Electrochemical Sensors For The Analysis Of Aflatoxin Production From Aspergillus Flavus

Aflatoxin is a highly toxic secondary metabolite produced by filamentous fungi such as Aspergillus flavus?A.flavus?during the growth process.A small amount of aflatoxin is not only harmful to human health,but also detrimental to the food and agricultural economy.Therefore,it is of great significance to control the growth of A.flavus,especially to inhibit aflatoxin production.In recent years,a lot of works have been carried out to study the mechanism of aflatoxin production from A.flavus,which provides a theoretical guidance for the effective inhibition of aflatoxin production.Reactive oxygen species?ROS?are the products of aerobic metabolism in organism,playing an important role in the process of cell signal transduction and the maintenance of oxidation-reduction banlance.It has been reported that ROS are closely related to the intracellular synthesis of aflatoxin,and the ROS level in the mycelium may be an important pre-requisite for aflatoxin synthesis.Therefore,it is highly demanded to monitor ROS levels and activities of anti-oxidative enzymes during the growth of A.flavus for better understanding mechanisms for aflatoxin production and antifungal drug-caused inhibition of aflatoxin synthesis.Although conventional molecular biology methods?such as gene knockout etc.?can build the relationship between genes and functions,real-time,rapid and sensitive detection still cannot be achieved.Electrochemical Sensors can real-time detect the biological targets rapidly with strong specificity and high sensitivity.If electrochemical sensors could be used to investigate the role of ROS in the aflatoxin production from A.flavus,real-time and in situ data might be obtained without affecting the activity of A.flavus.In this thesis,we developed a variety of nanomaterials-based electrochemical sensors for the analysis of catalase activity,superoxide anion?O₂^-?and hydrogen peroxide?H₂O₂?.Based on the data,the mechanism of aflatoxin production and the inhibitory mechanism of anti-fungal substances are analyzed.The main works are summarized as follows:1. Catalase activity-a potential indicator for aflatoxin productionA graphene-gold nanoparticles-based biosensor was constructed via an electrodeposition method for H₂O₂ detection.Field emission scanning electron microscope?FSEM?,Raman and X diffraction spectra were used to prove the successful construction of the biosensor.The electrochemical data show that the sensor could be applied to measure H₂O₂ with a linear range of 2.0-30?M and the detection limit of 0.2?M.The presence of catalase could degrade H₂O₂ molecules in the system,causing the reduction of H₂O₂ concentration.Therefore,by measuring the H₂O₂ concentration,the activity of catalase in the system could be estimated.After optimization,the biosensor can detect samples with the catalase activity in the range of 0-48 U/mg prot.In order to demonstrate the practical feasibility of the sensor,it was utilized to test the catalase activity in crude extract of A.flavus.Results indicate that the catalase activity in crude extract of A.flavus enhances along with the A.flavus growth.Moreover,the catalase activity is positively correlated to the production of aflatoxin.When A.flavus grows rapidly,the mycelium metabolism is also exuberant,followed by the increase of the aerobic metabolites ROS.Under such circumstances,catalase activity also enhances in order to balance the oxidative stress.Since the enhancement of ROS may promote the synthesis of aflatoxin,there could be a positive correlation between catalase activity and aflatoxin synthesis.2.Nanosilver-caused suppression of aflatoxin production from A.flavus and itspossible mechanismSilver nanoparticles?Ag NPs?were synthesized via chemical reduction method and characterized by transmission electron microscopy?TEM?and UV spectrophotometer?UV-VIS?.The products were confirmed to have spherical structures with a diameter of⁴.5 nm.Based on the anti-fungal assay,the concentration of 5?g/m L was chosen to study direct inhibiting effects of Ag NPs on aflatoxin production.Results show that the Ag NPs treatment could significantly decrease secretion of aflatoxin B₁ from A.flavus.By combining the multi-walled carbon nanotubes?CNTs?with artificial nano-enzyme Mn₃O₄ nanorods,we fabricated an electrochemical superoxide anion?O₂^-?sensor with a dynamic range of 57.5-862.5 n M and the detection limit of 57.5 n M.Real-time measurements of O₂^-with the sensor reveal that the Ag NPs could trigger the release of O₂^-from fungal mycelia.A mechanism involving O₂^-release is proposed to explain Ag NPs-caused suppression of aflatoxin production from A.flavus.3.Fabrication of a carbonized spores-based H₂O₂ biosensor for possibleapplication in fungal analysisInspired by the state-of-the-arts that use biomass as carbon sources to produce carbon materials,it may be possible to fabricate carbon materials with hollow and spherical morphologies using A.flavus conidia as bio-templates.In this work,the hollow carbon microspheres were prepared via carbonization of A.flavus conidia.Then,the Au NPs were in situ synthesized on carbon microspheres to fabricate a sensor for the detection of H₂O₂.After optimization,the sensor shows a linear dynamic range of 1?M-15?M and the detection limit of 0.05?M.In summary,the activity of catalase and the release of ROS from A.flavus were evaluated with the nanomaterials-based electrochemical biosensors.Based on the experimental data,the possible mechanisms for production and anti-fungal substances-caused suppression of aflatoxin in A.flavus were proposed.The implementation of the project may provide direct data and new methods for investigating the mechanism of aflatoxin production from fungi,further extending the applications of nanomaterials-based biosensor in microbial biochemical analysis.