| Mycotoxins are poisonous compounds produced by fungi,which can be generated by various toxigenic fungi.These mycotoxins,including deoxynivalenol(DON),T-2 toxin,and fumonisins B1(FB1),are major Fusarium toxins that coexist in food crops,posing a significant threat to the health of both humans and animals.Currently,quantitative detection methods for mycotoxins mainly rely on chromatographic and immunological techniques,which can be complex and costly or have low accuracy,respectively.Moreover,most of these methods target only a single toxin,limiting their usefulness.To address these limitations,this study developed a microfluidic chip-based platform for the quantitative detection of mycotoxins.This platform utilizes upconversion fluorescence-encoded microspheres as coded signal detection probes,with microfluidic chips as detection carriers.The optical signals obtained from the multi-color up-conversion fluorescent coded microspheres are collected under a dual-wave microscope system and analyzed by an algorithm to determine the types and concentrations of toxins present.To validate the effectiveness of this platform,corn flour was used as the food matrix,and DON,T-2,and FB1 toxins were analyzed.The research findings demonstrated the utility and feasibility of this platform for detecting multiple mycotoxins in a complex sample matrix.The primary research contents and results are presented below:(1)The present study details the preparation and characterization of upconverting fluorescence-encoded microspheres.Specifically,six types of core-shell upconversion nanoparticles(UCNPs)with varying colors were synthesized via high-temperature solvolysis and the layer-layer method,with Na YF4 serving as the matrix and the rare earth elements’doping type and ratio being altered.Characterization results reveal that the UCNPs boast uniform morphology,excellent dispersion,an average crystal core diameter of 28.81 nm,an average core-shell particle diameter of 41.26 nm,and differing fluorescence spectrum properties.Carboxyl microspheres were employed as encoding carriers,enabling the successful preparation of uniformly dispersed upconversion nanoparticles encoded microspheres(UCNMs).Characterization results confirmed that the UCNMs possess a 10μm diameter and the optical encoding capacity of UCNPs.With stable fluorescence performance,high fluorescence intensity,substantial coding capacity,and no photobleaching,UCNMs offer a material foundation for the subsequent detection of multiple mycotoxins in grains.(2)A multiple detection platform based on a microfluidic chip was constructed.The chip template parameters were optimized,and a template with a 15μm diameter,a 10μm depth,and a 7μm pitch was selected as the detection template.The material and characteristics of the chip were also optimized to obtain an opaque polydimethylsiloxane(PDMS)chip that addresses microsphere aggregation and light interference issues,while remaining cost-effective.The detection system used a dual-wave microscopy device with multi-color up-conversion fluorescence-encoded microspheres,which was capable of switching between near-infrared light excitation,visible light excitation,and white light source in a time sequence.This allowed for microscopic imaging of the same sample at the same position and angle of view,leading to the decoding of multi-color up-conversion coded microspheres and high-sensitivity imaging of detection signals.A self-written algorithm served as the analysis program to analyze the collected sample images.The program control system continuously collected sample images,identified the up-conversion coded microsphere images of different colors,and made a qualitative judgment of the type of toxin.The system also continued to identify and calculate the signal molecule image,eventually leading to the accurate results of the sample test.This platform provides a solid foundation for the subsequent detection of multiple mycotoxins in food samples.(3)A method has been developed for the simultaneous detection of DON,T-2,and FB1toxins in corn flour,based on the principle of indirect immunocompetition.This was achieved by preparing fluorescent probes,optimizing the amounts of antigen and antibody utilized in the detection process,and eliminating the interference between multiple detections.The method utilizes upconversion fluorescently encoded microspheres for the quantitative detection of various mycotoxins in corn flour.The linear fitting equation for DON within the range of 0.500-800 ng/m L is Y=-50.38Lg X+178.44,with an R~2 value of 0.9855,and a limit of detection(LOD)of 41.35 ng/m L.When spiked corn flour samples were detected,the recovery rate of standard addition was between 93.07%-106.12%,with a relative standard deviation lower than 1.81%,and a coefficient of variation between batches and batches lower than 5.12%.For T-2 within the linear range of 0.625-10.0 ng/m L,the linearity of the fitting equation is Y=-53.49(Lg X+2)+188.08,with an R~2 value of 0.9908,and a LOD of 0.5044 ng/m L.The spiked recoveries of the samples were between 96.83%-114.82%,with low relative standard deviations,and a coefficient of variation between batches and within batches lower than 5.43%.For FB1within the linear range of 0.500-800 ng/m L,the linear fitting equation is Y=-56.44Lg X+184.46,with an R~2 value of 0.9842,and a LOD of 35.45 ng/m L.The spiked recoveries were between90.76%-113.46%,with relative standard deviations all lower than 2.44%,and inter-batch and intra-batch coefficients of variation all lower than 4.97%.To summarize,compared with other recently reported mycotoxin detection methods,this detection platform offers a wider range of coding,detection,and high sensitivity at low cost.This provides a powerful technical tool for screening and quantitative analysis with greater accuracy. |
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