Electrochemical Detection Kit for Highly Sensitive Detection of AFB1 Based on eRAFT Aggregation Signal Amplification Strategy and Its Application

Abstract

The present invention discloses an electrochemical detection kit and application for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy. The kit includes a gold electrode, Aptamer, FcMMA, MCH, CPAD, C6H4BBrF4N2, Ab, DMF, KPF6, EDC, and NHS. In the present invention, the aptamer modified with a mercapto group at one end is connected to the gold electrode through an Au-S bond to specifically capture AFB1 in the sample. Then, Ab* specifically recognizes AFB1, and subsequently, the eRAFT reaction is initiated under the action of a constant negative potential to generate a large number of long-chain polymers formed by the electroactive probe FcMMA on the electrode surface. Under optimal conditions, there is a good linear relationship between the current intensity of this method and the logarithm of the AFB1 concentration, and the linear range is 2×10 ‑4 ~2×10 2 ng / mL, and the detection limit (LOD) is 37.34 fg / mL (S / N = 3). This kit has the characteristics of high sensitivity, good stability, and good reproducibility for the detection of AFB1, and can be used for the detection of AFB1 in actual samples.

Description

Technical Field

[0001] The present invention relates to an electrochemical detection kit for highly sensitive detection of AFB1 based on an electrochemically induced reversible addition-fragmentation chain transfer (eRAFT) polymerization signal amplification strategy, a using method and an application, belonging to the technical field of bioanalysis. Background Art

[0002] Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, which are widely present in foods, feeds and Chinese medicinal materials and pose a serious hazard to human and animal health. There are about more than 20 kinds of aflatoxins discovered at present, and the more common ones are AFB1, AFB2, AFG1, AFG2, etc. Among them, aflatoxin B1 (AFB1) has the strongest carcinogenicity and the greatest toxicity, and is listed as a class I carcinogen by the International Agency for Research on Cancer (IARC). AFB1 has genotoxicity, immunogenicity, hepatotoxicity and mutagenicity, and can cause Reye's syndrome, liver cancer, etc. At the same time, AFB1 has significant teratogenic potential in the prenatal life of humans and animals, especially for fetal development. Therefore, the highly sensitive detection of AFB1 is of great significance in the food and medical fields.

[0003] At present, the methods for detecting aflatoxin B1 mainly include high performance liquid chromatography (HPLC), thin layer chromatography (TLC), liquid chromatography-tandem mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), etc. However, these methods have defects such as complex operation, expensive equipment and high sample processing cost. Therefore, it is of great significance to develop a simple, rapid and specific method for detecting the contamination level of AFB1. Electrochemical sensors have the advantages of simple operation, low cost and high sensitivity, and have been widely used in the field of mycotoxin detection. However, the specificity and sensitivity of traditional electrochemical detection systems are still lacking, especially the detection ability for trace AFB1 in samples is insufficient.

[0004] A large number of signal molecules with electroactive labels are grafted onto the electrochemical sensor through various polymerization methods to achieve the purpose of signal amplification and improve the sensitivity of the sensing system. Among them, reversible addition-fragmentation chain transfer (RAFT) polymerization is a simple, economical and environmentally friendly method. RAFT polymerization is mediated by a dithiocarbonylthio RAFT reagent, which can tolerate a wide range of functional groups and solvent systems and can be used to synthesize complex compounds with well-defined structures, low dispersity and predetermined molecular weights. The initiation methods of RAFT polymerization include heating, light irradiation, redox reaction and electrochemical methods. Among them, electrochemically mediated RAFT polymerization (eRAFT) has mild reaction conditions, good biocompatibility and simple operation, and is a reliable method for preparing polymers with well-defined structures based on reversible radical exchange chain transfer agents (CTA). In-situ-initiated eRAFT polymerization can graft a large number of electroactive ferrocene-based polymers. This "graft from" method can graft polymers with a higher density than the "graft to" method, thereby achieving highly sensitive recognition of low-concentration targets and having great application prospects in bioanalysis and the detection of trace targets. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an electrochemical detection kit for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy, its use method and application, which are simple to operate, low in cost, high in sensitivity, and have good selectivity, specificity and stability.

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

[0007] An electrochemical detection kit for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy, comprising: a gold electrode, Aptamer, FcMMA, MCH, CPAD, C6H4BBrF4N2, Ab, DMF, KPF6, EDC, NHS, PBS buffer solution, LiClO4, ultrapure water.

[0008] Furthermore, some raw materials are first prepared into solutions with the following concentrations before use: the concentration of Aptamer is 1 μM, the concentration of FcMMA is 20 mM, the concentration of MCH is 2 mM, the concentration of CPAD is 20 mM, the concentration of C6H4BBrF4N2 is 5 μM, the concentration of Ab is 800 μg / mL, the concentration of KPF6 is 0.1 M, the concentration of EDC is 20 mM, the concentration of NHS is 5 mM, the concentration of LiClO4 is 1 M, the concentration of PBS buffer solution is 0.1 M, and pH = 7.4.

[0009] The sequence of the aptamer is 5′-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCTTCGCTAGGCCCACA-3′.

[0010] One of the technical solutions of the present invention is: a method for detecting AFB1 using the said kit, comprising the following steps:

[0011] (1) Drop the aptamer solution onto the gold electrode and react;

[0012] (2) Immerse the electrode from step (1) in the MCH solution and react;

[0013] (3) Drop the sample solution to be detected onto the electrode from step (2) and react;

[0014] (4) Drop the Ab* solution onto the electrode from step (3) and react;

[0015] (5) Immerse the electrode from step (4) in the eRAFT polymerization solution and react;

[0016] (6) Place the electrode from step (5) in the LiClO4 solution to measure the electrochemical signal.

[0017] Furthermore, first pre-treat the gold electrode, and the pre-treatment method is: polish the gold electrode to a mirror surface, clean it, dry it, and reserve it for use.

[0018] Furthermore, the reaction condition for step (1) is to react overnight at room temperature; the reaction temperature for step (2) is 37°C and the time is 30 min; the reaction temperature for step (3) is 37°C and the time is 30 min; the reaction temperature for step (4) is 37°C and the time is 1 h; the reaction temperature for step (5) is 37°C and the time is 2 h.

[0019] Furthermore, the preparation method of the Ab* solution is:

[0020] (1) Add the CPAD solution to the EDC / NHS mixed solution and react with shaking;

[0021] (2) Add the Ab solution and react with shaking to obtain the Ab* solution;

[0022] The volume ratio of the CPAD solution, the EDC / NHS mixed solution, and the Ab solution is 100:837.5:62.5; the EDC / NHS mixed solution is formed by mixing the EDC solution and the NHS solution in equal volumes;

[0023] The reaction temperature for step (1) is 37°C and the reaction time is 3 h;

[0024] The reaction temperature in step (2) is 37 °C and the reaction time is 12 h.

[0025] Furthermore, the eRAFT polymerization solution is prepared by mixing FcMMA solution, C6H4BBrF4N2 solution, KPF6 solution, and DMF at a volume ratio of 20:1:1600:379.

[0026] Furthermore, the measurement of the electrochemical signal is by square wave voltammetry, with the potential scanning range of -0.1 to 0.8 V, the rising potential of 4.0 mV, the pulse amplitude of 25 mV, and the frequency of 15 Hz.

[0027] One of the technical solutions of the present invention is: an application of the said kit in detecting AFB1.

[0028] The construction process of the electrochemical detection kit for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy is as Figure 1 shown. First, activate the carboxyl group of CPAD using EDC / NHS, then add Ab and activate its amino group, so that Ab forms a bioconjugate Ab* with CPAD. Then polish the gold electrode clean on suede and fix Ap on the gold electrode through Au-S bonds. Subsequently, add AFB1 to enable Ap to specifically capture AFB1. Next, the bioconjugate Ab* recognizes and forms a sandwich structure with AFB1 through antigen-antibody specific recognition. Finally, under the action of a constant negative potential Eapp (Eapp = -1.06 V), CPAD on Ab* serves as a chain transfer agent, BrPhN2 + serves as an initiator to provide the free radicals required for the reaction, initiate the eRAFT reaction, and form a long-chain electroactive polymer formed by ferrocene groups.

[0029] Advantages of the present invention:

[0030] 1. Using two recognition elements (aptamer and antibody) with strong affinity for AFB1 as probes greatly improves the selectivity of the kit, and at the same time makes it have good stability and reproducibility.

[0031] 2. Using eRAFT as a signal method means, in-situ grafts a high density of electroactive Fc markers, and at the same time avoids the use of artificial enzymes and the synthesis of nanomaterials, which is both economical and has good biocompatibility.

[0032] 3. In the present invention, an aptamer modified with a thiol group at one end is connected to a gold electrode through an Au-S bond to specifically capture AFB1 in a sample. Then, Ab* specifically recognizes AFB1. Subsequently, under the action of a constant negative potential, the eRAFT reaction is initiated, and a large number of long-chain polymers formed by electroactive probe FcMMA are generated on the electrode surface. Under optimal conditions, there is a good linear relationship between the current intensity of this method and the logarithm of the AFB1 concentration, with a linear range of 2×10 -4 ~2×10 2 ng / mL, and the linear equation is The limit of detection (LOD) is 37.34 fg / mL (S / N = 3). It has good stability. After being stored at 4°C for 14 days, the peak current of the fully modified gold electrode still remains at 97.04% of the initial value. This kit has the characteristics of high sensitivity, good stability, and good reproducibility for the detection of AFB1, and can be used for the detection of AFB1 in actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the principle of the detection method of the present invention.

[0034] Figure 2 In [the figure], A is the CV curve of a bare glassy carbon electrode in a 0.1 M TBAP solution containing 1.0 mM CPAD and 1.0 mM BrPhN2 + respectively, and B is the CV curve of Au / Ap / MCH / AFB1 / Ab* in a polymerization solution without FcMMA.

[0035] Figure 3 In [the figure], A is the SWV signal diagram of electrodes under different modification conditions, which are the electrodes lacking Ap (curve b), AFB1 (curve c), Ab* (curve d), BrPhN2 + (curve e), FcMMA (curve f), and the fully modified electrode (curve a); B is the CV curve of the modified electrode at different scanning rates and the linear relationship between the scanning rate and the redox current; C is the impedance change process of the electrode after each step of modification (curves a - f); D is the CV curve of the electrode after each step of modification (curves a - f).

[0036] Figure 4 They are AFM and WCA images before the eRAFT reaction (A, C) and after the eRAFT reaction (B, D).

[0037] Figure 5 In [the figure], A is the optimization of the reaction time of AFB1; B is the concentration optimization of BrPhN2 + ; C is the optimization of the reaction time of eRAFT.

[0038] Figure 6In A, it is the SWV response curves of AFB1 at different concentrations; in B, it is the linear relationship between the current signal and the logarithm of the AFB1 concentration.

[0039] Figure 7 In A, it is the comparison of the SWV response signals of AFB2, AFG1, AFG2, OTA, blank group, AFB1 and the mixed group at the same concentration under the same detection conditions; in B, it is the comparison of the initial signal of the successfully prepared electrode and the current signal after being placed at 4°C for 14 days. Specific implementation manners

[0040] The following further elaborates on the specific implementation manners of the present invention in conjunction with embodiments.

[0041] The used aflatoxin B1 aptamer was synthesized by Shanghai Sangon Biotech Co., Ltd. and purified by HPLC-CE. The aptamer sequence is as follows:

[0042] The sequence of Aptamer (Ap) is 5′-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCTTCGCTAGGCCCACA-3′ (SEQ ID NO.1).

[0043] The antibody (Ab) is rabbit anti-aflatoxin B1, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0044] Example 1: An electrochemical detection kit for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy

[0045] This kit includes: a gold electrode, aptamer (Aptamer), ferrocenylmethyl methacrylate (FcMMA), 6-mercapto-1-hexanol (MCH), 4-cyano-4-(phenylthiocarbonothioylthio) pentanoic acid (CPAD), 4-bromobenzenediazonium tetrafluoroborate (C6H4BBrF4N2, abbreviated as BrPhN2 + )、antibody (Ab), N,N-dimethylformamide (DMF), potassium hexafluorophosphate (KPF6), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), PBS buffer, ultrapure water, lithium perchlorate (LiClO4).

[0046] When using some raw materials, they are first prepared into solutions with the following concentrations: the concentration of Aptamer is 1 μM, the concentration of FcMMA is 20 mM, the concentration of MCH is 2 mM, the concentration of CPAD is 20 mM, BrPhN2 +The concentration of [substance] is 5 μM, the concentration of Ab is 800 μg / mL, the concentration of KPF6 is 0.1 M, the concentration of EDC is 20 mM, the concentration of NHS is 5 mM, the concentration of LiClO4 is 1 M, and the concentration of PBS buffer is 0.1 M, pH = 7.4.

[0047] Example 2: Construction of Detection Method

[0048] (1) Synthesis of Antibody 4-Cyano-4-(phenylthiocarbamoylthio)pentanoic Acid Bioconjugate (Ab*)

[0049] ① Mix 418.75 μL of 20 mM EDC solution and 418.75 μL of 5 mM NHS solution evenly.

[0050] ② Add 100 μL of 20 mM CPAD solution to the mixture in step ①, and react with shaking at 37 °C for 3 h to obtain a carboxyl-activated CPAD solution.

[0051] ③ Add 62.5 μL of 800 μg / mL Ab solution to the solution obtained in step ②, and react with shaking at 37 °C for 12 h to obtain a 50 μg / mL Ab* solution.

[0052] (2) Electrode Pretreatment

[0053] Place the bare gold electrode (diameter 2 mm) on the suede, polish it with 0.03 μm polishing powder until a polished mirror surface is obtained, then ultrasonically clean it with ultrapure water, anhydrous ethanol, and ultrapure water for 1 min in sequence, and dry it with nitrogen for standby.

[0054] (3) Electrode Modification

[0055] ① Drop 5 μL of 1 μM Aptamer solution onto the surface of the pretreated gold electrode, leave it at room temperature overnight, wash it with ultrapure water, and dry it with nitrogen.

[0056] ② Immerse the electrode in step ① in 300 μL of 2 mM MCH solution and react at 37 °C for 30 min to block non-specific sites, wash it with ultrapure water, and dry it with nitrogen.

[0057] ③ Drop 5 μL of the sample solution to be tested (containing AFB1) onto the gold electrode in step ②, incubate it at 37 °C for 30 min, and wash it with ultrapure water to remove unbound AFB1.

[0058] ④ Drop 5 μL of 50 μg / mL Ab* solution onto the surface of the gold electrode in step ③ and react at 37 °C for 1 h.

[0059] ⑤ Immerse the electrode in step ④ in 10 mL of eRAFT reaction solution (containing 0.1 mL of 20 mM FcMMA solution, 5 μL of 5 μM BrPhN2 + solution, 8 mL of 0.1 M KPF6 solution, 1.895 mL of DMF), and react at 37 °C for 2 h under the condition of -1.06 V.

[0060] (4) Square wave voltammetry (SWV) determination

[0061] Place the electrode in step (3) in 1 M LiClO4 solution, and measure its redox current by square wave voltammetry (SWV). The potential scanning range is -0.1 to 0.8 V, the rising potential is 4.0 mV, the pulse amplitude is 25 mV, and the frequency is 15 Hz. Calculate the AFB1 concentration according to the current signal.

[0062] Example 3, Redox properties of CPAD and BrPhN2 + of

[0063] To verify the redox properties of CPAD and BrPhN2 + in the strategy of the present invention, in 0.1 M tetrabutylammonium perchlorate (TBAP) (freshly prepared with DMF, containing 1 mM CPAD and 1 mM BrPhN2 + respectively) solution, use a glassy carbon electrode as the working electrode for CV scanning. The results are as Figure 2 shown in A. The irreversible reduction peaks of BrPhN2 + and CPAD are approximately -0.1 V and -1.0 V respectively. Obviously, the formation of the initial radical does not cause the cleavage of CPAD. When CPAD is used as a chain transfer agent for radical polymerization, BrPhN2 + can serve as an exogenous radical source to provide the initial radical. In addition, using Au / Ap / MCH / AFB1 / Ab* as the working electrode, the redox performance of BrPhN2 + in the eRAFT polymerization solution without FcMMA was studied by CV scanning method, and the optimal polymerization potential of eRAFT was determined. The results are as Figure 2 shown in B. There is a reduction peak at -1.06 V, indicating that at this potential, the diazonium salt is reduced to the starting radical BrPh · . However, due to the action of the constant negative potential (Eapp), a lower BrPh · concentration can maintain good eRAFT polymerization efficiency. Therefore, too low a potential is not conducive to eRAFT polymerization. Similarly, when the potential is too high, a lower concentration of BrPh · is produced, reducing the eRAFT polymerization rate. Therefore, -1.06 V is selected as the Eapp for eRAFT polymerization.

[0064] Example 4, Feasibility Analysis

[0065] To verify the feasibility of the kit, SWV was used to study the response signals of different modified electrodes. The results are as Figure 3 shown in A. When Ap (curve b), AFB1 (curve c), Ab* (curve d), BrPhN2 + (curve e) and FcMMA (curve f) are absent respectively, no obvious current signal can be observed. However, an obvious current signal can be observed for the fully modified electrode (curve a). The above experimental results indicate that this method is feasible for the detection of AFB1.

[0066] The fully modified electrode was scanned using different scan rates (scan rate: 0.01 - 1 V s -1 ), and the CV curves are as Figure 3 shown in B. The current values of the oxidation peak and the reduction peak have a good linear relationship with the scan rate. This indicates that FcMMA is connected to the electrode surface through covalent bonds rather than physical adsorption. This further indicates that the electroactive monomer FcMMA has been successfully grafted onto the electrode surface by polymerization.

[0067] Example 5, Characterization

[0068] In a solution containing potassium ferricyanide and potassium ferrocyanide, electrochemical impedance spectroscopy (EIS) was used to study the impedance of different modified electrodes. The diameter of the semicircle in the Nyquist plot represents the resistance (Rct). The results are as Figure 3 shown in C. The Rct of the bare gold electrode is the smallest (∼224.03 Ω, curve a), because the bare gold electrode has the smallest charge transfer resistance and the highest electron transfer ability; when the electrode is modified with Ap, the charge transfer of the redox probe [Fe(CN)6] 3- / 4- is inhibited by the PO4 3- in Ap, resulting in an increase in Rct (∼822.23 Ω, curve b); after being modified with MCH, a denser film is formed on the electrode surface, hindering the electron transfer ability on the electrode surface, thus increasing Rct again (∼1259.7 Ω, curve c); when Ap binds to AFB1, Rct further increases (∼1484 Ω, curve d), indicating that Ap has successfully captured AFB1; after the electrode is modified with Ab*, the steric hindrance on the electrode surface increases, hindering the electron transfer, making Rct increase significantly (∼1723.8 Ω, curve e); after eRAFT polymerization, Rct decreases significantly (∼740.09 Ω, curve f), because a high-density electroactive polymer has been successfully grafted onto the electrode surface, promoting the charge transfer on the electrode surface. The above results confirm the successful construction of the kit.

[0069] In addition, the cyclic voltammetry curve (CV curve) corresponding to the kit construction process is as Figure 3 shown in D. The bare gold electrode has the largest redox current (curve a) due to its smooth surface and fast electron transfer rate. After being modified with Ap and MCH, a dense non-conductive self-assembled monolayer (SAM) is formed on the electrode surface, and the redox current decreases (curves b and c). When the electrode is modified with AFB1 and Ab*, the steric hindrance gradually increases, the charge transfer number gradually decreases, and the redox current decreases (curves d and e). After eRAFT polymerization, a high-density electroactive polymer is grafted on the electrode surface, promoting charge transfer and resulting in an enhanced redox current signal (curve f). The above results further indicate that the kit is successfully constructed.

[0070] The surface morphology of the electrode before and after eRAFT polymerization was characterized by atomic force microscopy (AFM). Figure 4 A shows the AFM image of the electrode surface modified with Ap / MCH / AFB1 / Ab*, and the surface height is 19.3 nm. Figure 4 B shows the AFM image of the electrode surface after eRAFT polymerization, and the surface height increases to 38.2 nm. The increase in surface height indicates that eRAFT polymerization is successful, and a large amount of ferrocene-based polymer is modified on the electrode surface. In addition, as Figure 4 shown in C, the hydrophilicity change of the electrode surface before and after eRAFT polymerization was characterized by water contact angle (WCA). The WCA of the electrode surface before eRAFT polymerization is 73.3°, and the WCA of the electrode surface after eRAFT polymerization drops to 62.4°. This is because electroactive monomers with hydrophilic groups are successfully modified on the electrode surface, increasing the hydrophilicity and resulting in a decrease in WCA. This result further proves the success of the polymerization reaction.

[0071] Example 6. Condition Optimization

[0072] Several important conditions designed in the kit detection were optimized to achieve the best detection performance of the AFB1 electrochemical detection kit. The effects of the reaction time of AFB1, the concentration of BrPhN2 + and the eRAFT polymerization time on the detection performance were studied.

[0073] 1. Optimization of the reaction time of AFB1

[0074] As Figure 5 shown in A, sufficient reaction time can saturate the capture amount of AFB1, so that the antibody modification amount reaches the maximum. Within 10 - 30 min, as the reaction time of AFB1 increases, the current signal gradually increases. After 30 min, the current signal gradually stabilizes, and the AFB1 modification amount reaches saturation. Therefore, 30 min is selected as the optimal reaction time of AFB1.

[0075] 2. Concentration optimization of BrPhN2 + of

[0076] Reduction of BrPhN2 + can produce very active bromophenyl. It can quickly graft onto the electrode surface to form a multi-branched bromobenzene coating. In addition, some BrPh · can be dispersed in the reaction solution to initiate the eRAFT reaction. Since the presence of the bromobenzene coating can dissipate the applied voltage, high concentrations of BrPhN2 + will hinder the eRAFT polymerization, and low concentrations of BrPhN2 + will slow down the eRAFT polymerization rate. Therefore, the concentration of BrPhN2 + needs to be optimized. The results are as Figure 5 shown in B. When the concentration of BrPhN2 + increases from 1 μM to 5 μM, the current signal gradually increases. When the concentration of BrPhN2 + continues to increase, the current signal gradually weakens. This may be due to the bromobenzene coating consuming the external voltage, resulting in a decrease in the current signal. Therefore, 5 μM is selected as the optimal concentration of BrPhN2 + .

[0077] 3. Reaction time optimization of eRAFT

[0078] As Figure 5 shown in C, as the eRAFT polymerization time increases, the number of grafted FcMMA gradually increases, and the current signal also increases accordingly. When the polymerization time reaches 2 h, the current signal tends to be stable, and the number of grafted FcMMA reaches saturation. Therefore, 2 h is selected as the ideal reaction time for eRAFT.

[0079] Example 7. Performance analysis

[0080] According to the optimal experimental conditions obtained in Example 6, different concentrations of AFB1 were detected using the present invention, and the detection performance of the kit was analyzed. The results are as Figure 6 shown in A. In the range of 2×10 -4 ~2×10 2 ng / mL, the current signal increases with the increase in the concentration of AFB1. As Figure 6 shown in B, the current signal is positively correlated with the logarithm of the AFB1 concentration. The linear regression equation is I(μA) = 0.32×lg(C AFB1 ) + 1.59 (R 2= 0.998), and its detection limit is 37.34 fg / mL (S / N = 3). The results show that the method for detecting AFB1 based on the eRAFT polymerization signal amplification strategy proposed in the present invention has high sensitivity and low detection limit, and good performance. Compared with several methods for detecting AFB1, the kit of the present invention has a wider detection range and lower LOD. The specific comparison is shown in Table 1 as follows:

[0081] Table 1 Comparison of detection ranges and detection limits between the kit of the present invention and other detection methods

[0082]

[0083] Example 8, Selectivity, Stability and Reproducibility

[0084] To verify the selectivity of this strategy, the present invention tested the current response signals of the kit to aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), AFB1 and the mixture group (Mixture) under the same experimental conditions, and at the same time set a blank group (Blank). The results are as Figure 7 shown in A. AFB1 and the mixture group have obvious current response signals, while the current signals of other groups are lower. This result indicates that due to the use of two biological probes, Ap and Ab, which have strong affinity for AFB1, the kit has good selectivity.

[0085] To study the stability of the prepared kit for detection, the successfully prepared electrode was placed at 4 °C for 14 days. The results are as Figure 7 shown in B. After the electrode was stored for 14 days, the current signal remained 97.04% of the initial value. The results show that the kit has good stability.

[0086] In addition, the reproducibility of the successfully prepared electrode was also studied. When the concentration of AFB1 was 2 ng / mL, the RSD within the group and between groups were 3.06% and 2.68% (n = 5) respectively, indicating that the proposed strategy has good reproducibility.

[0087] Example 9, Detection of Actual Samples

[0088] To verify the effectiveness of this method in the detection of actual samples, corn, malt, quisqualis fruit, semen platycladi, astragalus membranaceus, oats, chili peppers, walnuts, and almonds were selected for the spiked recovery experiment. 2.00 g of the sample was accurately weighed, crushed, and passed through a No. 3 sieve, then 10 mL of 60% (v / v) methanol solution was added, and cold extraction was carried out for 24 h. Then the sample was further ultrasonically extracted for 2 h, after which the supernatant was aspirated and centrifuged for 5 min (8000 rpm), and finally the supernatant was taken and filtered through a 0.22 μm microporous filter membrane. After the filtrate was diluted 10 times with PBS buffer, AFB1 was added to make its concentrations 0.02, 0.2, and 2 ng / mL respectively for detection. The results are shown in Table 2, the recovery rate was 95.69% - 107.65%, and the RSD was 0.84% - 4.92%. In addition, the above spiked samples were detected by high performance liquid chromatography-fluorescence detection method (HPLC-FL), and the accuracy of the detection results of this kit was confirmed. The above results indicate that the kit developed by the present invention can be used for the detection of AFB1 in complex components of food and traditional Chinese medicine.

[0089] Table 2 Results of detecting AFB1 in actual spiked samples by the kit prepared in the present invention and HPLC-FL

[0090]

Claims

1. An electrochemical detection kit for highly sensitive detection of AFB1 based on the eRAFT polymerization signal amplification strategy, characterized in that, The kit includes: a gold electrode, Aptamer, FcMMA, MCH, CPAD, C6H4BBrF4N2, Ab, DMF, KPF6, EDC, NHS; The usage method of the kit is as follows: (1) Drop the Aptamer solution onto the gold electrode and react; (2) Immerse the electrode obtained in step (1) in the MCH solution and react; (3) Drop the sample solution to be detected onto the electrode obtained in step (2) and react; (4) Drop the Ab* solution onto the electrode obtained in step (3) and react; (5) Immerse the electrode obtained in step (4) in the eRAFT polymerization solution and react; (6) Place the electrode obtained in step (5) in the LiClO4 solution to measure the electrochemical signal; The preparation method of the Ab* solution is as follows: (1) Add the CPAD solution to the EDC / NHS mixed solution and react with shaking; (2) Add the Ab solution and react with shaking to obtain the Ab* solution; The eRAFT polymerization solution is prepared by mixing the FcMMA solution, C6H4BBrF4N2 solution, KPF6 solution, and DMF.

2. The kit according to claim 1, characterized in that, It also includes PBS buffer solution, LiClO4, and ultrapure water.

3. The kit according to claim 1 or 2, characterized in that, Some raw materials are first prepared into solutions with the following concentrations: the concentration of Aptamer is 1 μM, the concentration of FcMMA is 20 mM, the concentration of MCH is 2 mM, the concentration of CPAD is 20 mM, the concentration of C6H4BBrF4N2 is 5 μM, the concentration of Ab is 800 μg / mL, the concentration of KPF6 is 0.1 M, the concentration of EDC is 20 mM, the concentration of NHS is 5 mM, the concentration of LiClO4 is 1 M, the concentration of PBS buffer solution is 0.1 M, and pH = 7.4; The sequence of Aptamer is 5′-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCTTC GCTAGGCCCACA-3′.

4. A method for detecting AFB1 using the kit according to claim 1, characterized in that, It includes the following steps: (1) Drop the Aptamer solution onto the gold electrode and react; (2) Immerse the electrode obtained in step (1) in the MCH solution and react; (3) Drop the sample solution to be detected onto the electrode obtained in step (2) and react; (4) Drop the Ab* solution onto the electrode obtained in step (3) and react; (5) Immerse the electrode obtained in step (4) in the eRAFT polymerization solution and react; (6) Place the electrode obtained in step (5) in the LiClO4 solution to measure the electrochemical signal.

5. The method according to claim 4, wherein First, pre-treat the gold electrode. The pre-treatment method is: polish the gold electrode to a mirror surface, clean it, and dry it for standby.

6. The method according to claim 4, characterized in that, The reaction condition for step (1) is to react overnight at room temperature; the reaction temperature for step (2) is 37 °C and the time is 30 min; the reaction temperature for step (3) is 37 °C and the time is 30 min; the reaction temperature for step (4) is 37 °C and the time is 1 h; the reaction temperature for step (5) is 37 °C and the time is 2 h.

7. The method according to claim 4, wherein The preparation method of the Ab* solution is as follows: (1) Add the CPAD solution to the EDC / NHS mixed solution and react with shaking; (2) Add solution Ab, and react with shaking to obtain solution Ab*. The volume ratio of CPAD solution, EDC / NHS mixed solution and solution Ab is 100:837.5:62.5; the EDC / NHS mixed solution is formed by mixing equal volumes of EDC solution and NHS solution. The reaction temperature in step (1) is 37 °C, and the reaction time is 3 h. The reaction temperature in step (2) is 37 °C, and the reaction time is 12 h.

8. The method according to claim 4, wherein The eRAFT polymerization solution is prepared by mixing FcMMA solution, C6H4BBrF4N2 solution, KPF6 solution and DMF with a volume ratio of 20:1:1600:

379.

9. The method according to claim 4, wherein The measurement of the electrochemical signal is by square wave voltammetry, with a potential scanning range of -0.1 ~ 0.8 V, a rising potential of 4.0 mV, a pulse amplitude of 25 mV, and a frequency of 15 Hz.

10. Use of the kit according to claim 1 for detecting AFB1.

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