Aflatoxin B1 detection kit, detection method and application

Abstract

The present invention discloses an aflatoxin B1 detection kit, a detection method and an application. The kit includes: MBs, Apt, EDC, NHS, BSA, BMP, Ab, CuBr2, ME6TREN, AM, AA, TPP*. The present invention adopts an ARGETATRP signal amplification strategy. After the antigen is specifically recognized and combined with the aptamer and the antibody, the initiator BMP of ARGET ATRP is fixed on the surface of the magnetic beads. Under the catalytic action of CuBr2 / Me6TREN, the AGRET ATRP reaction is carried out on the surface of the magnetic beads to form a polymer chain with an amino group. Finally, a large amount of carboxyl porphyrin is connected to the polymer through an amide bond. Compared with other traditional polymerization methods, ARGET ATRP uses a strong reducing agent, which can greatly reduce the amount of transition state heavy metal catalysts, and makes the reaction oxygen tolerant, more efficient, environmentally friendly, and easy to operate. The kit designed by the present invention has a wider detection range, a lower detection limit, and a higher sensitivity.

Description

Technical Field

[0001] The invention relates to an aflatoxin B1 detection kit based on the fluorescence performance of porphyrin derivatives and in-situ triggered ARGET ATRP signal amplification, a detection method and an application thereof, and belongs to the technical field of bioanalysis. Background Art

[0002] Aflatoxins (AF) are a class of dihydrofuranocoumarin derivatives with similar structures produced by Aspergillus flavus and Aspergillus parasiticus strains in the secondary metabolism process. They are one of the most toxic and carcinogenic natural pollutants discovered so far. AF is widely distributed in nature and has a variety of species. Among them, aflatoxin B1 (AFB1) is the most toxic and carcinogenic.

[0003] At present, common analytical methods for AFB1 include thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), chromatography-mass spectrometry (LC-MS, GC-MS) and enzyme-linked immunosorbent assay (ELISA). These technologies provide strong support for AFB1 detection, but there are still problems such as difficulty in integration, difficulty in miniaturization of equipment and high cost. Compared with traditional AFB1 analysis and detection methods, biosensors constructed using biosensor technology have the advantages of short analysis time, fast detection speed, easy sample preparation, reusability and low cost.

[0004] As a means of signal amplification, the application of polymerization reaction in biosensors can not only improve the sensitivity of biosensing methods, but also has the advantages of mild reaction conditions, low cost and simple operation. Among them, the electron transfer regeneration activator atom transfer radical polymerization (ARGET ATRP) has oxygen tolerance due to the presence of a strong reducing agent, and greatly reduces the amount of transition state heavy metal catalysts. It has the advantages of simple operation and green economy, and is favored by many researchers. The purpose of the present invention is to introduce AM through ARGET ATRP, provide abundant binding sites for TPP*, effectively improve the intensity of the fluorescence signal, and provide a new method for high-sensitivity detection of AFB1. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an aflatoxin B1 detection kit, a detection method and an application thereof based on the fluorescence properties of porphyrin derivatives and in situ triggered ARGET ATRP signal amplification.

[0006] In order to achieve the above object, one of the technical solutions of the present invention is:

[0007] A detection kit for aflatoxin B1 based on the fluorescence properties of porphyrin derivatives and in situ triggered ARGET ATRP signal amplification comprises the following raw materials: MBs, Apt, EDC, NHS, BSA, BMP, Ab, CuBr2, ME6TREN, AM, AA, TPP*, DMSO, and PBS buffer.

[0008] Some raw materials need to be prepared into solutions when used, among which the concentration of Apt solution is 1μM, the concentration of EDC solution is 0.1M, the concentration of NHS solution is 0.025M, the concentration of BSA solution is 1%, the concentration of BMP solution is 3mM, the concentration of Ab solution is 2μM, the concentration of CuBr2 in CuBr2 / ME6TREN solution is 10mM, the concentration of ME6TREN is 12mM, the concentration of AM solution is 20-30mM, the concentration of AA solution is 2mM, the concentration of TPP* solution is 8-12mM, the concentration of PBS buffer is 0.1M, and the pH is 7.4.

[0009] Further, the synthesis method of the TPP* is:

[0010] ① Fully dissolve 4-hydroxybenzaldehyde and benzaldehyde in propionic acid and heat;

[0011] ② Add pyrrole to the solution in step ① under nitrogen protection, heat to reflux, and separate by silica gel column chromatography to obtain TPP-OH;

[0012] ③ Fully dissolve 2-bromoethanol, TPP-OH and K2CO3 in anhydrous acetone, heat to reflux under nitrogen protection, and separate by silica gel column chromatography to obtain TPP-C2-OH;

[0013] ④ Dissolve succinic anhydride, TPP-C2-OH and DMAP in THF, react, and separate by silica gel column chromatography to obtain the final product TPP*.

[0014] Furthermore, the molar ratio of 4-hydroxybenzaldehyde, benzaldehyde and pyrrole is 1:3:4.

[0015] The molar ratio of 2-bromoethanol, TPP-OH and K2CO3 is 3:2:4.

[0016] The molar ratio of succinic anhydride, TPP-C2-OH and DMAP is 28:7:1.1.

[0017] The heating reflux temperature of steps ① and ② is 140° C., and the heating reflux time of step ② is 4 h.

[0018] The heating reflux temperature of step ③ is 56°C and the time is 12h.

[0019] The reaction temperature of step ④ is room temperature and the reaction time is 12h.

[0020] Step ② The eluent for silica gel column chromatography separation is petroleum ether and dichloromethane in a volume ratio of 3:1.

[0021] Step ③ The eluent for silica gel column chromatography separation is petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0022] Step ④ The eluent for silica gel column chromatography separation is methanol and dichloromethane in a volume ratio of 1:60.

[0023] One of the technical solutions of the present invention is: a method for detecting aflatoxin B1, wherein magnetic beads are modified with Apt, BSA, a sample to be tested, Ab*, AM, and TPP* in sequence, and then the modified magnetic beads are subjected to fluorescence detection, and the content of aflatoxin B1 is analyzed according to the intensity of the fluorescence signal. Specifically, the following steps are included:

[0024] (1) Magnetic bead modification

[0025] ① Apt modified magnetic beads: Add Apt solution, EDC solution and NHS solution to the PBS buffer containing dispersed carboxyl magnetic beads for reaction;

[0026] ②BSA modified magnetic beads: add BSA solution and PBS buffer and react;

[0027] ③AFB1 modified magnetic beads: add the sample solution to be tested and PBS buffer solution, and react;

[0028] ④Ab* modified magnetic beads: add Ab* solution and PBS buffer and react;

[0029] ⑤AM modified magnetic beads: add CuBr2 / Me6TREN solution, AM solution, AA solution and PBS buffer solution, and react;

[0030] ⑥TPP* modified magnetic beads: add DMSO, PBS buffer and TPP* solution and react;

[0031] (2) Fluorescence detection

[0032] ① Wash the modified magnetic beads in step (1) and perform magnetic separation;

[0033] ② Add DMSO as a sample and detect the fluorescence signal intensity using a fluorescence spectrophotometer.

[0034] Furthermore, in step (1), the reaction conditions of ① are 37° C. overnight; the reaction temperature of ② is 37° C. and the time is 0.5 h; the reaction temperature of ③ is 37° C. and the time is 1 h; the reaction temperature of ④ is 37° C. and the time is 1 h; the reaction temperature of ⑤ is 37° C. and the time is 1 h; the reaction temperature of ⑥ is 37° C. and the time is 80 to 120 min.

[0035] The fluorescence detection conditions in step (2) are: excitation wavelength 420 nm, slit width 2.5 nm.

[0036] Further, the preparation method of Ab* solution is:

[0037] ①Mix equal volumes of EDC solution and NHS solution;

[0038] ② Add BMP solution and place on a constant temperature shaker for shaking;

[0039] ③ Add Ab solution and place on a constant temperature shaker to oscillate to obtain Ab* solution.

[0040] One of the technical solutions of the present invention is: an application of the above-mentioned kit in the detection of aflatoxin B1.

[0041] The principle of the kit of the present invention is as follows Figure 1 As shown in the schematic diagram of ARGET ATRP Figure 2 shown.

[0042] Beneficial effects of the present invention:

[0043] 1. The present invention successfully constructed a fluorescence detection kit that can accurately, sensitively and specifically detect AFB1. The in situ triggered ARGET ATRP signal amplification strategy provides abundant binding sites for carboxyporphyrin (TPP*), and then utilizes the fluorescence properties of carboxyporphyrin to greatly improve the sensitivity of detection.

[0044] 2. The present invention adopts the ARGET ATRP signal amplification strategy. After the antigen is specifically recognized and combined with the aptamer and the antibody, the initiator BMP of ARGET ATRP is fixed on the surface of the magnetic beads. Under the catalysis of CuBr2 / Me6TREN, the AGRETATRP reaction is carried out on the surface of the magnetic beads to form a polymer chain with an amino group. Finally, a large amount of carboxyl porphyrin is connected to the polymer through an amide bond. Compared with other traditional polymerization methods, ARGET ATRP uses a strong reducing agent, which can greatly reduce the amount of transition state heavy metal catalysts, and makes the reaction oxygen-tolerant, more efficient, environmentally friendly, and easy to operate.

[0045] 3. The present invention uses carboxyl porphyrin as a signal molecule to produce a fluorescence peak at 650 nm, which can avoid the influence of the spontaneous fluorescence of biological materials in the system.

[0046] 4. Under the optimal experimental conditions, the analytical performance of the kit within a certain range of AFB1 concentrations was studied. Magnetic beads were modified with different concentrations of AFB1 and the fluorescence signal intensity was detected at different wavelengths. The strongest emission wavelength was at 650nm. Through calculation, it was found that there was a good linear relationship between the logarithm of AFB1 concentration and the fluorescence signal intensity within a certain range. The linear range was 100fg / mL ~ 100ng / mL, and the linear equation was F(au) = 18932.92lgC AFB1 +112513.60, correlation coefficient (R 2 ) was 0.997, and the detection limit (LOD) of this method for detecting AFB1 was 91.5 fg / mL (S / N=3). Compared with the detection methods for detecting AFB1 in other literatures, the kit designed in this study has a wider detection range, lower detection limit and higher sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of the principle of the detection kit of the present invention.

[0048] Figure 2 This is the reaction principle of ARGET ATRP.

[0049] Figure 3 This is the synthesis step of TPP*.

[0050] Figure 4 For TPP* 1 HNMR spectrum.

[0051] Figure 5 The images of AFB1 blank modified magnetic beads (A) and AFB1 modified magnetic beads (B) under fluorescence confocal microscope. The SEM images of AFB1 blank modified magnetic beads (C) and AFB1 modified magnetic beads (D).

[0052] Figure 6 The feasibility of fluorescence detection.

[0053] Figure 7 The relationship between the fluorescence intensity and the concentration of AM (A), the concentration of TPP* (B), and the reaction time of TPP* (C) is shown in Figure 1.

[0054] Figure 8 The fluorescence intensity of the system after adding different concentrations of AFB1 (A) and the linear relationship between the fluorescence intensity and the logarithm of the AFB1 concentration (B).

[0055] Fig. 9 The fluorescence response of the kit to different mycotoxins (the error bar is the standard deviation of 3 parallel experiments). DETAILED DESCRIPTION

[0056] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0057] Carboxyl magnetic beads (MBs) were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0058] The Apt used was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC-CE. The sequence of Apt is:

[0059] 5'-NH2-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC ACA-3' (SEQ ID NO. 1).

[0060] The aflatoxin B1 antibody (Ab) used was anti-aflatoxin B1 rabbit antibody purchased from Sigma-Aldrich.

[0061] Example 1: Synthesis of carboxyporphyrin (TPP*)

[0062] TPP* synthesis steps Figure 3 As shown, the specific synthesis method is:

[0063] ① Dissolve 4-hydroxybenzaldehyde (2.2 g, 18 mM) and benzaldehyde (5.47 mL, 54 mM) in 150 mL of propionic acid and heat to 140 °C;

[0064] ② Under nitrogen protection, pyrrole (5 mL, 72 mM) was added to the solution in step ①, heated under reflux for 4 h, and separated by silica gel column chromatography using petroleum ether / dichloromethane (v / v=3:1) as the eluent to obtain TPP-OH;

[0065] ③ Dissolve 2-bromoethanol (17 μL, 0.24 mM), TPP-OH (0.1 g, 0.16 mM) and K2CO3 (44.20 mg, 0.32 mM) in 5 mL of anhydrous acetone, heat to 56 °C and reflux for 12 h under nitrogen protection, separate by silica gel column chromatography using petroleum ether / ethyl acetate (v / v=3:1) as eluent to obtain TPP-C2-OH;

[0066] ④ Dissolve succinic anhydride (28 mg, 0.28 mM), TPP-C2-OH (0.05 g, 0.07 mM) and 4-dimethylaminopyridine (DMAP) (1.34 mg, 0.011 mM) in 2 mL of tetrahydrofuran (THF), react at room temperature for 12 h, and separate by silica gel column chromatography using methanol / dichloromethane (v / v=1:60) as eluent to obtain the final product TPP*.

[0067] TPP* 1H NMR Figure 4 As shown: (500MHz,CDCl3)δ=8.99~8.76(m,8H),δ=8.21(t,J=5.4Hz,6H),δ=8.11(d,J=8.0Hz,2H),δ=7.82~7.63(m,9H),δ=7.24(s,2H),δ=4.61(t,J=4.7Hz,12H),δ=4.41(d,J=4.7Hz,2H),δ=2.77(q,J=4.1Hz,4H). The results show that TPP* was successfully synthesized.

[0068] Example 2: Kit

[0069] The invention discloses an aflatoxin B1 detection kit based on the fluorescence performance of porphyrin derivatives and in situ triggered ARGET ATRP signal amplification, comprising the following raw materials: carboxyl magnetic beads (MBs), AFB1 aptamer (Apt), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), 2-bromo-2-methylpropionic acid (BMP), aflatoxin B1 antibody (Ab), CuBr2, ligand tris-(N,N-dimethylaminoethyl)amine (ME6TREN), acrylamide (AM), ascorbic acid (AA), carboxyl porphyrin (TPP*), dimethyl sulfoxide (DMSO), and PBS buffer.

[0070] Some raw materials need to be prepared into solutions when used, among which the concentration of Apt solution is 1 μM, the concentration of EDC solution is 0.1 M, the concentration of NHS solution is 0.025 M, the solvent of EDC and NHS is PBS buffer, the concentration of BSA solution is 1%, the concentration of BMP solution is 3 mM, the concentration of Ab solution is 2 μM, the concentration of CuBr2 in CuBr2 / ME6TREN solution is 10 mM, the concentration of ME6TREN is 12 mM, the concentration of AM solution is 20-30 mM, the concentration of AA solution is 2 mM, the concentration of TPP* solution is 8-12 mM, the concentration of PBS buffer is 0.1 M, and pH is 7.4.

[0071] Example 3: Method of using the kit

[0072] A method for using a kit for detecting aflatoxin B1 based on the fluorescence properties of porphyrin derivatives and ARGET ATRP signal amplification technology, comprising the following steps:

[0073] (1) Modification of carboxyl magnetic beads

[0074] ① Apt modified magnetic beads: Take 20 μL of carboxyl magnetic beads (10 mg / mL) in a centrifuge tube, add 180 μL of PBS buffer to wash twice and remove the supernatant by magnetic separation, then disperse in 150 μL PBS buffer, add 10 μL of Apt solution (1 μM), 20 μL of EDC solution (0.1 M) and 20 μL of NHS solution (0.025 M), and react in a constant temperature oscillator at 37°C overnight.

[0075] ②BSA modified magnetic beads: Wash the Apt modified magnetic beads twice with PBS buffer and remove the supernatant by magnetic separation, add 20 μL 1% BSA solution and 180 μL PBS buffer, react in a constant temperature oscillator at 37°C for 0.5 h to block the unbound sites on the surface of the magnetic beads.

[0076] ③AFB1 modified magnetic beads: The Apt / BSA modified magnetic beads were washed twice with PBS buffer and magnetically separated to remove the supernatant, and 10 μL of the sample solution to be tested (containing AFB1) and 190 μL of PBS buffer were added, and the mixture was reacted in a constant temperature oscillator at 37°C for 1 h;

[0077] ④Ab* modified magnetic beads: Wash the Apt / BSA / AFB1 modified magnetic beads twice with PBS buffer and remove the supernatant by magnetic separation. Add 20 μL of AFB1 antibody Ab* solution that has been pre-connected with BMP and 180 μL of PBS buffer, and react in a constant temperature oscillator at 37°C for 1 hour.

[0078] ⑤AM modified magnetic beads (ATRP): The Apt / BSA / AFB1 / Ab* modified magnetic beads were washed twice with 200 μL PBS buffer and magnetically separated to remove the supernatant. 20 μL CuBr2 / Me6TREN solution, 20 μL AM solution (20 mM), 20 μL AA solution (2 mM) and 140 μL PBS buffer were added to a centrifuge tube and reacted in a constant temperature oscillator at 37°C for 1 h.

[0079] ⑥TPP* modified magnetic beads: Wash the Apt / BSA / AFB1 / Ab* / AM modified magnetic beads twice with 200 μL PBS buffer and remove the supernatant by magnetic separation. Add 80 μL DMSO, 100 μL PBS buffer and 20 μL TPP* solution (8 mM) and react in a constant temperature oscillator at 37°C for 80 min.

[0080] (2) Fluorescence detection

[0081] ① After completing the above reaction, wash the Apt / BSA / AFB1 / Ab* / AM / TPP* modified magnetic beads three times with 200 μL DMSO and separate them magnetically.

[0082] ② After washing, add 600 μL DMSO as a sample and use DMSO as a blank control for the experiment. Under the conditions of an excitation wavelength of 420 nm and a slit width of 2.5 nm, use a fluorescence spectrophotometer to detect the intensity of the fluorescence signal.

[0083] The preparation method of AFB1 antibody Ab* solution is as follows:

[0084] ① Mix 100 μL EDC solution (0.1 M) and 100 μL NHS solution (0.025 M) in equal volumes;

[0085] ② Add 100 μL BMP solution (3 mM) and place on a constant temperature shaker at 37°C for 2 h;

[0086] ③ Add 300 μL Ab solution (2 μM), place on a constant temperature shaker at 37°C for 2 to 6 hours to obtain Ab* solution.

[0087] Fluorescence confocal microscopy and scanning electron microscopy (SEM) were used to characterize the modified magnetic beads.

[0088] The fluorescence image of the modified magnetic beads observed under a fluorescence confocal microscope is Figure 5 A and Figure 5 B. As Figure 5 As shown in A, in the absence of AFB1-modified carboxyl magnetic beads, no obvious fluorescent spots can be observed by fluorescence confocal microscopy. Figure 5 In B, a large number of red fluorescent spots can be seen on the carboxyl magnetic beads modified with Apt / BSA / AFB1 / Ab* / AM / TPP* under a confocal microscope. The reason for this result is that AFB1 is modified on the surface of the carboxyl magnetic beads, and the "aptamer-antigen-antibody" sandwich structure is successfully constructed. Subsequently, the monomers are aggregated on the surface of the magnetic beads through the ARGET ATRP reaction and the porphyrin is successfully connected, and the fluorescence can be observed under a fluorescence confocal microscope.

[0089] The results of SEM are as follows Figure 5 C and 5D show that when AFB1 is not modified, the polymer and fluorescent molecule TPP* produced by the ATRP reaction cannot be connected to the MBs, so the surface of the MBs is relatively smooth ( Figure 5 C). On the contrary, after being modified with AFB1, Ab* binds to AFB1 and connects to MBs, and on the other hand, participates in initiating ARGET ATRP, so that the polymer can be grafted on the surface of MBs and further connected with a large number of fluorescent molecules TPP*. Therefore, the surface of MBs in SEM becomes rough, with many granular protrusions ( Figure 5 D).

[0090] The results of fluorescence confocal microscopy and SEM characterization demonstrated that the ARGET ATRP-based fluorescence amplification strategy can be used to detect AFB1.

[0091] Example 4: Feasibility Verification

[0092] In order to verify the feasibility of the constructed kit to detect AFB1, the fluorescence intensity of magnetic beads modified with different materials was compared. Figure 6 As shown in the figure, in the absence of AFB1 (curve b), Apt (curve c), Ab* (curve d), AM (curve e) or TPP* (curve f) modified magnetic beads, the fluorescence intensity of the reaction system is very weak. This is because without Apt or AFB1 modification, the polymer produced by the ATRP reaction cannot be connected to MBs; without initiator Ab* or monomer AM modification, the polymerization cannot be started and the polymer cannot be grafted on MBs; and without TPP* modification, the reaction system lacks fluorescent signal molecules and cannot produce fluorescent signals. On the contrary, when all modification materials are used to modify magnetic beads (curve a), a strong fluorescence intensity appears at 650nm. The above experimental results show the necessity of the existence of each step of modification and the feasibility of the kit to detect AFB1.

[0093] Example 5: Condition Optimization

[0094] During the construction of the kit, the concentration of AM, the concentration of TPP* and the TPP* reaction time will affect the fluorescence intensity of the system. In order to achieve the best detection performance of the kit, these conditions need to be optimized.

[0095] (1) AM concentration

[0096] The concentration of AM solution was set to 5, 10, 15, 20, 25, and 30 mM respectively, and the fluorescence intensity of the system was measured. The results are as follows Figure 7 As shown in A, within the AM concentration range of 5 to 20 mM, as the AM concentration increases, the fluorescence signal continues to increase. When the concentration reaches 20 mM, the fluorescence intensity of the kit reaches a maximum value and remains basically unchanged thereafter, indicating that the optimal concentration of the AM solution is 20 mM.

[0097] (2) TPP* concentration

[0098] At the optimal AM concentration, the concentration of TPP* solution was changed to 2, 4, 6, 8, 10, and 12 mM, and the fluorescence intensity of the system was measured. The results are as follows: Figure 7As shown in B, when the concentration of TPP* is in the range of 2 to 8 mM, the fluorescence intensity increases continuously with the increase of TPP* concentration. When the concentration of TPP* reaches 8 mM, the fluorescence intensity reaches the maximum and no longer changes with the increase of TPP* concentration, which indicates that the optimal concentration of TPP* solution is 8 mM.

[0099] (3)TPP* reaction time

[0100] The TPP* reaction time was set to 20, 40, 60, 80, 100, and 120 min, and the fluorescence intensity of the system was measured. The results are shown in Figure 7 As shown in Figure C, in the range of 20 to 80 min, the fluorescence intensity gradually increases with the extension of TPP* reaction time. When the TPP* reaction time exceeds 80 min, the fluorescence signal intensity no longer changes significantly, which indicates that the optimal reaction time of TPP* is 80 min.

[0101] Example 6: Performance Analysis

[0102] Under the optimal experimental conditions, a series of different concentrations of AFB1 were used to analyze the detection performance of the kit. Figure 8 As shown in Figure 2, within the range of 100 fg / mL to 100 ng / mL, the fluorescence intensity and the logarithmic value of the AFB1 concentration showed a good linear relationship, and the linear regression equation was F(au)=18932.92lgC AFB1 +112513.60(R 2 =0.997), and its detection limit is 91.5 fg / mL (S / N=3). Compared with the AFB1 detection methods reported in the literature in Table 1, the kit for detecting AFB1 based on the fluorescence properties of porphyrin derivatives and ARGET ATRP signal amplification fluorescence has a lower detection limit and a wider detection range.

[0103] Table 1 Comparison of analytical detection performance of the method of the present invention and other AFB1 detection methods

[0104]

[0105] Example 7: Selectivity and reproducibility

[0106] In order to verify the selectivity of the kit for AFB1 detection, the fluorescence signal intensities of AFB1, aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA) and blank group (Blank) were detected and compared under the same conditions. Fig. 9As shown, the fluorescence intensities of AFB2, AFG1, AFG2, OTA and blank groups were 16.33%, 10.78%, 9.27%, 6.85% and 3.60% of the fluorescence response value of AFB1, respectively. This is because the Apt modified on the MBs can specifically recognize and connect to AFB1, and connect to Ab* on AFB1 to form a sandwich structure. However, AFB2, AFG1, AFG2 and OTA cannot cause Apt recognition, making the subsequent steps impossible. The experimental results show that the kit based on the ARGET ATRP signal amplification strategy has a high selectivity for AFB1.

[0107] In addition, under the same conditions, parallel experiments within and between groups (n=5) were set up to study the reproducibility of the kit. The results showed that the coefficient of variation within the group was 2.05% and the coefficient of variation between the groups was 3.21%, indicating that the strategy has good reproducibility.

[0108] Example 8: Detection of AFB1 in Traditional Chinese Medicine Samples

[0109] In order to further evaluate the accuracy and potential application of the kit in the analysis of traditional Chinese medicine samples, three different concentrations of AFB1 standards (0.01 ng / mL, 1.00 ng / mL, 100 ng / mL) were added to four traditional Chinese medicine samples (Chinese yam, Rehmannia root, Chrysanthemum, Malt) for fluorescence detection, and the recovery was calculated and analyzed. The results are shown in Table 2. The recovery of this method in actual samples was 90.52% to 98.45%; the RSD was 0.94 to 5.60%. These results show that the kit can be used for the detection of AFB1 in traditional Chinese medicine samples and can be used to evaluate the exposure level of mycotoxins in traditional Chinese medicine.

[0110] Table 2 Detection results of AFB1 in actual samples and its recovery rate

[0111] Sequence Listing <110> Henan University of Traditional Chinese Medicine <120> Aflatoxin B1 detection kit, detection method and application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <400> 1 gttgggcacg tgttgtctct ctgtgtctcg tgcccttcgc taggcccaca 50

Claims

1. A detection kit for aflatoxin B1 based on the fluorescence properties of porphyrin derivatives and in situ triggered ARGET ATRP signal amplification, characterized in that: The invention comprises the following raw materials: carboxyl magnetic beads MBs, AFB1 aptamer Apt, EDC, NHS, BSA, 2-bromo-2-methylpropionic acid BMP, aflatoxin B1 antibody Ab, CuBr2, ME6TREN, acrylamide AM, ascorbic acid AA, carboxyl porphyrin TPP*; the synthesis method of the TPP* is: ① Fully dissolve 4-hydroxybenzaldehyde and benzaldehyde in propionic acid and heat; ② Add pyrrole to the solution in step ① under nitrogen protection, heat to reflux, and separate by silica gel column chromatography to obtain TPP-OH; ③ Fully dissolve 2-bromoethanol, TPP-OH and K2CO3 in anhydrous acetone, heat to reflux under nitrogen protection, and separate by silica gel column chromatography to obtain TPP-C2-OH; ④ Dissolve succinic anhydride, TPP-C2-OH and DMAP in THF, react, and separate by silica gel column chromatography to obtain the final product TPP*; Some raw materials need to be prepared into solutions when used, among which the concentration of Apt solution is 1 μM, the concentration of EDC solution is 0.1 M, the concentration of NHS solution is 0.025 M, the concentration of BSA solution is 1%, the concentration of BMP solution is 3 mM, the concentration of Ab solution is 2 μM, the concentration of CuBr2 in CuBr2 / ME6TREN solution is 10 mM, the concentration of ME6TREN is 12 mM, the concentration of AM solution is 20 ~30 mM, the concentration of AA solution is 2 mM, the concentration of TPP* solution is 8 ~ 12 mM, the concentration of PBS buffer is 0.1 M, and pH = 7.

4.

2. The kit according to claim 1, characterized in that Also included are DMSO and PBS buffer.

3. The kit according to claim 1, characterized in that The molar ratio of 4-hydroxybenzaldehyde, benzaldehyde and pyrrole is 1:3:4; The molar ratio of 2-bromoethanol, TPP-OH and K2CO3 is 3:2:4; The molar ratio of succinic anhydride, TPP-C2-OH, and DMAP was 28:7:1.1; The heating reflux temperature of steps ① and ② is 140°C, and the heating reflux time of step ② is 4 h; The heating reflux temperature in step ③ is 56°C and the time is 12 h; The reaction temperature of step ④ is room temperature and the reaction time is 12 h; Step ② The eluent for silica gel column chromatography separation is petroleum ether and dichloromethane in a volume ratio of 3:1; Step ③ The eluent for silica gel column chromatography separation is petroleum ether and ethyl acetate in a volume ratio of 3:1; Step ④ The eluent for silica gel column chromatography separation is methanol and dichloromethane in a volume ratio of 1:

60.

4. A method for detecting aflatoxin B1 for non-diagnostic purposes, characterized in that: The kit according to claim 1 is used for detection, and the magnetic beads are modified with Apt, BSA, the sample to be tested, Ab*, AM, and TPP* in sequence, and then the modified magnetic beads are subjected to fluorescence detection, and the content of aflatoxin B1 is analyzed according to the intensity of the fluorescence signal.

5. The detection method according to claim 4, characterized in that: The following steps are involved: (1) Magnetic bead modification ① Apt modified magnetic beads: Add Apt solution, EDC solution and NHS solution to the PBS buffer containing dispersed carboxyl magnetic beads for reaction; ②BSA modified magnetic beads: add BSA solution and PBS buffer and react; ③AFB1 modified magnetic beads: add the sample solution to be tested and PBS buffer solution, and react; ④Ab* modified magnetic beads: add Ab* solution and PBS buffer and react; ⑤AM modified magnetic beads: add CuBr2 / Me6TREN solution, AM solution, AA solution and PBS buffer solution, and react; ⑥TPP* modified magnetic beads: add DMSO, PBS buffer and TPP* solution and react; (2) Fluorescence detection ① Wash the modified magnetic beads in step (1) and perform magnetic separation; ② Add DMSO as a sample and detect the fluorescence signal intensity using a fluorescence spectrophotometer.

6. The detection method according to claim 5, characterized in that: The reaction conditions of step (1) ① are 37°C overnight; the reaction temperature of ② is 37°C for 0.5 h; the reaction temperature of ③ is 37°C for 1 h; the reaction temperature of ④ is 37°C for 1 h; the reaction temperature of ⑤ is 37°C for 1 h; the reaction temperature of ⑥ is 37°C for 80-120 min; The fluorescence detection conditions in step (2) are: excitation wavelength 420 nm, slit width 2.5 nm.

7. The detection method according to claim 5, characterized in that: The preparation method of Ab* solution is: ①Mix equal volumes of EDC solution and NHS solution; ② Add BMP solution and place on a constant temperature shaker for shaking; ③ Add Ab solution and place on a constant temperature shaker to oscillate to obtain Ab* solution.

8. Use of the kit according to any one of claims 1 to 3 in the detection of aflatoxin B1.

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