CN111999502B - Aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation multimode signal output - Google Patents
Aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation multimode signal output Download PDFInfo
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Abstract
A PBNPs-based in-situ growth regulation multimode signal output aflatoxin B1 detection kit and method are characterized in that MNPs are used as carriers and precursors, target AFB1 nucleic acid aptamer is coupled with Cy 5-marked cDNA to form double-stranded DNA by hybridization, a fluorescence quenching substrate detection probe is obtained, and then the fluorescence quenching substrate detection probe is arranged on a 96 polystyrene micro-pore plate, and the MNPs are used as precursors to carry out K under the condition of hydrochloric acid digestion 4 Fe(CN) 6 The reaction yields in situ grown PBNPs. The PBNPs can raise the temperature of the substrate solution under 808 and nm laser irradiation to realize thermal signal detection; at the same time can catalyze colorless TMB in H 2 O 2 Oxidizing in the presence of (2) to form a blue product, and realizing colorimetric signal detection; finally, in the in-situ growth process of the PBNPs, the MNPs are etched by acid to fall off DNA double chains containing fluorescent groups, so that fluorescent signal detection is realized. The method has high sensitivity, simple operation, high speed and portability, high accuracy and can be applied to the detection of AFB1 in contaminated food.
Description
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing detection, and particularly relates to an aflatoxin B1 detection kit and a detection method based on PBNPs in-situ growth regulation and control multimode signal output.
Background
Aflatoxin B1 (AFB 1 for short) is a derivative of dihydrofuran oxanaphthalen-o-ne, containing a bisfuran ring and an oxanaphthalen-o-ne (coumarin), which is mainly present in soil, animals, plants and nuts, wherein foods most easily contaminated with AFB1 are peanut, corn, walnut and rice. AFB1 is one of the most carcinogenic of known chemicals, and has strong toxicity to both humans and animals, and its toxic effect is mainly severe damage to the liver, so it is an important factor for causing food safety accidents. Because extremely low AFB1 can induce more significant physiological toxicity, a highly sensitive, selective and accurate method is needed for its detection.
Currently, methods for detecting AFB1 mainly include high performance liquid chromatography, liquid chromatography/mass spectrometry, immunochromatography and traditional enzyme-linked immunoassay. Although the high performance liquid chromatography and the liquid chromatography/mass spectrometry have higher detection accuracy, the high performance liquid chromatography/mass spectrometry has high instrument cost, requires professional operators and is not suitable for on-site rapid analysis and detection; although immunochromatography has the advantages of simplicity, rapidness and low cost, it has poor sensitivity and precision; the traditional ELISA method has the defects of complicated operation process, high cost, long detection period, easy occurrence of false positive and the like. Therefore, it is becoming increasingly important to achieve simple, sensitive and accurate determination of AFB1 content in contaminated food by improved ELISA methods.
Disclosure of Invention
One of the purposes of the invention is to overcome the defects of the prior art, and provide an aflatoxin B1 detection kit based on PBNPs (Prussian blue nano particles (Prussian blue nanoparticles) abbreviated as PBNPs) in-situ growth regulation and control multimode signal output, so as to realize the on-site instant semi-quantitative and fluorescence accurate quantitative detection of AFB 1.
In order to achieve the above purpose, the present invention adopts the following technical scheme: an aflatoxin B1 detection kit based on PBNPs in-situ growth regulation multimode signal output comprises an AFB1 monoclonal antibody (Ab 1), a biotin-marked AFB1 nucleic acid aptamer (bio-aptamer), cy 5-marked cDNA and streptavidin-type MNPs (SA-MNPs); the streptavidin MNPs are coupled with double-stranded DNA formed by hybridization of biotin-marked AFB1 aptamer and Cy 5-marked cDNA, and then the detection probe is obtained.
The kit also comprises 96-well polystyrene microwell plates, PBS buffer solution and chromogenic substrates.
Specifically, the kit comprises the following reagents:
(1) 96-well polystyrene microwell plates;
(2) AFB1 monoclonal antibody (Ab 1), commercially available product;
(3) Biotin-labeled AFB1 nucleic acid aptamer (bio-aptamer): the concentration is 1 mu M, and the nucleotide sequence is as follows: 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3', as shown in SEQ ID No. 1;
(4) Cy 5-labeled cDNA: concentration was 1. Mu.M, sequence: 5 '-GGCCTAGCG-3' as shown in SEQ ID No. 2;
(5) Streptavidin-formed MNPs (SA-MNPs, MNPs being ferroferric oxide nanoparticles): the concentration is 0.1-1mg/mL, and the products are purchased in the market;
(6) PBS buffer: the concentration was 0.01M, a commercially available product; preparing PBS buffer solution with concentration of 1mg/mL Bovine Serum Albumin (BSA) according to a conventional technology;
(7) Chromogenic substrate: solution A, H with concentration of 5.88 mM 2 O 2 A solution; solution B, TMB solution at a concentration of 1.25 mM.
The invention also aims to provide an aflatoxin B1 detection method based on PBNPs in-situ growth regulation multimode signal output, which comprises the following steps:
(1) The biotin-labeled AFB1 aptamer and Cy 5-labeled cDNA were mixed in a volume ratio of 1:1, hybridizing to form double-stranded DNA, adding the double-stranded DNA into a streptavidin MNPs solution, and modifying the double-stranded DNA onto the surface of the MNPs through the specific combination of biotin and streptavidin to obtain detection probes aptamer@cDNA@MNPs;
(2) Diluting an AFB1 monoclonal antibody with PBS buffer solution, and then adding the diluted AFB1 monoclonal antibody into a 96 polystyrene micro-pore plate to finish antibody coating; and blocking nonspecific active sites on the microwell plate with PBS buffer solution containing bovine serum albumin;
(3) Adding a sample solution and a concentration gradient AFB1 standard solution into the microplate respectively, and then adding the sample solution and the concentration gradient AFB1 standard solution into the microplate to obtain the final productThe volume ratio of the detection probe is 1:3 and K 4 Fe(CN) 6 Carrying out ultrasonic reaction on the solution;
(4) And drawing a standard curve according to the detection results of the AFB1 standard solution according to different detection requirements, and correspondingly drawing the standard curve according to the detection results of the sample solution, so that the AFB1 in the sample solution can be detected and analyzed.
Specifically, the detection method comprises the following steps:
(1) Bio-aptamer and Cy5 marked cDNA are taken according to the volume ratio of 1:1 hybridizing to form double-stranded DNA, adding the double-stranded DNA into SA-MNP solution of 0.1-1mg/mL, incubating for 0.5-2h at room temperature to modify aptamer/cDNA to the surface of MNPs through specific binding of biotin and streptavidin, centrifuging for 8-15 min at 8000-12000 r/mm, and washing for 3 times to obtain detection probe aptamer@cDNA@MNPs;
(2) Diluting the AFB1 monoclonal antibody (Ab 1) to 0.01 mg/mL with 0.01M PBS buffer solution, adding 200 mu L into 96 polystyrene microwell plates, reacting overnight at 4 ℃, washing 3 times with 0.01M PBS buffer solution, and finishing antibody coating; adding 200 mu L of PBS buffer solution with the concentration of 1mg/mL Bovine Serum Albumin (BSA) into the microplate, incubating for 1h at room temperature in a dark place to seal non-specific active sites on the microplate, and washing 3 times by using 0.01M PBS solution;
(3) 200. Mu.L of the sample solution and 200. Mu.L of the sample solution having a concentration of 10 were added to the microplate, respectively -14 -10 -7 g/mL and 0 g/mL of AFB1 standard solution, incubating for 0.5-2h at room temperature in the dark, and flushing the redundant AFB1 with 0.01M PBS solution; adding the detection probe prepared in the step (1) into a microplate, incubating for 0.5-2h at room temperature in a dark place, flushing unreacted probe by using a PBS solution, and adding the solution into the microplate in a volume ratio of 1:3 and K 4 Fe(CN) 6 Carrying out ultrasonic reaction on the solution for 20-60min;
(4) And drawing a standard curve according to the detection results of the AFB1 standard solution according to different detection requirements, and correspondingly drawing the standard curve according to the detection results of the sample solution, so that the AFB1 in the sample solution can be detected and analyzed.
The sample solution is obtained by crushing a sample, taking 2-8g of the sample, soaking the sample in methanol with the volume concentration of 40% of 20-40 mL%, uniformly stirring, centrifuging, taking supernatant, and diluting by 5 times. Wherein the centrifugation is carried out at 3000-6000 r/min for 5-10 min.
The detection requirements mentioned in the step (4) are: photo-thermal detection, colorimetric semi-quantitative detection and fluorescence detection, specifically:
photo-thermal detection: and (3) irradiating the microwell plate subjected to ultrasonic reaction in the step (3) by using infrared laser of 808 and nm, and drawing a standard curve by using a signal reader based on a thermometer according to the relation between the temperature and the concentration of the AFB1 standard solution, so as to realize rapid and portable detection of the AFB 1.
Colorimetric detection; hydrogen peroxide solution and TMB solution were added to the microwell plate after the ultrasonic reaction in step (3) irradiated with infrared laser of 808 nm, after the reaction, the color change was observed, and the ultraviolet absorption value of the solution thereof at 652 nm was recorded with an enzyme-labeled instrument. And drawing a standard curve through the relation between the ultraviolet absorption value and the concentration of the AFB1 standard solution, so as to realize the rapid semi-quantitative detection of the AFB 1.
Fluorescence detection: rapidly centrifuging the solution of the microplate subjected to the ultrasonic reaction in the step (3), and taking supernatant; the supernatant was photographed using a fluorescence imager. And (3) analyzing the relationship between the fluorescence intensity and the concentration of the AFB1 standard solution by image Lab software to establish a standard curve, so as to realize high-sensitivity accurate quantitative detection of the AFB 1.
The invention firstly adopts streptavidin MNPs as a carrier and a precursor, couples a target object AFB1 nucleic acid aptamer and a Cy5 marked DNA to form double-stranded DNA, obtains a fluorescence quenching detection probe due to stronger fluorescence quenching capability and separation efficiency of the MNPs, and then carries out immune reaction on a 96 polystyrene micro-pore plate, and then jointly recognizes and captures the target object through an antibody and the nucleic acid aptamer, namely takes the MNPs as the precursor, and releases Fe under hydrochloric acid digestion 3+ ，K 4 Fe(CN) 6 With Fe 3+ Prussian blue nanoparticles (Prussian blue nanoparticles, abbreviated as PBNPs) grown in situ are obtained after complete reaction to regulate and control multimode signal output (temperature-colorimetric-fluorescent signals) so as to complete portable-visual semi-quantification and experiments of AFB1Indoor accurate quantitative determination specifically does: the in-situ grown PBNPs have excellent near infrared light (NIR) driven light-heat conversion characteristics (photo-heat effect), heat generated in the light-heat signal transduction process can be used as a signal reader of a quantification target by using a thermometer, and the temperature of a substrate solution is increased through photon-electron-phonon coupled plasma photo-heat conversion, so that the rapid and portable heat signal detection of the AFB1 is realized; meanwhile, the PBNPs have the catalytic performance similar to that of natural peroxidase, and can catalyze colorless 3,3', 5' -tetramethyl benzidine (TMB) in H 2 O 2 Oxidizing in the presence of (a) to form a blue product (ox-TMB, oxidized TMB), and realizing rapid visual colorimetric signal detection of AFB1; in addition, MNPs are used as a probe substrate, and DNA double chains containing fluorescent groups are etched and separated by hydrochloric acid in the process of growing PBNPs in situ, so that fluorescence is recovered, and the fluorescence accurate quantitative detection of AFB1 is realized. The three-mode immunoassay method designed by the invention has high sensitivity, simple operation, rapidness, portability and high accuracy, and can be applied to the detection of AFB1 to obtain good detection results; has important application value for detecting aflatoxin B1 in contaminated food.
Compared with the prior art, the invention has the beneficial effects that:
(1) The multi-mode immunodetection method combines the advantages of different single-mode technologies, has the advantages of portability of temperature signals, visualization of colorimetric signals and high sensitivity of fluorescent signals, and is expected to synchronously realize the portability-visualization on-site instant semi-quantitative detection and high-sensitivity accurate quantitative detection in a laboratory.
(2) The detection probe is constructed by using the nucleic acid aptamer as a target object identification element, so that the detection specificity can be remarkably improved, meanwhile, the method is endowed with a wider application range and stronger expansibility, and the corresponding nucleic acid aptamer is found, so that the method can be applied to detection of other food harmful substances.
(3) The MNPs are used as the probe substrate, so that the Prussian blue load is greatly increased, and the colorimetric, fluorescent and photothermal signals are further improved.
Drawings
FIG. 1 is a graph showing experimental properties of MNPs@PBNPs prepared in the presence of hydrochloric acid solutions of different concentrations according to the present invention.
Wherein: a is an ultraviolet spectrogram, and b is an absorbance value change chart.
FIG. 2 shows K of different concentrations according to the invention 4 Fe(CN) 6 Experimental characterization of mnps@pbnps prepared in solution.
Wherein: a is an ultraviolet spectrogram, and b is an absorbance value change chart.
FIG. 3 is a graph showing experimental characteristics of MNPs@PBNPs generated under different reaction time conditions.
Wherein: a is an ultraviolet spectrogram, and b is an absorbance value change chart.
FIG. 4 is a photo-thermal assay of the kit of the invention for AFB 1.
Wherein a is a trend graph of the temperature of the solution changing with the concentration of AFB1 with time; b is the thermal signal detection standard curve of AFB 1.
FIG. 5 is a colorimetric detection scheme for AFB1 using the kit of the invention.
Wherein: a is a change trend chart of ultraviolet absorption spectrum of the solution changed along with the concentration of AFB1; b is a colorimetric detection standard curve of AFB 1.
FIG. 6 is a fluorescence detection chart of the kit for AFB1, showing a change trend chart of fluorescence intensity of a solution according to concentration of AFB1 and a fluorescence detection standard curve of AFB 1.
FIG. 7 is an experimental graph of the recovery rate of AFB1 using the kit of the present invention, showing the comparison of the detection values of AFB1 added to peanut samples at different concentrations in three detection modes with the theoretical addition amount.
Wherein: 1-7 are peanut samples of the same concentration.
Detailed Description
The invention is described in further detail below with reference to specific embodiments and figures.
Example 1
Taking 8-branch separation tube, adding 100 μL SA-MNPs and 100 μ L K respectively 4 Fe(CN) 6 As precursors, hydrochloric acid solutions of different concentrations (0, 1,2. 4, 8, 16, 32, 64 mmol/L), and after reaction at room temperature for 40min, the color change of the solution was observed. The prepared particles are MNPs@PBNPs, and have obvious absorption peaks around 780 and nm. The change in the ultraviolet spectrum in the wavelength range of 200-1000 nm is recorded with an ultraviolet spectrophotometer. And the absorbance at 780 nm wavelength with different concentrations of hydrochloric acid solution and the absorbance at 780 nm wavelength for the blank sample (without SA-MNPs added) were recorded.
As can be seen from FIG. 1, when the concentration of hydrochloric acid is less than 16 mmol/L, the absorbance at 780 and nm gradually increases as the concentration of hydrochloric acid increases, the solution gradually changes to blue, and PBNPs are already generated. When the concentration of hydrochloric acid is more than 16 mmol/L, the absorbance at 780 and nm is slowly reduced, and the color of the solution is gradually reduced.
Example 2
Taking 8-branch separation tubes, adding 100 μL SA-MNP, and adding K with concentration of 0, 0.1, 0.2, 0.75, 1, 2, 5 mmol/L 4 Fe(CN) 6 The solution was then added with 16 mmol/L hydrochloric acid solution, allowed to react at room temperature for 40min, and the color change of the solution was observed. With K 4 Fe(CN) 6 The color of the prepared MNPs@PBNPs composite particles is different due to the change of the concentration of the solution. Referring to FIG. 2, when K 4 Fe(CN) 6 At a solution concentration of 1 mmol/L, the absorbance value at 780 and nm wavelengths was highest, and the solution color was blue. Thus, it can be said that K 4 Fe(CN) 6 The concentration of the solution was 1 mmol/L, which was the optimum concentration.
Example 3
Taking 1 branch off the tube, adding 100. Mu.L SA-MNP, and then adding K at a concentration of 1 mmol/L 4 Fe(CN) 6 Adding 16 mmol/L hydrochloric acid solution, reacting at room temperature, reacting for 0, 10, 20, 30, 40, 50 and 60min, and recording ultraviolet spectrogram change within 200-1000 nm wavelength range. The absorbance value of the prepared MNPs@PBNPs composite particle at 780 and nm changes along with the change of the reaction time. Referring to fig. 3, the absorbance value at 780 nm wavelength was highest when the reaction was performed for 40min. Thus, 40min can be the optimal reactionTime concentration.
Example 4 preparation and detection of the kit of the present invention
(1) Hybridization is carried out on 50 mu L of 1 mu M bio-aptamer and 50 mu L of 1 mu McDNA, double-stranded DNA is formed, then the double-stranded DNA is added into 0.1 mg/mL SA-MNP solution of 1 mL, incubation is carried out for 1h time at room temperature, centrifugation is carried out for 8 min at 10000 r/mm, and washing is carried out for 3 times, thus obtaining detection probe aptamer@cDNA@MNPs;
(2) Diluting the AFB1 monoclonal antibody (Ab 1) to 0.01 mg/mL with 0.01M PBS buffer solution, adding 200 mu L into 96 polystyrene microwell plates, reacting overnight at 4 ℃, washing 3 times with 0.01M PBS buffer solution, and finishing antibody coating;
(3) Adding 200 mu L of PBS buffer solution with the concentration of 1mg/mL Bovine Serum Albumin (BSA) into the step (2), incubating for 1h at room temperature in a dark place, blocking nonspecific active sites on the microplate, and washing 3 times by using 0.01M PBS solution;
(4) 200. Mu.L of the sample solution and 200. Mu.L of the sample solution having a concentration of 10 were added to the step (3), respectively -7 g/mL、10 -8 g/mL、10 -9 g/mL、10 -10 g/mL、10 -11 g/mL、10 -12 g/mL、10 -13 g/mL、10 -14 g/mL and 0 g/mL of AFB1 standard solution, incubating for 1h at room temperature in the absence of light, and washing off the excess AFB1 with 0.01M PBS solution;
(5) And (3) adding the detection probe prepared in the step (1) into the step (4), incubating the detection probe in a dark place at room temperature for 1h, and flushing the unreacted probe by using a PBS solution. To the microplate, 16. Mu.L of 0.1. 0.1M strength hydrochloric acid solution and 40. Mu.L of 5. 5mM strength K were added 4 Fe(CN) 6 The solution was subjected to ultrasonic reaction for 40min. After the MNPs are etched by hydrochloric acid, fe is released 3+ ，K 4 Fe(CN) 6 With Fe 3+ In situ grown PBNPs are obtained after complete reaction.
Photo-thermal detection: the microwell plate after the ultrasonic reaction in step (5) was irradiated with the infrared laser of 808 nm, and the temperature was measured as a signal reader, and it can be seen from fig. 4a that the temperature increased with the increase in the concentration of AFB 1. A standard curve is plotted according to the relationship between temperature and AFB1 standard solution concentration, as shown in fig. 4b, y=40.6+5.26 lgX.
Colorimetric detection: to the solution irradiated with the laser of 808. 808 nm in temperature detection, 50. Mu.L of a solution containing hydrogen peroxide and 50. Mu.L of a solution B containing TMB were added, after the reaction, a color change was observed, and the ultraviolet absorption value of the solution was recorded at 652. 652 nm with an enzyme-labeled instrument. From fig. 5a it can be seen that the solution color deepens with increasing concentration of AFB1 and the absorbance value at 652 nm increases gradually. A standard curve is drawn according to the relationship between the uv absorbance and the AFB1 standard solution concentration, as shown in fig. 5b, y=0.398+0.069 lgC.
Fluorescence detection: rapidly centrifuging the solution of the microplate subjected to the ultrasonic reaction in the step (5), and taking supernatant; the supernatant was photographed using a fluorescence imager. The relationship between fluorescence intensity and AFB1 standard solution concentration was analyzed by image Lab software to establish a standard curve, as shown in fig. 6, y= 2625.01 + 330.68lgX. From fig. 6, it can be seen that the fluorescence intensity increases with increasing concentration of AFB 1.
Example 5 recovery test
Preparation of peanut sample solution: peanut samples were crushed, 5 g was soaked in 25 mL 40% methanol, stirred for 10 min, centrifuged at 4000 r/min for 5 min, and the supernatant 1 mL was diluted 5-fold.
Firstly adding AFB1 antibody into 96-hole polystyrene micro-porous plate, after reacting overnight at 4 ℃, washing 3 times with PBS buffer solution, adding BSA blocking agent for reacting 1h, washing 3 times with PBS solution, adding 50 mu L of peanut sample (1-7) solution with the same concentration and AFB1 (0.05-1 ng/mL) with different concentrations for reacting 1h, washing 3 times with PBS solution, adding 0.01 mg/mL SA-MNPs for reacting 1h, washing 3 times with PBS solution, and then adding K of 1 mM 4 Fe(CN) 6 And 16 mM in HCl, sonicated for 40min, and its temperature/colorimetric/fluorescent signal observed. As shown in FIG. 7, the theoretical addition value is consistent with the actual detection values of the three detection methods, which indicates that the aflatoxin B1 detection kit based on PBNPs in-situ growth regulation and control multimode signal output has good accuracy.
The foregoing is merely exemplary of the present invention, and those skilled in the art should not be considered as limiting the invention, since modifications may be made in the specific embodiments and application scope of the invention in light of the teachings of the present invention.
Sequence listing
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<120> aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation and control multimode signal output
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Claims (10)
1. A PBNPs-based in-situ growth regulation multimode signal output aflatoxin B1 detection kit is characterized by comprising an AFB1 monoclonal antibody, a biotin-labeled AFB1 nucleic acid aptamer, cy 5-labeled cDNA and streptavidin MNPs, a hydrochloric acid solution and K 4 Fe(CN) 6 The method comprises the steps of carrying out a first treatment on the surface of the The streptavidin MNPs are coupled with double-stranded DNA formed by hybridization of biotin-marked AFB1 nucleic acid aptamer and Cy 5-marked cDNA to obtain a detection probe; MNPs are taken as precursors, and Fe is released under the digestion of hydrochloric acid 3+ ，K 4 Fe(CN) 6 With Fe 3+ In situ grown PBNPs are obtained after complete reaction.
2. The PBNPs in-situ growth regulation multimode signal output-based aflatoxin B1 detection kit of claim 1, wherein the nucleotide sequence of the biotin-labeled AFB1 nucleic acid aptamer is as set forth in SEQ ID NO: 1.
3. The PBNPs in situ growth regulation multimode signal output-based aflatoxin B1 detection kit of claim 1, wherein the Cy 5-labeled cDNA has a sequence as set forth in SEQ ID NO: 2.
4. The PBNPs in-situ growth regulation multimode signal output-based aflatoxin B1 detection kit of claim 1, further comprising a 96-well polystyrene microwell plate, PBS buffer, stop buffer, and chromogenic substrate.
5. The aflatoxin B1 detection method based on PBNPs in-situ growth regulation multimode signal output is characterized by comprising the following steps:
(1) Hybridizing the biotin-marked AFB1 aptamer with Cy 5-marked cDNA according to the volume ratio of 1:1, forming double-stranded DNA, adding the double-stranded DNA into a streptavidin MNPs solution, and modifying the aptamer/cDNA onto the surface of the MNPs through the specific combination of biotin and streptavidin to obtain detection probes aptamer@cDNA@MNPs;
(2) Diluting an AFB1 monoclonal antibody with PBS buffer solution, and then adding the diluted AFB1 monoclonal antibody into a 96 polystyrene micro-pore plate to finish antibody coating; and blocking nonspecific active sites on the microwell plate with PBS buffer solution containing bovine serum albumin;
(3) Respectively adding a sample solution and a concentration gradient AFB1 standard solution into the micro-pore plate, and then adding the detection probe prepared in the step (1), a hydrochloric acid solution and K in a volume ratio of 1:3 4 Fe(CN) 6 Carrying out ultrasonic reaction on the solution;
(4) And drawing a standard curve according to the detection results of the AFB1 standard solution according to different detection requirements, and correspondingly drawing the standard curve according to the detection results of the sample solution, so that the AFB1 in the sample solution can be detected and analyzed.
6. The method for detecting aflatoxin B1 based on PBNPs in-situ growth regulation multimode signal output according to claim 5, wherein the detection requirements in the step (4) comprise photo-thermal detection, colorimetric semi-quantitative detection and fluorescence detection.
7. The method for detecting aflatoxin B1 based on PBNPs in-situ growth regulation multimode signal output according to claim 6, wherein in the photo-thermal detection, 808 nm infrared laser is used for irradiating the microplate after ultrasonic reaction in the step (3), and a standard curve is drawn through the relation between the temperature and the concentration of an AFB1 standard solution.
8. The method for detecting aflatoxin B1 based on PBNPs in-situ growth regulation multimode signal output according to claim 6, wherein in the colorimetric semi-quantitative detection, hydrogen peroxide solution and TMB solution are added into a microplate subjected to ultrasonic reaction in the step (3) after 808 and nm infrared laser irradiation, and a standard curve is drawn according to the relation between the ultraviolet absorption value of the reaction solution at 652 nm and the concentration of the AFB1 standard solution.
9. The method for detecting aflatoxin B1 based on PBNPs in-situ growth regulation multimode signal output according to claim 6, wherein in the fluorescence detection, the solution in the microwell plate after the ultrasonic reaction in the step (3) is centrifuged, and the supernatant is taken; a standard curve was established by the relationship between fluorescence intensity of the supernatant and the concentration of AFB1 standard solution.
10. The method for detecting aflatoxin B1 based on PBNPs in-situ growth regulatory multimode signal output according to claim 5, wherein the gradient concentration of the AFB1 standard solution in the step (3) is 10 -14 -10 -7 g/mL。
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