A preparation method of an electrochemiluminescence detection AFB1 probe, a test strip for electrochemiluminescence detection of AFB1, and an AFB1 detection method

Abstract

The present invention provides a preparation method of an electrochemiluminescence (ECL) detection probe for aflatoxin B1 (AFB1), the construction of an electrochemiluminescence test strip, and an AFB1 detection method. The AFB1 detection method of the present invention uses the combination of ECL imaging and immunochromatographic test strip method to detect AFB1. This method has a low detection limit, high sensitivity and accuracy. The linear detection range of the colorimetric signal for AFB1 detection is 1 ng / mL to 100 ng / mL, the colorimetric detection limit (V LOD) is 0.103 ng / mL, the linear detection range of the ECL imaging signal is 0.1 ng / mL to 100 ng / mL, and the ECL imaging detection limit (ECL LOD) is 0.012 ng / mL. The colorimetric signal can be used to quickly predict and screen AFB1, and then the target can be accurately quantified by ECL imaging.

Description

Technical Field

[0001] The present invention relates to the technical field of aflatoxin B1 detection, and particularly to a preparation method of an electrochemiluminescence detection AFB1 probe, a test strip for electrochemiluminescence detection of AFB1, and an AFB1 detection method. Background Art

[0002] Aflatoxin B1 (AFB1) is a secondary metabolite produced by specific fungal strains such as Aspergillus flavus, which contains a bifuran ring and an o-naphthopyrone structure. In 1993, AFB1 was listed as a class I chemical carcinogen by the International Agency for Research on Cancer (IARC). It has strong carcinogenic, teratogenic, mutagenic, immunosuppressive, and liver-damaging effects on humans and animals even at extremely low levels. Therefore, it is necessary to develop highly sensitive and rapid AFB1 detection techniques. Traditional lateral flow immunoassay test strips (LFI) use colorimetric or fluorescently labeled antigens or antibodies, and rapid determination is achieved by observing the color change on the test line. This method has the advantages of portable device, simple operation, short time consumption, low cost, etc., and has been widely used in medical diagnosis and food safety risk assessment. However, for ultra-trace AFB1 monitoring, this method still has problems such as insufficient detection sensitivity and difficulty in avoiding false positives / negatives.

[0003] Currently, to improve the sensitivity of traditional test strips, various technology couplings based on test strips have been started to establish multifunctional test strips for quantitative detection of mycotoxins. Existing LFI-based coupling technologies include LFI-fluorescence method (FLFI) coupling technology, LFI-chemiluminescence method (CLLFI) coupling technology, LFI-Raman scattering sensing method (SLFI) coupling technology, LFI-electrochemistry (ELFI) coupling technology, etc. Although these coupling technologies have effectively improved the detection sensitivity to a certain extent, they still have problems such as background light interference (self-fluorescence and scattered light) or poor environmental stability.

[0004] Based on the defects existing in the current AFB1 rapid detection technology, it is necessary to improve it. Summary of the Invention

[0005] In view of this, in view of the above deficiencies in the prior art, the present invention provides a preparation method of an electrochemiluminescence detection AFB1 probe, the construction of an electrochemiluminescence imaging test strip, and an AFB1 detection method to solve or at least partially solve the defects existing in the prior art.

[0006] In the first aspect, the present invention provides a preparation method of an electrochemistry detection AFB1 probe, including the following steps:

[0007] Dissolve [Ru(dcbpy)3] 2+and PEI solution as a precursor to react and obtain Ru-PEI complex;

[0008] The Ru-PEI complex, water, ammonia water and ethanol were mixed and stirred, and then TEOS was added and stirred in the dark to react to obtain a Ru-PEI@SiO2 solution;

[0009] Add the gold-containing compound to the Ru-PEI@SiO2 solution, stir at 50-70°C, then add sodium citrate, continue stirring, centrifuge and wash, then disperse in water to obtain Ru-PEI@SiO2@Au;

[0010] Mixing Ru-PEI@SiO2@Au with AFB1 antibody to obtain a mixed solution;

[0011] The mixed solution was incubated at 20-25°C for 1.5-3h, and then the BSA solution was added and incubated for another 20-40min. The anti-AFB1-Ru-PEI@SiO2@Au conjugate was collected and resuspended in Tris-HCl to obtain an electrochemiluminescence detection probe for AFB1.

[0012] Preferably, [Ru(dcbpy)3] 2+ The solution and the EDC / NHS mixed solution need to be mixed and stirred for 20 to 40 minutes, and the preparation of the Ru-PEI complex needs to react at 2 to 6°C for 4 to 8 hours;

[0013] Preferably, [Ru(dcbpy)3] 2+ The solution concentration is 0.005-0.02M, the PEI solution concentration is 15-25mg / mL, and the EDC concentration in the EDC / NHS mixed solution is 0.001-0.003M, and the NHS concentration is 0.004-0.012M.

[0014] Preferably, the Ru-PEI complex, water, ammonia water and ethanol are mixed and stirred for 10 to 30 minutes, and after adding TEOS, the mixture is stirred and reacted for 10 to 15 hours in the dark at 20 to 25° C., and the mass concentration of ammonia water is 25 to 28%.

[0015] Preferably, the gold-containing compound includes at least one of HAuCl4, KAuCl4, and NaAuCl4. After the gold-containing compound solution is added to the Ru-PEI@SiO2 solution, it needs to be stirred at 50-70°C, the centrifugal speed is 8000-12000r / min, the mass concentration of the gold-containing compound solution is 5-15%, and the mass concentration of the sodium citrate solution is 0.5-2%.

[0016] Preferably, the concentration of AFB1 antibody in the Ru-PEI@SiO2@Au and AFB1 antibody mixed solution is 0.05 - 0.2 mg / mL, the mass concentration of the BSA solution is 5 - 20%, and the concentration of Tris-HCl used for resuspending the anti-AFB1-Ru-PEI@SiO2@Au conjugate in Tris-HCl is 0.05 - 0.2 M, with a volume of 1 - 3 mL.

[0017] In a second aspect, the present invention also provides a test strip for electrochemiluminescence detection of AFB1, comprising:

[0018] A substrate, on the surface of which a working electrode, a reference electrode, and a counter electrode are provided;

[0019] An NC membrane, on the surface of which AFB1-BSA and goat anti-mouse IgG are respectively sprayed to form a test line and a quality control line;

[0020] The surface of the NC membrane facing away from the test line is attached to the surface of the substrate where the working electrode is provided, and the test line is disposed opposite to the working electrode;

[0021] A sample pad, one side of which is attached to the NC membrane and the other side is attached to the substrate;

[0022] An absorption pad, one side of which is attached to the NC membrane and the other side is attached to the substrate;

[0023] The sample pad and the absorption pad are respectively located on both sides of the NC membrane;

[0024] A PDMS solution pool, which is attached to the NC membrane, and through holes are opened on the PDMS solution pool corresponding to the working electrode, the reference electrode, and the counter electrode of the substrate to add PBS buffer solution into the PDMS solution pool.

[0025] Preferably, the materials of the working electrode, the reference electrode, and the counter electrode on the substrate of the test strip for electrochemiluminescence detection of AFB1 are all conductive carbon ink.

[0026] In a third aspect, the present invention also provides a method for electrochemiluminescence detection of AFB1, comprising the following steps:

[0027] Step 1: Mix the solution to be tested with the prepared electrochemiluminescence detection AFB1 probe to obtain a sample to be tested, and drop it on the sample pad of the test strip for electrochemiluminescence detection of AFB1, and qualitatively detect AFB1 in the sample to be tested by the color change of the test line;

[0028] Step 2: Add PBS buffer to the test line area of the NC membrane of the electrochemiluminescence detection AFB1 test strip. Connect the working electrode, reference electrode, and counter electrode to the electrochemical workstation, and quantitatively detect AFB1 in the test sample added in Step 1 through the electrochemiluminescence imaging signal intensity.

[0029] Preferably, the preparation method of the sample pad is as follows: Immerse the sample pad in the buffer solution for 20 - 40 min, and then dry it to obtain the sample pad. The buffer solution includes Tris-HCl, TritonX-100, and NaCl. The concentration of Tris-HCl in the buffer solution is 0.01 - 0.03 M, the mass concentration of TritonX-100 is 0.1 - 0.4%, and the concentration of NaCl is 0.1 - 0.3 M.

[0030] The preparation method of the electrochemiluminescence detection AFB1 probe, the construction of the electrochemiluminescence imaging test strip, and the AFB1 detection method of the present invention have the following technical effects compared with the prior art:

[0031] 1. For the preparation method of the electrochemiluminescence detection AFB1 probe of the present invention, the Ru-PEI complex is synthesized by using an amide reaction. Specifically, mix the aqueous solution of [Ru(dcbpy)3] 2+ with the aqueous solution of PEI and stir to finally obtain the Ru-PEI complex; then, synthesize Ru-PEI@SiO2 by hydrolysis method, and in-situ reduce HAuCl4 on its surface to obtain Ru-PEI@SiO2@Au; add the AFB1 antibody to Ru-PEI@SiO2@Au and stir to obtain the anti-AFB-Ru-PEI@SiO2@Au conjugate, and block the excess sites with BSA to obtain the AFB1-responsive dual-signal probe for the detection of AFB1.

[0032] 2. For the electrochemiluminescence imaging test strip of the present invention, a test strip integrated with the electrode is constructed. By outputting two visual signals of electrochemiluminescence imaging and colorimetry, the mutual verification between the two signals of the test strip of the present invention is realized, ensuring the accuracy of the signal. At the same time, due to the advantages of low background signal, high sensitivity, and controllable potential of electrochemiluminescence, the electrochemiluminescence imaging signal is used to improve the sensitivity of the traditional lateral flow chromatography test strip.

[0033] 3. The AFB1 detection method of the present invention combines electrochemiluminescence imaging with immunochromatographic strip method to detect aflatoxin B1. This method has a low detection limit, high sensitivity and accuracy. The linear detection range of the colorimetric signal for aflatoxin B1 detection is 1 ng / mL to 100 ng / mL, the colorimetric detection limit (VLOD) is 0.103 ng / mL, the linear detection range of the ECL imaging signal is 0.1 ng / mL to 100 ng / mL, and the ECL imaging detection limit (ECL LOD) is 0.012 ng / mL. The colorimetric signal can be used to quickly predict and screen AFB1, and then the target can be accurately quantified by ECL imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0035] Figure 1 Schematic diagram of the preparation method principle of the electrochemiluminescence detection AFB1 probe of the present invention;

[0036] Figure 2 Schematic diagram of the structure of the substrate of the present invention;

[0037] Figure 3 Schematic diagram of the connection of the NC membrane, sample pad, and absorption pad of the present invention;

[0038] Figure 4 Schematic diagram of the structure of the PDMS solution pool of the present invention;

[0039] Figure 5 Schematic diagram of the structure of the immunochromatographic strip for electrochemiluminescence detection of AFB1 of the present invention;

[0040] Figure 6 Schematic diagram of the preparation method of the immunochromatographic strip for electrochemiluminescence detection of AFB1 of the present invention;

[0041] Figure 7 Linear relationship diagram of colorimetric signal and different concentrations of aflatoxin B1 (AFB1);

[0042] Figure 8 Linear relationship diagram of ECL imaging intensity and different concentrations of aflatoxin B1 (AFB1). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0044] It should be noted that the description order of the following embodiments does not limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". The various embodiments of the present invention may exist in a range form; it should be understood that the description in a range form is only for convenience and simplicity, and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the counted range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0045] The embodiment of the present application provides a preparation method of an electrochemiluminescence detection AFB1 probe, including the following steps:

[0046] S1. Mix a [Ru(dcbpy)3] 2+ solution with an EDC / NHS mixed solution, stir, add a PEI solution, and react to obtain a Ru-PEI complex;

[0047] S2. Mix the Ru-PEI complex, water, ammonia water, and ethanol, stir, and then add TEOS, and stir and react in the dark to obtain a Ru-PEI@SiO2 solution;

[0048] S3. Add a gold-containing compound to the Ru-PEI@SiO2 solution, stir at 50-70 °C, then add sodium citrate, continue to stir, centrifugally wash and disperse in water to obtain Ru-PEI@SiO2@Au;

[0049] S4. Mix Ru-PEI@SiO2@Au with an AFB1 antibody to obtain a mixed solution;

[0050] S5. Incubate the mixed solution at 20 - 25 °C for 1.5 - 3 h, then add the BSA solution and continue to incubate for 20 - 40 min. Collect the anti-AFB1-Ru-PEI@SiO2@Au conjugate and resuspend it in Tris-HCl to obtain the electrochemiluminescence detection probe for AFB1.

[0051] The preparation method of the probe of the present invention synthesizes the Ru-PEI complex by using an amide reaction. Specifically, mix and stir the aqueous solution of [Ru(dcbpy)3] 2+ with the aqueous solution of PEI, and finally obtain the Ru-PEI complex; then, synthesize Ru-PEI@SiO2 by hydrolysis method, and in-situ reduce HAuCl4 on its surface to obtain Ru-PEI@SiO2@Au; add the AFB1 antibody to Ru-PEI@SiO2@Au and stir to obtain the anti-AFB-Ru-PEI@SiO2@Au conjugate, and block the excess sites with BSA; obtain the AFB1-responsive dual-signal probe for the detection of AFB1.

[0052] Specifically, the solution of [Ru(dcbpy)3] 2+ is the aqueous solution of tris(4,4'-dicarboxy-2,2'-bipyridine) ruthenium chloride, and the chemical formula of [Ru(dcbpy)3] 2+ is as follows:

[0053]

[0054] The EDC / NHS mixed solution is the mixed solution of the aqueous solution of EDC (EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and the aqueous solution of NHS (N-hydroxysuccinimide);

[0055] The PEI solution is the aqueous solution of PEI. Specifically, PEI is polyethyleneimine, and its chemical formula is as follows:

[0056]

[0057] In some embodiments, mix the solution of [Ru(dcbpy)3] 2+ with the EDC / NHS mixed solution and stir for 20 - 40 min to activate the carboxyl group of [Ru(dcbpy)3] 2+ , add the PEI solution, and react at 2 - 6 °C for 4 - 8 h to obtain the Ru-PEI complex;

[0058] Among them, [Ru(dcbpy)3] 2+The solution concentration is 0.005 - 0.02 M (i.e., mol / L), and the PEI solution concentration is 15 - 25 mg / mL. Specifically, the preparation method of the EDC / NHS mixed solution is to mix the EDC aqueous solution and the NHS aqueous solution. Among them, the volume ratio of the EDC aqueous solution to the NHS aqueous solution is 1:4, the concentration of the EDC aqueous solution is 0.01 M, and the concentration of the NHS aqueous solution is 0.01 M. The concentration of EDC in the obtained EDC / NHS mixed solution is 0.001 - 0.003 M, and the concentration of NHS is 0.004 - 0.012 M.

[0059] In some embodiments, Ru-PEI complex, water, ammonia water, and ethanol are mixed and stirred for 10 - 30 min, then TEOS is added, and the mixture is stirred and reacted at 20 - 25 °C in the dark for 10 - 15 h. The product obtained from the reaction is centrifuged and dispersed in water to obtain the Ru-PEI@SiO2 solution, which is stored at 4 °C;

[0060] Among them, the mass concentration of ammonia water is 25 - 28%;

[0061] Specifically, TEOS is tetraethyl orthosilicate, also known as tetraethyl silicate, which is an organic compound with the chemical formula C8H 20 O4Si.

[0062] In some embodiments, the gold-containing compound includes at least one of HAuCl4·3H2O, HAuCl4, KAuCl4, and NaAuCl4, and preferably HAuCl4.

[0063] In some embodiments, the HAuCl4 solution is added to the Ru-PEI@SiO2 solution, and the mixture is stirred at 50 - 70 °C, then the sodium citrate solution is added, and stirring is continued. After centrifugal washing at a rate of 8000 - 12000 r / min, it is dispersed in water to obtain Ru-PEI@SiO2@Au, which is stored at 4 °C;

[0064] Among them, the mass concentration of the HAuCl4 solution is 5 - 15%, and the mass concentration of the sodium citrate solution is 0.5 - 2%.

[0065] The HAuCl4 solution specifically refers to the HAuCl4 aqueous solution, and the sodium citrate solution specifically refers to the sodium citrate aqueous solution.

[0066] In some embodiments, Ru-PEI@SiO2@Au is mixed with the AFB1 antibody to obtain a mixed solution; among them, the concentration of the AFB1 antibody in the mixed solution is 0.05 - 0.2 mg / mL.

[0067] In some embodiments, the mass concentration of the BSA solution is 5 - 20%. Specifically, the BSA solution is the BSA aqueous solution.

[0068] In some embodiments, the anti-AFB1-Ru-PEI@SiO2@Au conjugate is resuspended in 1 - 3 mL of Tris-HCl to obtain an electrochemical AFB1 detection probe; wherein the concentration of Tris-HCl is 0.05 - 0.2 M.

[0069] Specifically, BSA is bovine serum albumin.

[0070] In some embodiments, Ru-PEI@SiO2@Au is mixed with an AFB1 antibody to obtain a mixed solution; the mixed solution is incubated at 20 - 25 °C for 1.5 - 3 h, then a BSA solution is added to block the excess binding sites, and incubation is continued for 20 - 40 min. The anti-AFB1-Ru-PEI@SiO2@Au conjugate is collected, resuspended in Tris-HCl, and stored at 4 °C for later use to obtain an electrochemiluminescence AFB1 detection probe.

[0071] Further, as shown in Figure 1 which shows a schematic diagram of the preparation method of the electrochemical AFB1 detection probe of the present invention.

[0072] Based on the same inventive concept, the present invention also provides a test strip for electrochemiluminescence detection of AFB1, as shown in Figures 2 to 5 which includes:

[0073] A substrate 1, on the surface of which a working electrode 11, a reference electrode 12, and a counter electrode 13 are provided;

[0074] An NC membrane (i.e., nitrocellulose membrane) 2, on the surface of which AFB1-BSA and goat anti-mouse IgG are respectively sprayed to form a test line 21 and a quality control line 22;

[0075] The surface of the NC membrane 2 facing away from the test line is attached to the surface of the substrate 1 provided with the working electrode 11, and the test line 21 is disposed opposite to the working electrode 11;

[0076] A sample pad 3, one side of which is attached to the NC membrane 2 and the other side is attached to the substrate 1;

[0077] Among them, the solution to be measured is mixed with the electrochemiluminescence AFB1 detection probe prepared by the above preparation method to obtain a sample to be measured;

[0078] The sample to be measured is dropped onto the sample pad 3, and qualitative detection of AFB1 in the sample to be measured is achieved by the color change of the test line;

[0079] A PBS buffer solution is dropped onto the NC membrane 2 corresponding to the test line;

[0080] The working electrode, the reference electrode, and the counter electrode are connected to an electrochemical workstation;

[0081] The electrochemiluminescence imaging signal intensity is used to quantitatively detect AFB1 in the sample to be tested.

[0082] Specifically, during the test, first, it is determined whether AFB1 is present in the sample to be tested by the color change of the test line; when there is no AFB1 in the sample to be tested, when the sample to be tested flows through the test strip, the signal probe will bind to AFB1-BSA modified on the T line, forming an obvious red band; when AFB1 is present in the sample to be tested, the specific AFB1 antibody on the signal probe will bind to AFB1, resulting in fewer binding sites of AFB1-BSA on the T line when the test solution flows through the T line. As the concentration of AFB1 in the sample to be tested increases, the color on the T line gradually fades; if it is determined that AFB1 is present in the sample to be tested by the color change of the test line, at this time, PBS buffer solution is dropped onto the corresponding test line on the NC membrane 2, and the three electrodes are connected to the electrochemical workstation, and the electrochemiluminescence imaging signal intensity is used to quantitatively detect AFB1 in the sample to be tested.

[0083] In some embodiments, it further includes:

[0084] An absorbent pad 4, one side of which is attached to the NC membrane 2 and the other side is attached to the substrate 1; the sample pad 3 and the absorbent pad 4 are respectively located on both sides of the NC membrane 2;

[0085] A PDMS solution pool 5, which is attached to the NC membrane 2, and through holes 51 are opened on the PDMS solution pool 5 corresponding to the working electrode, reference electrode and counter electrode of the substrate 1 to add PBS buffer solution into the PDMS solution pool.

[0086] In some embodiments, the NC membrane 2 is a non-backing NC membrane. Using a non-backing NC membrane facilitates the permeation of the solution through the NC membrane to contact the electrode, enabling the electrochemiluminescence reaction to occur.

[0087] In some embodiments, the preparation method of the sample pad 3 is: soaking the sample pad in a buffer solution for 20 - 40 min, and then drying to obtain the sample pad;

[0088] The buffer solution includes Tris-HCl, TritonX-100 and NaCl. Among them, the concentration of Tris-HCl in the buffer solution is 0.01 - 0.03 M, the mass concentration of TritonX-100 is 0.1 - 0.4%, and the concentration of NaCl is 0.1 - 0.3 M.

[0089] In some embodiments, the materials of the working electrode, reference electrode and counter electrode are all conductive carbon ink.

[0090] In some embodiments, refer to Figure 6 , the preparation method of the test strip for electrochemiluminescence detection of AFB1 according to the present invention includes the following steps:

[0091] S1. The substrate 1 is a PVC substrate. Design the pattern 10 of three electrodes (i.e., working electrode 11, reference electrode 12, and counter electrode 13) using drawing software, then engrave the hollow self-adhesive pattern 10 with a cutting plotter and paste it on the PVC substrate. Then evenly apply 30 μL of conductive carbon ink and dry it at 50 °C for 20 min. Finally, peel off the self-adhesive pattern, and the working electrode 11, reference electrode 12, and counter electrode 13 can be formed on the PVC substrate and stored at room temperature for standby.

[0092] S2. Preparation of the immunochromatographic strip: The sample pad is soaked in the buffer solution for 30 min and then dried naturally. Then the sample pad and the absorbent pad are cut into lengths and widths of (2.3 cm × 0.5 cm) and (1.7 cm × 0.5 cm) respectively. Spray 60 μL of AFB1-BSA and 60 μL of goat anti-mouse IgG on the NC membrane 2 at a speed of 2 μL / cm to form a test line 21 (T line) and a quality control line 22 (C line), and dry it at 37 °C for 2 h. Cut the prepared NC membrane into a width of 5 mm. The NC membrane 2 is a non-backing NC membrane to enable the reagent solution to contact the electrode and achieve ECL measurement.

[0093] S3. Device assembly: Paste the NC membrane 2 on the PVC substrate 1, align the test line 21 (T line) vertically with the working electrode 11 on the PVC substrate 1, and then paste the absorbent pad 4 and the sample pad 3 on the PVC substrate respectively, overlapping by about 2 mm to ensure good migration of the sample solution. Finally, cover the PDMS solution pool 5 above the working electrode to complete the manufacture of the integrated electrochemiluminescence imaging test strip.

[0094] Specifically, AFB1-BSA is bovine serum albumin conjugated with aflatoxin B1. When in use, dilute AFB1-BSA with PBS solution to prepare a solution with a concentration of 1 mg / mL; when in use, dilute goat anti-mouse IgG with PBS solution to prepare a solution with a concentration of 1 mg / mL.

[0095] The detection principle of the present invention is as follows: The anti-AFB-Ru-PEI@SiO2@Au signal probe has AFB1 specificity. When there is no AFB1 in the test solution, as the test solution flows through the test strip, the signal probe will bind to AFB1-BSA modified on the T line, forming an obvious red band, and the ECL signal intensity is also high after applying the corresponding voltage; when there is AFB1 in the test solution, the specific AFB1 antibody on the signal probe will bind to AFB1, resulting in fewer binding sites of AFB1-BSA on the T line when the test solution flows through the T line. As the concentration of AFB1 in the test solution increases, the color on the T line gradually fades, and the ECL signal intensity will also weaken accordingly. In addition, regardless of whether there is a target AFB1 in the test solution, the signal probe will bind to non-specific goat anti-mouse IgG on the C line, showing a red band. Qualitative detection of AFB1 can be carried out by observing the color depth of the T line first, and then quantitative detection of AFB1 can be achieved by using the ECL intensity. The test strip for detecting AFB1 by electrochemiluminescence imaging of the present invention can achieve rapid detection of AFB1. Ru-PEI@SiO2@Au is used as a signal probe to provide electrochemiluminescence signals and colorimetric signals. This method can achieve rapid screening, pre-judgment and accurate quantification of aflatoxin B1.

[0096] Based on the same inventive concept, the present invention also provides a method for integrated detection of AFB1, comprising the following steps:

[0097] S1. Provide the test strip for electrochemiluminescence detection of AFB1 as described above;

[0098] S2. Mix the test solution with the electrochemiluminescence detection AFB1 probe prepared by the preparation method as described above to obtain a test sample;

[0099] S3. Drop the test sample onto the sample pad, and perform qualitative detection of AFB1 in the test sample by observing the color change of the test line;

[0100] S4. Drop PBS buffer onto the corresponding test line on the NC membrane;

[0101] S5. Connect the working electrode, reference electrode and counter electrode to an electrochemistry workstation;

[0102] S6. Perform quantitative detection of AFB1 in the test sample by the electrochemiluminescence imaging signal intensity.

[0103] Electrochemiluminescence (ECL) has shown superior performance in the highly sensitive detection of mycotoxins due to its significant advantages of low background signal, high sensitivity, wide linear range, good stability, and easy control. The integration of ECL and LFI in this invention can combine the inherent high sensitivity of ECL with the simplicity and rapidity of LFI to construct an integrated ECL imaging test strip. On the one hand, it is expected to overcome matrix interference and reduce the background signal. On the other hand, it can further improve the sensing sensitivity. The invention integrates a three-electrode system (this three-electrode system includes a working electrode, a reference electrode, and a counter electrode, and the three electrodes in this system are all manufactured by curing conductive carbon ink on a PVC bottom plate using screen printing technology) between the NC membrane and the PVC substrate of the test strip, integrating the three-electrode system and the lateral flow immunoassay test strip into one device. This sensor utilizes the feedback of two different signals, improving the accuracy and reliability of detection and having broad application prospects. The above method shows a wide linear range and low sensitivity for the detection of AFB1, confirming its potential application prospects in the field of rapid food safety testing and providing a feasible detection idea for the on-site detection of AFB1.

[0104] Specifically, the detection process includes: mixing AFB1 standard solutions with different concentrations with the anti-AFB1-Ru-PEI@SiO2@Au signal probe to prepare test solutions with different AFB1 concentrations, and dropping 100 μL of the above test solutions onto the sample pad. Due to capillary action, the test solutions migrate from the sample pad towards the absorbent pad. Red bands (T line, C line) can be visually observed on the NC membrane within 15 minutes. Qualitative judgment can be first made by observing the color depth of the T line band. Subsequently, connect the three-electrode system to the electrochemical workstation, add 10 μL of PBS buffer solution to the PBS buffer solution addition hole on the PDMS pad, and apply a constant potential of 1.25 V to the three-electrode system. 2+ [Ru(dcbpy)3] is oxidized to generate [Ru(dcbpy)3] on the surface of the working electrode. 3+ , and PEI is oxidized to generate PEI. +· , and then it quickly deprotonates to form PEI. · , and reacts with [Ru(dcbpy)3]. 3+ to undergo an oxidation-reduction reaction to generate the excited state [Ru(dcbpy)3]. 2+* species, which generates ECL during the process of returning to the ground state. After collecting the ECL image and performing intensity analysis, a linear standard curve is obtained by fitting, which can be used for regression analysis of the quantitative judgment of AFB1 in the sample to achieve qualitative and quantitative analysis of the sample.

[0105] The following further illustrates the electrochemiluminescence detection AFB1 probe, the preparation method of the electrochemiluminescence imaging test strip, and the AFB1 detection method of the present application with specific examples. This part further illustrates the content of the present invention in combination with specific examples, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0106] In the following examples, the AFB1 antibody was purchased from Sigma-Aldrich (MO, USA); AFB1-BSA was purchased from Sigma-Aldrich (MO, USA)

[0107] PEI was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China);

[0108] BSA was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China);

[0109] Goat anti-mouse IgG was purchased from Sigma-Aldrich (MO, USA);

[0110] The NC membrane is a backless NC membrane and was purchased from Whatman (UK)

[0111] Example 1

[0112] This example provides a preparation method of an electrochemiluminescence detection AFB1 probe, including the following steps:

[0113] S1. Mix 0.5 mL of 0.01 M 2+ aqueous solution of [Ru(dcbpy)3] with the EDC / NHS mixed solution, stir for 30 min, add 2 mL of 20 mg / mL aqueous solution of PEI, and react at 4 °C for 6 h to finally obtain the Ru-PEI complex; the EDC / NHS mixed solution is obtained by mixing 0.1 mL of 0.01 M aqueous solution of EDC and 0.4 mL of 0.01 M aqueous solution of NHS;

[0114] S2. Mix 1 mL of the Ru-PEI complex, 1 mL of ultrapure water, 1.6 mL of ammonia water (mass concentration of 25%), and 50 mL of ethanol, stir for 20 min, then add 1.5 mL of TEOS, stir and react at 25 °C in the dark for 12 h. After the reaction is completed, centrifuge the reaction product and disperse it in 2 mL of ultrapure water to obtain the Ru-PEI@SiO2 solution;

[0115] S3. Add 10 μL of an aqueous solution of HAuCl4 with a mass concentration of 10% to 2 mL of the Ru-PEI@SiO2 solution, stir at 60 °C, then add 100 μL of an aqueous solution of sodium citrate with a mass concentration of 1%, continue stirring for 2 min, centrifuge and wash at 10000 r / min, and disperse in 2 mL of ultrapure water to obtain Ru-PEI@SiO2@Au;

[0116] S4. Mix the Ru-PEI@SiO2@Au in S3 with the AFB1 antibody to obtain a mixed solution with an AFB1 antibody concentration of 0.1 mg / mL;

[0117] S5. Incubate the mixed solution at 25 °C for 2 h, then add 100 μL of an aqueous solution of BSA with a mass concentration of 10%, continue incubating for 30 min, collect the anti-AFB1-Ru-PEI@SiO2@Au conjugate, resuspend it in 1 mL of 0.1 M Tris-HCl (pH 7.5) to obtain the electrochemical detection AFB1 probe, and store it at 4 °C for later use.

[0118] Example 2

[0119] This example provides a test strip for electrochemiluminescence detection of AFB1, as Figure 6 shown, including:

[0120] A substrate 1, on the surface of which a working electrode 11, a reference electrode 12, and a counter electrode 13 are provided;

[0121] An NC membrane 2, on the surface of which AFB1-BSA and goat anti-mouse IgG are respectively sprayed to form a test line 21 and a quality control line 22;

[0122] The surface of the NC membrane 2 facing away from the test line is attached to the surface of the substrate 1 provided with the working electrode 11, and the test line 21 is disposed opposite to the working electrode 11;

[0123] A sample pad 3, one side of which is attached to the NC membrane 2 and the other side is attached to the substrate 1;

[0124] An absorption pad 4, one side of which is attached to the NC membrane 2 and the other side is attached to the substrate 1; the sample pad 3 and the absorption pad 4 are respectively located on both sides of the NC membrane 2;

[0125] A PDMS solution pool 5, which is attached to the NC membrane 2, and through holes are opened in the PDMS solution pool 5 corresponding to the working electrode, reference electrode, and counter electrode of the substrate 1 to add PBS buffer solution into the PDMS solution pool.

[0126] The preparation method of the above-mentioned test strip for electrochemiluminescence detection of AFB1 includes the following steps:

[0127] S1. The substrate 1 is a PVC substrate. Design a three - electrode pattern 10 (i.e., working electrode 11, reference electrode 12, and counter electrode 13) using CorelDRAW software. Then, engrave a hollowed - out self - adhesive pattern 10 with a cutting plotter and paste it on the PVC substrate. Then, evenly coat 30 μL of conductive carbon ink and dry it at 50 °C for 20 min. Finally, peel off the self - adhesive pattern, and the working electrode 11, reference electrode 12, and counter electrode 13 can be formed on the PVC substrate, and store it at room temperature for standby;

[0128] S2. Preparation of the immunochromatographic strip: The sample pad is soaked in a buffer solution (the buffer solution includes Tris - HCl, TritonX - 100, and NaCl, where the concentration of Tris - HCl in the buffer solution is 0.02 M, the mass concentration of TritonX - 100 is 0.25%, and the concentration of NaCl is 0.15 M) for 30 min and then dried naturally. Then, cut the sample pad and the absorbent pad into lengths and widths of (2.3 cm×0.5 cm) and (1.7 cm×0.5 cm) respectively. Spray 60 μL of AFB1 - BSA (prepared into a solution with a concentration of 1 mg / mL after dilution with PBS solution) and 60 μL of goat anti - mouse IgG (prepared into a solution with a concentration of 1 mg / mL after dilution with PBS solution) on the NC membrane 2 at a speed of 2 μL / cm to form a test line 21 (T - line) and a quality control line (C - line) 22, and dry it at 37 °C for 2 h. The NC membrane is used to enable the reagent solution to contact the electrode to achieve ECL measurement. Cut the prepared NC membrane into a width of 5 mm.

[0129] S3. Device assembly: Paste the NC membrane 2 on the PVC substrate 1, align the test line 21 (T - line) vertically with the working electrode 11 on the PVC substrate 1, then paste the absorbent pad 4 and the sample pad 3 on the PVC substrate respectively, with an overlap of about 2 mm to ensure good migration of the sample solution. Finally, cover a PDMS pad 5 above the working electrode to complete the manufacture of the integrated electrochemiluminescence imaging test strip.

[0130] Example 3

[0131] This example provides a method for detecting AFB1 by electrochemiluminescence, including the following steps:

[0132] Mix the test solution with the electrochemiluminescence detection AFB1 probe prepared in Example 1 to obtain a test sample;

[0133] Drop 100 μL of the test sample onto the sample pad; Drop the test sample onto the sample pad, and qualitatively detect AFB1 in the test sample by the color change of the test line;

[0134] Add 10 μL of PBS buffer solution (pH = 7.4) to the through - hole of the PDMS solution pool;

[0135] Connect the working electrode, reference electrode and counter electrode to the electrochemical workstation, and apply a constant voltage of 1.25 V to the three-electrode system;

[0136] Quantitatively detect AFB1 in the sample to be tested by the signal intensity of electrochemiluminescence imaging.

[0137] Specifically, mix the solution to be tested with known concentration with the AFB1 probe for electrochemical detection in Example 1 to obtain the sample to be tested; the concentrations of AFB1 in the sample to be tested are 0 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL respectively.

[0138] Figure 7 It is a linear relationship diagram of the colorimetric signal and aflatoxin B1 (AFB1) with different concentrations;

[0139] Figure 8 It is a linear relationship diagram of the ECL intensity and aflatoxin B1 (AFB1) with different concentrations.

[0140] From Figures 7 to 8 In the linear relationship diagram of the ECL signal / colorimetric signal and the target aflatoxin B1, based on the detected ECL signal / colorimetric signal, the concentration of aflatoxin B1 (AFB1) can be calculated.

[0141] Specifically, from Figure 7 it can be seen that when the test line (T line) is lighter in color from left to right, it indicates that the concentration of AFB1 in the sample to be tested is higher; from Figure 8 it can be seen that the higher the signal intensity of electrochemiluminescence imaging (the darker the color from left), the higher the concentration of AFB1 in the sample to be tested.

[0142] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An integrated AFB1 detection method, characterized in that, Including the following steps: Step 1: Mix the solution to be tested with the electrochemiluminescence detection AFB1 probe to obtain a sample to be tested, and drop it on the sample pad of the electrochemiluminescence detection AFB1 test strip. Qualitatively detect AFB1 in the sample to be tested by the color change of the test line; Step 2: Drop PBS buffer on the corresponding test line area of the NC membrane of the electrochemiluminescence detection AFB1 test strip. Connect the working electrode, reference electrode and counter electrode to the electrochemistry workstation, and realize the quantitative detection of AFB1 in the sample to be tested dropped in Step 1 through the electrochemiluminescence imaging signal intensity; The preparation method of the electrochemiluminescence detection AFB1 probe includes the following steps: Step 1: Use [Ru(dcbpy)3] 2+ and the PEI solution as precursors to react and obtain the Ru-PEI complex; Step 2: Mix and stir Ru-PEI complex, water, ammonia water, and ethanol, and then add TEOS for reaction to obtain Ru-PEI@SiO2 solution; Step 3: Add the gold-containing compound to the Ru-PEI@SiO2 solution, add sodium citrate for reduction, fully react, centrifuge, wash and separate, and then disperse it in water to obtain Ru-PEI@SiO2@Au; Step 4: Mix Ru-PEI@SiO2@Au with the AFB1 antibody to obtain a mixed solution. After incubation, add BSA solution and continue to incubate for a period of time to obtain the anti-AFB1-Ru-PEI@SiO2@Au conjugate; resuspend it in Tris-HCl to obtain the electrochemiluminescence detection AFB1 probe; The preparation method of the Ru-PEI complex in Step 1 includes: mixing 0.5 mL of 0.01 M [Ru(dcbpy)3] 2+ aqueous solution with an EDC / NHS mixed solution, stirring for 30 min, adding 2 mL of 20 mg mL –1 PEI aqueous solution, reacting at 4 °C for 6 h to finally obtain the Ru-PEI complex; the EDC / NHS mixed solution is obtained by mixing 0.1 mL of 0.01 M EDC aqueous solution and 0.4 mL of 0.01 M NHS aqueous solution; In Step 2, 1 mL of Ru-PEI complex, 1 mL of ultrapure water, 1.6 mL of ammonia water, and 50 mL of ethanol are mixed and stirred for 20 min, then 1.5 mL of TEOS is added, and the reaction is stirred at 25 °C in the dark for 12 h. After the reaction is completed, the reaction product is centrifuged and dispersed in 2 mL of ultrapure water to obtain Ru-PEI@SiO2 solution; Among them, the mass concentration of the ammonia water is 25%; In Step 3, add 10 μL of 10% HAuCl4 aqueous solution to 2 mL of Ru-PEI@SiO2 solution, stir at 60 °C, then add 100 μL of 1% sodium citrate aqueous solution, continue to stir for 2 min, centrifuge and wash at 10000 r / min, and disperse in 2 mL of ultrapure water to obtain Ru-PEI@SiO2@Au; In Step 4, mix Ru-PEI@SiO2@Au in S3 with the AFB1 antibody to obtain a mixed solution with an AFB1 antibody concentration of 0.1 mg / mL; incubate the mixed solution at 25 °C for 2 h, then add 100 μL of 10% BSA aqueous solution, continue to incubate for 30 min, collect the anti-AFB1-Ru-PEI@SiO2@Au conjugate, resuspend it in 1 mL of 0.1 M Tris-HCl with a pH of 7.5 to obtain the electrochemiluminescence detection AFB1 probe, and store it at 4 °C for standby.

2. The integrated AFB1 detection method according to claim 1, wherein, The electrochemiluminescence detection AFB1 test strip includes: A substrate, on the surface of which a working electrode, a reference electrode and a counter electrode are provided; NC membrane, on the surface of which AFB1-BSA and goat anti-mouse IgG are respectively sprayed to form a test line and a quality control line; The surface of the NC membrane facing away from the test line is attached to the surface of the substrate where the working electrode is provided, and the test line is arranged opposite to the working electrode; Sample pad, one side of which is attached to the NC membrane and the other side is attached to the substrate; Absorbent pad, one side of which is attached to the NC membrane and the other side is attached to the substrate; The sample pad and the absorbent pad are respectively located on both sides of the NC membrane; PDMS solution pool, which is attached to the NC membrane, and through holes are opened on the PDMS solution pool corresponding to the working electrode, reference electrode and counter electrode of the substrate to add PBS buffer solution into the PDMS solution pool.

3. The integrated AFB1 detection method according to claim 2, wherein The materials of the working electrode, reference electrode and counter electrode are all conductive carbon ink.

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