CN114994155A - Electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts as well as preparation method and application of electrochemical immunosensor - Google Patents
Electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts as well as preparation method and application of electrochemical immunosensor Download PDFInfo
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Abstract
The invention belongs to the field of sensors, relates to an electrochemical immunosensor, and particularly relates to an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts, and a preparation method and application thereof, wherein the electrochemical immunosensor comprises the following steps: 1) preparing a composite material: obtaining a composite material capable of amplifying a sensor signal by bonding Zn/Ni-ZIF-8-800, graphene and chitosan; 2) modification of sensor surface function: and (3) dripping a composite material on the surface of the electrode, depositing gold nanoparticles at a constant potential, and adding an AFB1 antibody to obtain the modified electrochemical immunosensor for enhancing the signal. The sensitivity of the sensor is improved through the composite material, the AFB1 is used as an antigen, and the sensor has high-efficiency selectivity by introducing an AFB1 antibody antigen recognition method; meanwhile, the composite material is combined with a sensor to improve the adsorption capacity of AFB1 antibodies, improve the adsorption process of antigens and increase the detection efficiency, and the detection limit is 0.18 ng/mL.
Description
Technical Field
The invention belongs to the field of mycotoxin detection of an electrochemical immunosensor, relates to preparation of the electrochemical immunosensor, and particularly relates to an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts as well as a preparation method and application of the electrochemical immunosensor.
Background
Aflatoxin B1 (AFB 1) is a mycotoxin which is extremely harmful to human health and has the highest toxicity, is a polypeptide compound produced by various aspergillus, is mainly produced by aspergillus flavus and aspergillus parasiticus, and can stably exist at the temperature of more than 100 ℃. Widely produced in food products such as cereals and their by-products, spices, nuts, milk and dried fruits. According to the data of Food and Agricultural Organization (FAO) of the United nations, about 25% of agricultural products are polluted to different degrees every year worldwide, and huge economic losses are caused to agriculture. Peanut oil is a food material which is basic in life, and AFB1 is very easy to grow due to the abnormality of climate, storage environment and the like in the processes of peanut production, harvesting, storage and oil refining. And the peanut oil in the peanuts is polluted by AFB1 because the thermal stability is strong and the structure is not easy to damage.
The existing detection technology aiming at AFB1 mainly comprises an enzyme-linked immunosorbent assay, a test strip method, a gas chromatography-mass spectrometry combined method, a liquid chromatography and the like. However, the methods have limitations of the methods, wherein the enzyme-linked immunosorbent assay and the test strip assay have high sensitivity and good specificity, but are easy to cause false positive results; the gas chromatography-mass spectrometry combined method and the liquid chromatography usually need complicated sample pretreatment and expensive detection instruments, have higher requirements on the quality of operators and experimental conditions, and are not suitable for on-site rapid detection.
English literature discloses an electrochemical immunosensor based on AuNPs/Zn/Ni-ZIF-8-800@ graphene composite materials, which is used for detecting monensin in milk. However, there is a problem that the current signal is not ideal (Hu M et al. Label-free electrochemical sensor based on AuNPs/Zn/Ni-ZIF-8-800@ graphene compounds for sensitive detection of monensin mill [ J ]. Sensors & initiators B Chemical, 2019).
Therefore, the preparation of the detection method which is simple in operation, rapid in detection, high in sensitivity, good in repeatability and low in cost is an urgent problem to be solved in the field, and no method for preparing the electrochemical immunosensor from the peanut to detect the AFB1 by using the Zn/Ni-ZIF-8-800@ graphene composite material exists in the current market.
Disclosure of Invention
Aiming at the problems of high cost, false positive, single material, high detection limit and the like of the conventional technology for detecting AFB1 in peanuts, the invention provides a preparation method and application of an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts. The invention can realize the rapid detection of AFB1 in peanuts, and has the advantages of low manufacturing cost, convenience in carrying, simplicity in operation, good specificity and the like.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a preparation method of an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts comprises the following steps:
(1) preparing Zn/Ni-ZIF-8-800: by solvothermal method, reacting Ni (NO) 3 ) 2 ·6H 2 O、Zn (NO 3 ) 2 ·6H 2 Dissolving O and 2-methylimidazole (HMelm) in methanol, continuously stirring, centrifuging, collecting precipitate, washing with methanol, and vacuum drying; finally, calcining at high temperature in a tube furnace to obtain black powder Zn/Ni-ZIF-8-800;
(2) preparation of Zn/Ni-ZIF-8-800@ graphene: respectively adding the Zn/Ni-ZIF-8-800 obtained in the step (1) and graphene into a chitosan solution, and performing ultrasonic dispersion until the mixture is uniform to obtain a composite material, namely Zn/Ni-ZIF-8-800@ graphene;
(3) deposition of gold nanoparticles: dripping the composite material obtained in the step (2) on the surface of the electrode, and then electrodepositing gold nanoparticles to obtain the electrode in which the composite material is dripped and deposited by the gold nanoparticles;
(4) and (4) soaking the electrode obtained in the step (3) in a mixed detection solution of potassium ferricyanide, potassium ferrocyanide and potassium chloride with the pH value adjusted, detecting a current signal, and then sequentially dropwise adding an AFB1 antibody solution and a BSA solution on the surface of the electrode for incubation to obtain the aflatoxin B1 electrochemical immunosensor.
Further, Ni (NO) in the step (1) 3 ) 2 ·6H 2 O、Zn (NO 3 ) 2 ·6H 2 The molar ratio of O to HMelm is 1: 1: (10 to 20)
Further, in the step (1), the stirring temperature is 20-30 ℃, the stirring time is 20-30 hours, the vacuum drying time is 10-15 hours, the high-temperature calcination temperature is 700-900 ℃, and the high-temperature calcination time is 1-2 hours.
Further, the mass fraction of chitosan in the chitosan solution in the step (2) is 0.25-1%
Further, the solution used for electrodepositing the gold nanoparticles in the step (3) is 0.5-1% of chloroauric acid solution by mass ratio; the chloroauric acid solution with the mass ratio of 0.5-1% contains 0.5M sulfuric acid solution, the mixing volume ratio of the chloroauric acid to the sulfuric acid is 1 (10-50), the potential in the electrodeposition is-0.2V, and the deposition time is 100-500 s.
Further, in the step (4), the pH value is adjusted to be 5.0-9.0; the concentration of the AFB1 antibody is 4.125-66 mug/mL, the incubation temperature of the AFB1 antibody is 37 ℃, and the incubation time is 10-60 min; the mass fraction of BSA is 0.5% -1%, the incubation temperature is 37 ℃, and the incubation time is 20-40 min.
Further, the electrochemical immunosensor for rapidly detecting the aflatoxin B1 in the peanuts, which is prepared by the method.
Further, the electrochemical immunosensor for rapidly detecting the aflatoxin B1 in the peanuts is applied to rapidly detecting the aflatoxin B1 in the peanuts.
Further, the application steps are as follows:
a. diluting the AFB1 standard solution with the concentration of 1mg/mL into an AFB1 solution with the concentration of 0.01-100ng/mL by using a PBS buffer solution, and storing the AFB1 solution in a refrigerator at the temperature of 4 ℃;
b. b, dropwise coating the AFB1 solution with the concentration of 0.01-100ng/mL obtained in the step a on the surface of a constructed electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts, taking a mixed solution of 5mM potassium ferricyanide and 5mM potassium ferrocyanide (the volume ratio is 1: 1) containing 1M potassium chloride as an analysis solution for DPV detection, carrying out DPV test in the detection solution, and drawing a standard curve according to the relation between the obtained peak current and the concentration of AFB 1;
c. and (c) pretreating a sample to be detected, dripping the pretreated sample on the surface of the modified aflatoxin B1 electrochemical immunosensor, detecting according to the operation B, and substituting the detected current signal into the standard curve obtained in the step B to obtain the concentration of AFB1 in the sample to be detected.
The invention has the following beneficial effects:
1. according to the invention, the composite material with the ZIF-8 standard simulation card closer is prepared by exploring the proportioning ratio of the composite material, the deposition condition of the nanogold and optimizing the experimental condition, the crystal structure is closer to the theoretical crystal structure, the sensitivity of the sensor is improved by using the composite material, and the method is more suitable for detecting the AFB1 in the peanut. Meanwhile, researches show that after the dilute sulfuric acid and a 1% chloroauric acid solution are mixed for use, the current signal can be obviously enhanced, the sensitivity is high, and the detection limit is lower, wherein the dilute sulfuric acid mainly dissolves a metal oxide layer so as to achieve the purpose of activating the metal surface.
2. The method comprises the steps of preparing a composite material, modifying the surface function of the sensor and the like, improves the sensitivity of the sensor by using composite nano materials Zn/Ni-ZIF-8-800 and Graphene, leads the sensor to have high-efficiency selectivity by using AFB1 as an antigen and introducing AFB1 antibody to recognize specific antigen, and improves the adsorption quantity of AFB1 antibody and the adsorption process of AFB1 by combining the composite material and the sensor, thereby increasing the detection efficiency.
3. The electrochemical immunosensor can realize the rapid detection of AFB1 in peanuts, the detection limit is 0.18ng/mL, the standard recovery rate is in the range of 80.26-109.60%, and the RSD of 5-time repeated determination is less than 11%.
4. The electrochemical immunosensor method for rapidly detecting AFB1 in peanuts, which is prepared by the invention, overcomes the defects of high cost, time and labor consumption and easy occurrence of false positive in the traditional method, and thus, the AFB1 in peanuts can be efficiently and rapidly detected.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of the preparation process of the composite material of example 1 of the present invention.
Fig. 2 is a schematic flow chart of the surface functional modification of the electrochemical immunosensor for rapidly detecting AFB1 in peanuts in the embodiment 1 of the present invention.
FIG. 3 is a scanning electron microscope characterization result diagram of the composite material of example 1, wherein (A) is a Zn/Ni-ZIF-8 SEM diagram, (B) is a Zn/Ni-ZIF-8-800 SEM diagram, (C) is Graphene, (D) is a Zn/Ni-ZIF-8-800@ Graphene SEM diagram, and (E) is a Zn/Ni-ZIF-8-800@ Graphene SEM-EDS diagram.
FIG. 4 is a scanning electron microscope characterization result chart of the comparative example composite material of the present invention, wherein (A) the SEM images of Zn/Ni-ZIF-8, (B) Zn/Ni-ZIF-8-800, (C) graphene, (D) Zn/Ni-ZIF-8-800@ graphene mixed material, (E) the SEM-EDS images of Zn/Ni-ZIF-8-800 in chitosan solution, and (F) the SEM-EDS images of AuNPs/Zn/Ni-ZIF-8-800@ graphene composite material.
FIG. 5 is a graph of the Fourier transform infrared spectrum characterization result of example 1 of the present invention, wherein a is dimethylimidazole and b is Zn/Ni-ZIF-8.
FIG. 6 is a graph showing the results of X-ray photoelectron spectroscopy characterization in example 1 of the present invention, wherein the left graph shows an XPS measurement spectrum of Zn/Ni-ZIF-8-800; the right panel shows the fine spectra of Zn/Ni-ZIF-8-800, wherein (A) carbon 1s XPS spectrum, (B) nitrogen 1s XPS spectrum, (C) nickel 2p XPS spectrum, and (D) zinc 2p XPS spectrum.
FIG. 7 is a diagram showing the result of characterization of X-ray diffraction spectrum of example 1 of the present invention, wherein the left diagram is XRD spectrum of example 1, and the right diagram is XRD spectrum of comparative example.
FIG. 8 is a graph comparing current signals of the composite materials prepared in example 1 of the present invention and the comparative example.
FIG. 9 is a graph comparing the current signals of 1% chloroauric acid mixed with sulfuric acid deposited gold of example 1 of the present invention with 1% chloroauric acid deposited gold prepared in the comparative example.
FIG. 10 is a DPV test chart (a) of the AFB1 concentration measurement and a standard graph (b) of the AFB1 concentration measurement, which are examples of the application of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without inventive step, are within the scope of the present invention.
Preparation of AFB1 antibodies reference: yaojing, Hucellufei, Hanjunling, Xufan, Tengchen, Chenyunrui, Sunining, Dendrough, Zhangping, the preparation of aflatoxin B1 monoclonal antibody and the establishment of aflatoxin B1 immunological detection method based on the antibody [ J ] animal nutrition bulletin, 2019,31(03):1405 Amphoa. 1414).
BSA was purchased from Gibco, Inc. in the animal immunization Key laboratory of agricultural sciences, Henan province.
Example 1
The preparation method of the electrochemical immunosensor for rapidly detecting AFB1 in peanuts in the embodiment comprises the following steps:
(1) preparing Zn/Ni-ZIF-8-800: by solvothermal method, adding Ni (NO) 3 ) 2 ·6H 2 O、Zn(NO 3 ) 2 ·6H 2 O, HMelm measured at a rate of 1: 1: dissolving the mixture in methanol at a ratio of 14, continuously stirring for 25h at 25 ℃, centrifuging, taking the precipitate, washing with methanol, and vacuum-drying for 10 h; finally, calcining the mixture for 1 hour at the high temperature of 800 ℃ in a tubular furnace to obtain black powder Zn/Ni-ZIF-8-800 as shown in figure 1;
(2) preparing a composite material: mixing Zn/Ni-ZIF-8-800 prepared in the step (1) with graphene in a ratio of 1: 5, adding the mixture into 5mL of 0.75% chitosan solution, and performing ultrasonic dispersion until the mixture is uniform to obtain Zn/Ni-ZIF-8-800@ graphene;
(3) and (3) gold nanoparticle deposition: inserting the GCE electrode surface coated with the composite material into a solution containing 0.7% chloroauric acid and 0.5M sulfuric acid, depositing for 300s at a potential of-0.2V, and depositing a layer of gold nanoparticles (AuNPs) on the surface of the working electrode;
(4) after the surface of the sensor is washed by double distilled water, the pH value is adjusted to 6.0, and then an antibody of AFB1 with the concentration of 4.125-66 mu g/mL is dripped to incubate for 30min at 37 ℃; specifically, 66. mu.g/ml of AFB1 antibody was diluted to 16.5. mu.g/ml with PBS, and the resulting solution was stored in a-20 ℃ refrigerator;
(5) and dropwise adding a BSA solution with the mass fraction of 0.7% on the sensor surface, and incubating at 37 ℃ for 40min to block the specific recognition sites. Specifically, 0.7g of BSA solid was accurately weighed and dissolved in 100ml of BST, and the resulting solution was stored in a refrigerator at-20 ℃ as shown in FIG. 2.
Example 2
The preparation method of the electrochemical immunosensor for rapidly detecting AFB1 in peanuts in the embodiment comprises the following steps:
(1) preparing Zn/Ni-ZIF-8-800: by simple solvothermal method, Ni (NO) 3 ) 2 ·6H 2 O、Zn (NO 3 ) 2 ·6H 2 O, HMelm measured at a rate of 1: 1: dissolving 18 parts of the precipitate in methanol, continuously stirring for 25 hours at 20 ℃, centrifuging, taking the precipitate, cleaning with methanol, and vacuum-drying for 14 hours; finally, calcining the mixture for 2 hours at the high temperature of 700 ℃ in a tubular furnace to obtain black powder Zn/Ni-ZIF-8-800;
(2) preparing a composite material: mixing Zn/Ni-ZIF-8-800 prepared in the step (1) with graphene in a ratio of 1: 1, adding the mixture into 5mL of 0.25% chitosan solution, and performing ultrasonic dispersion until the mixture is uniform to obtain Zn/Ni-ZIF-8-800@ graphene;
(3) and (3) gold nanoparticle deposition: inserting the GCE electrode surface coated with the composite material into a 0.9% chloroauric acid solution containing 0.5M sulfuric acid, and depositing for 500s at a potential of-0.2V, wherein a layer of gold nanoparticles (AuNPs) is deposited on the surface of the working electrode;
(4) after the surface of the sensor is washed by double distilled water, the pH value is adjusted to 9, and then an antibody of AFB1 with the concentration of 66 mu g/mL is dripped to incubate for 60min at 37 ℃; specifically, 66. mu.g/mL of AFB1 antibody was diluted to 4.125. mu.g/mL with PBS, and the resulting solution was stored in a-20 ℃ refrigerator;
(5) dropwise adding a BSA solution with the mass fraction of 0.5% on the surface of the sensor, and incubating at 37 ℃ for 20min to block the specific recognition site; specifically, 0.5g of BSA solid was accurately weighed and dissolved in 100ml of BST, and the resulting solution was stored in a refrigerator at-20 ℃.
Example 3
The preparation method of the electrochemical immunosensor for rapidly detecting AFB1 in peanuts in the embodiment comprises the following steps:
(1) preparing Zn/Ni-ZIF-8-800: by simple solvothermal method, Ni (NO) 3 ) 2 ·6H 2 O、Zn(NO 3 ) 2 ·6H 2 O, HMelm is measured at a speed of 1: 1: dissolving 20 in methanol, continuously stirring at 30 deg.C for 20 hr, centrifuging, collecting precipitate, washing with methanol, and vacuum drying for 15 hr; finally, calcining the mixture for 1 hour at the high temperature of 900 ℃ in a tubular furnace to obtain black powder Zn/Ni-ZIF-8-800;
(2) preparing a composite material: mixing Zn/Ni-ZIF-8-800 prepared in the step (1) with graphene in a ratio of 1:3, adding the mixture into 5mL of 1% chitosan solution, and performing ultrasonic dispersion until the mixture is uniform to obtain Zn/Ni-ZIF-8-800@ graphene;
(3) and (3) gold nanoparticle deposition: inserting the GCE electrode surface coated with the composite material into a solution containing 0.5M sulfuric acid in 1% chloroauric acid, depositing for 100s at a potential of-0.2V, and depositing a layer of gold nanoparticles (AuNPs) on the surface of the working electrode;
(4) after the surface of the sensor is washed by double distilled water, the pH value is adjusted to 5, and then antibody of AFB1 with the concentration of 66 mu g/mL is dripped to incubate for 10min at 37 ℃; specifically, 66. mu.g/ml of AFB1 antibody was diluted to 16.5. mu.g/ml with PBS. The resulting solution was stored in a-20 ℃ freezer;
(5) dropwise adding a BSA solution with the mass fraction of 1% on the surface of the sensor, and incubating at 37 ℃ for 40min to seal the specific recognition site; specifically, 1g of BSA solid was accurately weighed and dissolved in 100ml of BST, and the resulting solution was stored in a refrigerator at-20 ℃.
Comparative example
The electrochemical immunosensor in English literature is taken as a comparative example, and the preparation steps are as follows:
(1) preparing Zn/Ni-ZIF-8-800: 2.910gNi (NO) 3 ) 2 ·6H 2 O、2.978gZn(NO 3 ) 2 ·6H 2 O and 3.296g HMelm were dissolved in 80mL methanol and stirred continuously at 25 ℃ for 24h, the precipitate was centrifuged and washed with methanolWashing and drying at 60 ℃ for 12 h; finally, calcining the mixture for 1 hour at the high temperature of 800 ℃ in a tubular furnace to obtain black powder Zn/Ni-ZIF-8-800;
(2) preparing a composite material: mixing 5mg of Zn/Ni-ZIF-8-800 prepared in the step (1) and 10mg of graphene into 5mL of 0.5% chitosan solution, and performing ultrasonic dispersion for 1h to obtain a composite material Zn/Ni-ZIF-8-800@ graphene;
(3) and (3) gold nanoparticle deposition: dripping 5 mu LZn/Ni-ZIF-8-800@ graphene composite material suspension on a pretreated GCE electrode, drying at room temperature for 4h, then inserting Zn/Ni-ZIF-8-800@ graphene/GCE into 5mL of 1% chloroauric acid solution, depositing for 15s at a potential of-0.2V, and washing with distilled water to obtain AuNPs/Zn/Ni-ZIF-8-800@ graphene/GCE.
FIGS. 3 and 4 are scanning electron microscope characterization results of the composite materials of example 1 and comparative example, respectively, and as shown in FIG. 3A, Zn/Ni-ZIF-8 is in a monodisperse crystal state. FIG. 3B is a morphology of Zn/Ni-ZIF-8 after calcination, showing that significant shrinkage distortion has occurred. As can be seen from fig. 3C, the graphene is purchased to meet the experimental requirements and has a typical folded layered structure. FIG. 3D is a morphology of the composite, and it can be clearly observed that Zn/Ni-ZIF-8-800 is attached to the surface of graphene. As shown in FIG. 3E, the main elements of the selected region of the Zn/Ni-ZIF-8-800@ Graphene composite material comprise C, O, Zn and Ni, and the successful synthesis of the composite material is preliminarily proved. In fig. 4, (a) Zn/Ni-ZIF-8, (B) Zn/Ni-ZIF-8-800, (C) graphene, (D) SEM images of Zn/Ni-ZIF-8-800@ graphene hybrid materials, (E) Zn/Ni-ZIF-8-800 in chitosan solution, and (F) AuNPs/Zn/Ni-ZIF-8-800@ graphene composite materials. As can be seen from the comparison of fig. 3 and 4, the microstructure of the example and the microstructure of the comparative example are not significantly different.
FIG. 5 is a graph of the Fourier transform infrared spectrum characterization of example 1, where a is dimethylimidazole, b is Zn/Ni-ZIF-8, and 2671cm for dimethylimidazole -1 、1441cm -1 、1112cm -1 And 757cm -1 The absorption peaks at (a) can be attributed to-NH stretching, imidazole ring stretching, -CH bending, and-CH torsional vibration, respectively. Zn/Ni-ZIF-8 at 1441cm -1 And is 757cm -1 Peak at lower wavelength 1382cm -1 And 755cm -1 Move and in1112cm -1 Peak at the longer wavelength of 1145cm -1 Movement, which is a result of coordination of the organic ligand to the metal ion.
FIG. 6 is a graph of the results of X-ray photoelectron spectroscopy characterization of example 1, wherein the Zn/Ni-ZIF-8-800 composite material is composed of elements C, N, Zn and Ni as shown in the left graph. In the right panel A, the peaks at 284.7eV and 286eV are attributable to carbonization of organic matter in Zn/Ni-ZIF-8-800, with C-C bonds at 284.7eV and C-O bonds at 286 eV. The two strong peaks observed at 398.4eV and 400eV in the right panel B belong to the pyrrole-nitrogen. The peaks appearing at 854.7eV and 872.2eV in the right panel C can be attributed to the splitting of the Ni 2p spin orbit into Ni 2p 3/2 And Ni 2p 1/2 Thereby, the effect is achieved. The peaks appearing at 1021.8eV and 1044.8eV in the right panel D pertain to the cleavage of the Zn 2p spin orbit into Zn 2p 3/2 And Zn 2p 1/2 So that the effect is achieved. FIG. 6 shows the successful synthesis of Zn/Ni-ZIF-8-800.
FIG. 7 is a diagram of the characterization result of X-ray diffraction spectrum, wherein the left diagram is the XRD spectrum of example 1, and the right diagram is the XRD spectrum of comparative example. In the left figure, the main peaks of Zn/Ni-ZIF-8 at 7.2 °, 10.3 °, 12.7 °, 14.6 °, 16.4 °, 18.0 °, 22.0 °, 24.4 ° and 29.7 ° are respectively corresponding to the diffraction peaks (011), (002), (112), (022), (013), (222), (114), (233) and (004), and the main strong peaks are consistent with those of the ZIF-8 standard simulation card. In addition, the peak intensities of the Zn/Ni-ZIF-8 diffraction peaks (002), (112) are significantly enhanced as compared with those of ZIF-8, while (013) is relatively weakened, which is attributable to the substitution of a part of Zn element by Ni element in the material. In the right figure, several characteristic diffraction peaks are found at 10.4 degrees, 12.7 degrees, 14.7 degrees, 16.3 degrees and 18.1 degrees, and the XRD pattern of the prepared Zn/Ni-ZIF-8 is basically consistent with the simulated pattern. As can be seen from the comparison of the diffraction peaks of the relevant characteristics of the left and right graphs, the diffraction peak intensities, the diffraction angles and the like of the example 1 and the comparative example are slightly different, and are particularly closer to the simulated graph at 30-40 degrees, which shows that the crystal structure of the experimental example 1 is closer to the crystal structure designed by theory, and the purity of the sensor composite material is higher.
FIG. 8 is a graph comparing current signals obtained from electrochemical characterization of example 1 and comparative composites tested at 5.0 mM K with 1M KCl 3 Fe(CN) 6 /K 4 Fe(CN) 6 In solution. It can be seen from the figure that: the current signal of the composite material prepared in example is significantly stronger than that of the comparative example.
FIG. 9 is a graph comparing current signals of electrochemical characterization results after deposition of example 1 and comparative gold nanoparticles, and it can be seen from FIG. 9 that: compared with a comparative example, the dilute sulfuric acid has an activation effect on 0.1% chloroauric acid, and dissolves a nano-gold surface oxide layer, so that the effect of activating a metal surface is achieved, and a current signal of the sensor after gold deposition can be obviously enhanced.
Application example
The application of the electrochemical immunosensor for rapidly detecting the aflatoxin B1 in the peanuts in rapidly detecting the aflatoxin B1 in the peanuts comprises the following steps:
1) drawing a standard curve:
the electrochemical immunosensor provided by the invention is used for detecting electrochemical signals in AFB1 with different concentrations and drawing a standard curve of a peak current change value and AFB1 concentration, and the specific detection steps are as follows:
AFB1 in the range of 0.01-100ng/mL is dripped on the interface of an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts for incubation, and then a DPV test is carried out in a mixed detection solution containing 5mM potassium ferricyanide and 5mM potassium ferrocyanide (volume ratio of 1: 1) of 1M potassium chloride, and since AFB1 antibody exists on the surface of the sensor, the AFB1 antibody is specifically combined when the AFB1 is incubated. In the DPV test, AFB1 molecules are subjected to reduction reaction in a detection solution to generate a reduction peak, the current value of the reduction peak is changed along with the change of the concentration of AFB1, a standard curve is established according to the current peak value (shown in figure 10A) and the concentration of AFB1 at the moment, and a linear regression equation delta I =21.695lgC is obtained AFB1 +86.959（R 2 = 0.998), as shown in fig. 10B, the detection limit thereof was 0.18 ng/mL. When the concentration of AFB1 exceeded 100ng/mL, the sensor began to lose sensitivity due to the saturating behavior of the antigen-antibody reaction.
2) Repeatability and spiking recovery test:
in the DPV assay, each sample was measured five times, with the average of the five readings being the final record. The data measured for the samples are shown in table 1: the recovery rate of the added standard is in the range of 80.26-109.60%, and RSD of 5 times of repeated measurement is less than 11%. The sensor designed by the invention has the advantages of low detection limit, high sensitivity, wide measurement range and the like, and can realize detection and application of AFB1 in peanuts.
TABLE 1 detection results and recovery of AFB1
3) Calculating the concentration of AFB1 in the sample to be tested according to the established standard curve
Peanuts are provided by the laboratory. The samples were processed according to the prior reference. Specifically, the method comprises the following steps: grinding 5g of peanut, placing in a beaker, adding 20mL of petroleum ether, transferring the sample to a 125mL separating funnel in portions, adding 25mL of methanol water (1: 1), adding a plug, shaking for 5min, standing for layering, discharging a lower layer methanol water extract (sample extract), and diluting with PBS for later use.
And (3) dripping the pretreated sample to be detected on the surface of the modified electrode, capturing AFB1 molecules, generating reduction current in DPV detection due to the existence of AFB1 molecules on a sensor interface, and calculating the concentration of AFB1 in the sample to be detected through a peak current value based on the established current value and concentration standard curve.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A preparation method of an electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts is characterized by comprising the following steps:
(1) preparing Zn/Ni-ZIF-8-800: by solvothermal method, reacting Ni (NO) 3 ) 2 ·6H 2 O、Zn (NO 3 ) 2 ·6H 2 Dissolving O and 2-methylimidazole in methanol, continuously stirring, centrifuging, collecting precipitate, washing with methanol, and vacuum drying; finally, in the tube typeCalcining at high temperature in a furnace to obtain black powder Zn/Ni-ZIF-8-800;
(2) preparation of Zn/Ni-ZIF-8-800@ graphene: respectively adding Zn/Ni-ZIF-8-800 and graphene obtained in the step (1) into a chitosan solution, and performing ultrasonic dispersion until the mixture is uniform to obtain a composite material, namely Zn/Ni-ZIF-8-800@ graphene;
(3) deposition of gold nanoparticles: dripping the composite material obtained in the step (2) on the surface of the electrode, and then electrodepositing gold nanoparticles in chloroauric acid solution containing dilute sulfuric acid to obtain the electrode on which the composite material is dripped and deposited by the gold nanoparticles;
(4) and (4) soaking the electrode obtained in the step (3) in a mixed detection solution of potassium ferricyanide, potassium ferrocyanide and potassium chloride with the pH value adjusted, measuring a current signal, and then dropwise adding an AFB1 antibody and BSA (bovine serum albumin) on the surface of the electrode in sequence for incubation to obtain the aflatoxin B1 electrochemical immunosensor.
2. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: ni (NO) in the step (1) 3 ) 2 ·6H 2 O、Zn(NO 3 ) 2 ·6H 2 The molar ratio of O to 2-methylimidazole is 1: 1: (10-20).
3. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: in the step (1), the stirring temperature is 20-30 ℃, the stirring time is 20-30 h, the vacuum drying time is 10-15 h, the high-temperature calcining temperature is 700-900 ℃, and the high-temperature calcining time is 1-2 h.
4. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: in the step (2), the mass ratio of Zn/Ni-ZIF-8-800 to graphene is 1: (1-3), wherein the mass fraction of chitosan in the chitosan solution is 0.25-1%.
5. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: the solution used for electrodepositing the gold nanoparticles in the step (3) is 0.5-1% of chloroauric acid solution by mass ratio; the chloroauric acid solution with the mass ratio of 0.5-1% contains 0.5M sulfuric acid solution, the mixing volume ratio of the chloroauric acid to the sulfuric acid is 1 (10-50), the potential in the electrodeposition is-0.2V, and the deposition time is 100-500 s.
6. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: in the step (4), the pH value is adjusted to be 5.0-9.0; the concentration of the AFB1 antibody is 4.125-66 mu g/mL, the incubation temperature of the AFB1 antibody is 37 ℃, and the incubation time is 10-60 min.
7. The method for preparing the electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts according to claim 1, wherein the electrochemical immunosensor comprises: in the step (4), the mass fraction of BSA is 0.5-1%, the incubation temperature is 37 ℃, and the incubation time is 20-40 min.
8. An electrochemical immunosensor for rapid detection of aflatoxin B1 in peanuts, prepared by the method of any one of claims 1-7.
9. The use of the electrochemical immunosensor of claim 8 for the rapid detection of aflatoxin B1 in peanuts in the rapid detection of aflatoxin B1 in peanuts.
10. Use according to claim 8, characterized in that the steps are as follows:
a. diluting the AFB1 standard solution with the concentration of 1mg/mL into an AFB1 solution with the concentration of 0.01-100ng/mL by using a PBS buffer solution, and storing the AFB1 solution in a refrigerator at the temperature of 4 ℃;
b. b, dropwisely coating the AFB1 solution with the concentration of 0.01-100ng/mL obtained in the step a on the surface of the constructed electrochemical immunosensor for rapidly detecting aflatoxin B1 in peanuts, and using 5mM potassium ferricyanide and 5mM potassium ferrocyanide containing 1M potassium chlorideTaking the mixed solution (volume ratio is 1: 1) as an analysis solution for DPV detection, carrying out DPV detection in the detection solution, drawing a standard curve according to the relation between the obtained peak current and the concentration of AFB1, and obtaining delta I =21.695lgC AFB1 +86.959（R 2 =0.998）；
c. And (c) pretreating a sample to be detected, dripping the sample to the surface of the modified aflatoxin B1 electrochemical immunosensor, detecting according to the operation B, and substituting the detected current signal into the standard curve obtained in the step B to obtain the concentration of AFB1 in the sample to be detected.
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| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
| CN112920268A (en) * | 2021-03-19 | 2021-06-08 | 河南农业大学 | ASFV-p54 protein specific nano antibody-HRP fusion protein, preparation method and application |
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| CN101655473A (en) * | 2009-09-24 | 2010-02-24 | 浙江大学 | Preparation method of nanogold immunoelectrode |
| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
| CN112920268A (en) * | 2021-03-19 | 2021-06-08 | 河南农业大学 | ASFV-p54 protein specific nano antibody-HRP fusion protein, preparation method and application |
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