Colorimetric Sensor For Detection Of Aflatoxin B₁ In Grain

Aflatoxin B₁（AFB₁）is a kind of aflatoxin mainly produced by Aspergillus flavus and Aspergillus parasiticus.It is the most widely distributed toxin with the strongest toxicity and the highest pollution level.It is recognized as IA carcinogen by the World Health Organization and seriously endangers human safety.At present,the traditional detection methods of AFB₁,such as mass spectrometry,have the problems of difficult operation,high detection cost,professional training,and inability to quickly detect on-site.To overcome the weaknesses of the above methods,many rapid assays have been developed,among which the enzyme-linked immunosorbent assay（ELISA）has become the most widely used immunoassay technique due to its high throughput,good specificity,robustness and ease of automation.However,ELISA has the problems of weak colorimetric signal,limited selection of chromogenic substrates and unstable signal markers,which limit its further application.Therefore,in this thesis,three novel,simple,rapid and sensitive colorimetric sensors were constructed to improve the above-mentioned problems of ELISA method and used for the trace detection of AFB₁in grain.In order to solve the problem of weak colorimetric signal in the AFB₁ELISA detection method,a simple,sensitive and specific colorimetric immunoassay based on gold nanoparticles labeled with glucose oxidase（GOx）/Rabbit anti mouse immunoglobulin G was developed quantitative detection method for AFB₁contamination.In this strategy,Prussian blue was produced by a GOx catalyst-assisted Fe³⁺and Fe（CN）₆^3-system,which was designed and employed as a probe.Under the catalyzation of GOx,the H₂O₂generated from glucose oxidation reduced Fe³⁺to Fe²⁺,resulting in a visible color change from yellow to blue.Moreover,with an increase in AFB₁concentration,the absorbance at 706nm gradually decreased linearly from 0.5 to 5 ng/m L,with a limit of detection（LOD）is350 pg/m L.Thus,acceptable recovery rates（87.60–106.00%）were achieved in the spiked rice flour samples.The results of commercial AFB₁ELISA kit were compared with those of the proposed method.The results were consistent with those of the proposed method,which proved the accuracy and practicability of the proposed colorimetric strategy.In order to solve the problems of low selectivity of chromogenic substrates and unfriendly chromogenic substrates to the environment in AFB₁ELISA,an indirect competitive ELISA with curcumin as the ratiometric probe for the detection of AFB₁in grain was established in this paper by selecting curcumin as a safe and non-toxic chromogenic substrate.The determination was based on the hydrolysis of the substrate urea by urease to produce ammonia,leading to the increase of p H in the solution.The phenolic hydroxyl group of curcumin ionized into phenolic oxygen anion under alkaline conditions,which strengthened the synergistic effect of electron supply and absorption in curcumin.As a result,the color of curcumin changed from yellow to reddish brown,presenting a visible color change.Under optimal conditions,AFB₁can be determined qualitatively with bare eyes,and quantitatively by measuring the absorbance ratio at the wavelengths of 550 nm and 428 nm.The change in the ratio of absorbanceΔ₅₅₀/Δ₄₂₈decreased linearly in a range from 0.01 to 5 ng/m L,and the LOD was 10 pg/m L.The results of commercial AFB₁ELISA kit were compared with those of the proposed method.The results of commercial AFB₁ELISA kit were compared with those of the proposed method.The results were consistent with those of the proposed method,which proved the accuracy and practicability of the proposed colorimetric strategy.To solve the problem of poor stability of signal markers in the rapid detection method of AFB₁,an enzyme-linked immunoassay based on metal organic frameworks（MOFs）was established to detect AFB₁in rice flour.Horseradish peroxidase（HRP）was encapsulated in zeolite imidazole frameworks 8（ZIF-8）by biomimetic mineralization.The surface of HRP@ZIF-8 was modified with dopamine（DA）to form a polydopamine（PDA）layer.Using ZIF-8 as template,HRP@ZIF-8/PDA complex with richπelectrons was prepared by coupling with immunoglobulin G（IgG）to synthesize signal marker HRP@ZIF-8/PDA/IgG.3,3’5,5’-tetramethylbenzidine（TMB）was catalyzed by the complex.It was found that HRP@ZIF-8 composites could maintain stable catalytic activity under extreme conditions such as alkaline condition and high temperature.On this basis,indirect competitive immunocolorimetry was established to detect AFB₁,and samples containing AFB₁could be directly distinguished by naked eyes.Under the optimal conditions,with the increase of AFB₁concentration,the absorbance decreased within0.01-20 ng/m L,with an obvious linear relationship,the LOD was 72 pg/m L,and the precision and specificity were acceptable.The recoveries of AFB₁in rice flour and flour were 96.00-105.00%and 82.00-110.00%,respectively,with relative standard deviations less than 5%.The results of commercial AFB₁ELISA kit were compared with those of the proposed method.The results were consistent with those of the proposed method,which proved the accuracy and practicability of the proposed colorimetric strategy.These three new rapid detection methods in this paper enhanced the colorimetric signal,selected the non-toxic and environment-friendly curcumin as the color substrate,enhanced the stability of signal markers,and realized the high efficiency of AFB₁detection and wider range,expanded its applications in separation and clinical diagnosis of research.The three fast,simple,low cost and high selectivity detection methods provided new technical support and research prospects for AFB₁analysis in food security.