Researches On Microchannel Resistance Homogeneous Immunosensing Method For The Detection Of Deoxynivalenol In Wheats

Deoxynivalenol（DON）is an important cause of major diseases such as cancer and chronic diseases,which seriously endangers human health and easily pollutes food crops and causes huge economic losses.Due to the possibility of contamination of grains during production,processing and storage,accurate and rapid detection of DON in grains is one of the necessary means to reduce its hazard.At present,the commonly used detection method for DON is high performance liquid chromatography-mass spectrometry（HPLC-MS）.Although this method has good accuracy,it has the disadvantages of cumbersome operation steps,long time,and expensive equipment,which cannot meet the needs of on-site The need for rapid detection.Therefore,it is of great significance to construct a detection method with simple and efficient pretreatment,fast signal readout,sensitivity and convenience.In this project,Fe₃O₄nanomagnetic particles and polydopamine（PDA）were combined to construct Fe₃O₄@PDA nanomagnetic enrichment pretreatment material,which was used for the efficient separation of DON in grains.At the same time,based on the Coulter resistance principle,polystyrene（PS）microspheres are signal probes,combined with immune response,change the state of insulating microspheres in the microchannel,and then cause the change of current,thus constructing a microchannel resistance sensor（Microchannel resistance sensor）with simple operation,low cost and high sensitivity.resistance sensor,MCRS)for efficient detection of DON in wheat.Finally,by coupling antibodies on the surface of Fe₃O₄@PDA,the organic integration of pretreatment and MCRS immunosensors was realized for one-step rapid and accurate detection of DON in wheat.This study aims to provide new ideas for the detection of mycotoxins in food matrices.The specific research contents are as follows:1.Preparation and characterization of DON-enriched material Fe₃O₄@PDAFe₃O₄nanoparticles with uniform size were synthesized by solvothermal method,and a layer of polydopamine（PDA）was coated on the surface to form Fe₃O₄@PDA nanoparticles with shell-core structure.Due to the large number of functional groups on the surface of PDA,it has a high affinity for DON in the sample.In addition,Fe₃O₄@PDA can be rapidly separated under an external magnetic field,so the rapid enrichment of DON in complex samples can be achieved.The experimental results show that 3 mg of Fe₃O₄@PDA can adsorb about 100 ng of DON standard,the enrichment effect is good,and the enrichment is rapid,and the whole process can be completed in about 15 min.In this study,the enrichment method based on Fe₃O₄@PDA was further compared with the commercial immunoaffinity column method,and the results showed that the recovery rates of both methods were between 83 and 103%,and the performance was similar.Therefore,Fe₃O₄@PDA has a good enrichment effect on DON,and has a good application prospect in the pretreatment of mycotoxins.2.Homogeneous detection of DON in wheat by microchannel resistance sensorIn this chapter,polystyrene microspheres were used as signal probes,combined with immunoreaction,to construct an easy-to-operate microchannel resistance sensor for the rapid detection of DON.The complete DON（BSA-DON）and antibody（Ab）were coupled to the carboxyl-functionalized PS microspheres to prepare PS-BSA-DON and PS-Ab.Through a competitive immune response,the aggregation of PS microspheres is caused.When passing through the microchannel under the action of electroosmotic flow,the resistance increases and the current decreases due to the blocking effect.Due to the fixed number of PS-Ab immune sites,the correlation between DON and current can be established.The results show that MCRS is extremely sensitive to the state changes of PS microspheres,which makes the method have good sensitivity.The detection range of DON is 10 pg/m L～1μg/m L,and the detection limit is 768 pg/m L.Compared with large-scale instrument analysis methods,MCRS is composed of basic electronic components and capillaries,and the instrument cost is low;compared with enzyme-linked immunosorbent assay,MCRS homogeneous detection method requires only one step of immune reaction,and the detection time is 30 minutes,which avoids the complicated process.Separating the washing process,the operation is easier.3.Fe₃O₄@PDA-Ab-mediated microchannel resistive sensor for the detection of DON in wheatIn order to combine MCRS with effective pretreatment methods,this chapter utilizes the good biocompatibility of PDA to directly couple antibodies on the surface of Fe₃O₄@PDA to make Fe₃O₄@PDA-Ab specific enrichment materials,thereby combining pretreatment with MCRS.The sensor is organically integrated for fast and accurate detection of DON in wheat.Fe₃O₄@PDA-Ab has a specific adsorption effect on DON in the sample.The enrichment of DON can be achieved by magnetic separation after immunoreaction.The entire enrichment process takes only 15 minutes.Ab-DON can directly immunoreact with PS-BSA-DON,causing the aggregation of PS microspheres,which in turn causes the current to decrease.Since the number of immune sites is fixed,the concentration of DON is directly related to the degree of aggregation,which enables quantitative detection of DON.The experimental results show that only 10μg of Fe₃O₄@PDA-Ab can adsorb about 100 ng of DON.Meanwhile,the detection limit of Fe₃O₄@PDA-Ab-mediated MCRS for the detection of DON was 431.7 pg/m L,which was about 21 times more sensitive than traditional ELISA.The method does not require the elution step of organic reagents,and while shortening the detection time,it also successfully integrates the pretreatment and detection methods,avoids the influence of the complicated pretreatment process on the detection results,and greatly improves the detection accuracy.It provides a good rapid detection platform for the sensitive and accurate detection of mycotoxins in food.