, M1-2-8, and M1-2-9 are TAGCAGT, AAATCTC, ATGTGGA, AATCTC, AAATCTCT, and AAAATCTCT, respectively; The nucleic acid sequences of O-1, O-2, O-3, O-3-10, O-3-14 and O-3-16 are shown in SEQ ID No. 1 to SEQ ID NO.

6.

4. A sensor for detecting multiple mycotoxins according to claim 1, characterized in that: The Au@Ir@SH-PolyA-cDNA nanoprobe was prepared by the "low pH" method: Au@IrNPs were mixed with SH-PolyA-cDNA, and sodium citrate buffer was added for the first incubation. Then HEPES buffer was added several times to adjust the pH of the solution to neutral, and the second incubation was performed, followed by centrifugation.

5. A test strip for simultaneously detecting multiple mycotoxins, characterized in that: The test strip comprises the sensor according to any one of claims 1 to 4; the test strip contains a plurality of detection channels.

6. The test strip according to claim 5, characterized in that: The plurality of detection channels is at least 3.

7. The test strip according to claim 5, characterized in that: The test strip comprises a bottom plate, a sample pad, and a plurality of binding pads, a nitrocellulose membrane and a water absorbent pad connected to the sample pad in sequence. The plurality of binding pads are radially distributed outwards with the sample pad as the center; the nitrocellulose membrane is provided with a detection line and a control line.

8. The test strip according to claim 7, characterized in that: The Au@Ir@SH-PolyA-cDNA nanoprobe is dropped onto the binding pad.

9. The test strip according to claim 7, characterized in that: The control line is provided with SA-Bio-DNA C The detection line is provided with SA-Bio-DNA T .

10. The test strip according to claim 7, characterized in that: The distance between the detection line and the control line is 4-5 mm.

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