| Ochratoxin is one of the important mycotoxins,which widely exists in grape and its products.However,there are few studies on the removal of ochratoxin A(OTA)in grape products,which has some problems,such as great influence on product quality,difficulty in practical application and the complex operation.In addition,the detection of OTA always depends on large-scale instruments,with the drawbacks of high cost,complicated pretreatment.In this study,two kinds of microporous ceramic membrane of nano positive charged material were prepared by impregnation-thermal decomposition.The removal conditions and mechanism on OTA in grape products,and the effect on the quality and flavor of grape product were studied.Moreover,a label-free impedimetric aptasensor has been developed.The modification performance and electrochemical properties of amino aptamer and thiolated aptamer was studied.The preparation conditions were optimized to construct label-free aptasensor for rapid detection of OTA in grape and its products.The results showed as follow:1.The microporous nano MgO and nano Y2O3 diatomite ceramic membrane with high positive charge was prepared by impregnation-thermal decomposition.Scanning electron microscopy showed that nano-MgO and nano-Y2O3 were uniform Ly coated on the surface of the ceramic membrane.As the results of transmission electron microscope showed,the diameter of nano MgO particles was about 80 nm,which was cubic crystal structure.The diameter of nano Y2O3 particles was from 30 nm to 60 nm,which were the mixture of sphere and cubic crystal.The zeta potential of the diatomite ceramic membrane was negative,while the nano MgO and nano Y2O3 diatomite ceramic membrane were positive charged in the range of p H 2-12.8 and 2-6.5,respectively.The electric properties and coating conditions of nano-MgO/diatomite ceramic membrane were better than nano-Y2O3/diatomite ceramic membrane.2.The solution was injected with peristaltic pump dynamic to removed OTA by nano-MgO/diatomite ceramic membrane.The optimal conditions for OTA removal were as follows:the flow rate at 141?L/min,solution p H 5,the ceramic membrane 2.0g,the solution at 25 OC.The removal rate of the charged membrane reached 88%,much higher than 20%of diatomite ceramic membrane.The charged membrane also showed good regeneration capability according desorption experiments.The isotherm adsorption data accorded with the Langmuir model,with a maximum adsorption capacity of 806 ng/g at 25 OC.The separation factor was between 0-1,the changes of adsorption enthalpy(?H),adsorption free energy(?G)and adsorption entropy(?S)were all negative.The combination of nano-MgO/diatomite ceramic membrane and OTA was a spontaneous exothermic process,which was non-specific physical adsorption.3.Nano-MgO/diatomite ceramic membranes were applied to remove OTA in wine and grape juice,which was 5-10 times of the maximum levels of OTA prescribed by European Commission.The concentration of OTA in treated products was lower than 2?g/kg,and the removal rate was over 91%.The quality of four kind of grape products were determined before and after removal of OTA.In grape products it was found that,the contents of phenolic substances,reducing sugar and soluble solids decreased after treated with charged membrane,while the transmittance increased,and the color value kept unchanged.After the removal of OTA,the components and relative contents of aroma substances in grape products changed little.The relative contents of esters decreased with the alcohols increased,and the overall flavor was preserved.4.Gold nanoparticles played an important role in signal amplification by increasing the effective gold surface to fix more aptamers in the process of sensor preparation.There are obvious differences in the modification performance and electrochemical properties between amino aptamer and thiolated aptamer when they combined to the electrochemical impedimetric sensors.The results showed that the initial modification density of amino aptamers(6.85×1013 cm-2)was higher than that of thiol aptamers(4.19×1013 cm-2).But the modification density of amino aptamer decreased with the immersion time prolonging in 1m M HCl which suggested the stability of this sensor was poor.However,thiolated aptamer maintained constant density relatively and could be reused.Thus,the thiolated aptasensor had good reproducibility and stability.5.A simple and sensitive label-free impedimetric aptasensor for rapid determination of OTA has been developed,which was based on the combination between thiolated aptamer and gold nanoparticles.Because of the interaction between aptamer and OTA,the relative normalized electron-transfer resistance(?Rct)values was proportional to the concentration of OTA and showed a good linear relationship from0.1 to 10.0 ng/m L,with a lower detection limit(0.030 ng/m L).The label-free impedimetric aptasensor based on layer-by-layer self-assembly exhibited a good specificity and stability,high sensitivity,time-efficient.6.The established method was successfully applied to detect OTA in wine and grape juice,the recovery was 90.56%-104.21%when PVP effective removed of phenolic substances.The label-free impedimetric aptasensor was used for rapid detection and quantitation of OTA in the inoculated grapes with the Aspergillus nigri(H1)and the production of OTA(62.4?g/kg,20?g/kg)were far exceeded the maximum levels of 2?g/kg after 3 days of inoculation.It showed the label-free impedimetric aptasensor could be applied to detect the OTA concentration in grape and its'commodities.To sum up,the surface of nano-MgO/diatomite ceramic membranes are uniformly distributed with the positive charged nano MgO particles,and regeneration of the membrane is good,which shows good removal effect on OTA standard solution and grape products.The quality and flavor of grape products change little after the treatment.The removal of OTA is accomplished by electrostatic adsorption between nano-MgO particles and OTA,which is a spontaneous exothermic process.Moreover,the surface modification density of the layer by layer self-assembly sensor based on thiolated aptamer is high,and with simple preparation and good stability.The application of gold nanoparticles could increase the amount of aptamer modification,improve the detection sensitivity and obtain a good detection limit.OTA in grape and its products could be directly determined before PVP treatment,and this method has a good recovery. |
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