Research Of The Sensitive Detection Methods For Microbial Toxins Based On Upconversion Nanoparticals As Labels

Due to the unique property of rare earth doped UCNPs that emitted shorter wavelength(ultraviolet or visible light) under excitation by longer wavelength (usually near-infrared lightor infrared light) via a two-photon or multi-photon mechanism, the autofluorescence ofbiosamples could be effectively avoided. It had a profound significance on improving thesensitivity and accuracy of detection methods. Microbial toxins contamination are the primaryunsafety factors which causing food security problem, and the related detections were veryimportant to food safety analysis. In this dissertation, rare-earth-doped upconversionnanoparticles (UCNPs) were applied in microbial toxins analysis detection. The applicationsof UCNPs as a kind of novel fluorescent nano-labels were researched in quantitative analysisof microbial toxins in foods. Based on advantages of UCNPs, a series of sensitive, rapid,reliable and simultaneous analytical methods were developed, and those methods couldprovide a guarantee for food safety.Firstly, based on the solvothermal technology and optimization condition, therare-earth-doped nanoscale UCNPs which had good fluorescent property and uniformmorphology were synthesized. And the silanization groups or ligand exchange were thensuccessfully modified on the surface of UCNPs. Furthermore, the modified UCNPs wereapplicable for bio-functionalization due to their biological functional groups andwell-dispersed in aqueous solution. The synthesis and modified of UCNPs were characterizedby transmission electron microscope (TEM) image, fluorescence spectrum, X-ray diffraction(XRD), fourier transform infrared (FT-IR) and ultraviolet spectrophotometer (UV).Secondly, the applications of UCNPs in the field of laser induced fluorescence (LIF)detection were researched in this section. In brief, aptamers were immobilized onavidin-conjugated magnetic nanoparticles (MNPs), and their complementary DNA werelinked to avidin-conjugated UCNPs, then the aptamers hybridized with the complementaryDNA to form the duplex structure, therefore were assembled on to the surface of MNPsgiving a background fluorescent signal. In the presence of analyte, the aptamer preferentiallybond with analyte and caused the dissociation of some complementary DNA, liberating someUCNPs-labeled complementary DNA. Then the remaining of UCNPs-MNPs compoundswere separated with an external magnet, and excited by980nm infrared laser. Based onUCNPs labeling, aptamer affinity and magnetic separation, a novel analytical method hasbeen successfully applied to the detection of ochratoxin A (OTA) related to the intensity ofUCNPs and the detection limit was0.0001ng·mL-1.Thirdly, the applications of dual-colour UCNPs labeling were researched in this sectionbased on the UCNPs successfully used in single LIF detection. Two sets of emission peakswhich could be easily distinguished in spectrum by980nm laser, belonged to differentUCNPs were chosen as dual-colour labels for determination two kinds of analytes. In onedesign pattern, two kinds of UCNPs were used to conjunct with antibodies of aflatoxin B1(AFB1) and ochratoxin A (OTA) respectively. A novel competitive fluorescent immunoassayfor the simultaneous detection of AFB1and OTA using UCNPs as dual-colour labels was developed. The detection limit of AFB1and OTA were0.01ng·mL-1. In another designpattern, two kinds of UCNPs were firstly used to conjunct with the signal nucleic acids, andthen hybridized with target nucleic acids. A novel sandwich type fluorescence analysis forsensitive and selective detection of enterovirus71(EV-71) and coxsackievirus A16(CV-A16)by dual-colour UCNPs labeling technology was also successfully established, and thedetection limit was20pmol/L and25pmol/L, respectively. The results suggested thatdual-colour UCNPs labeling-based fluorescent assay was applicable and promising for thesensitive detection. This assay could also be extended to the detection for various analytesbased on multicolour labeling of UCNPs.Fourthly, the applications of UCNPs as fluorescent energy donors in fluorescenceresonance energy transfer (FRET) were researched in this section. In one design pattern, wepresented a new aptasensor for fumonisin B1(FB1) based on FRET between UCNPs and goldnanoparticles (AuNPs). The quenchers (AuNPs) were attached to the5’ end of the molecularbeacon (MB), and the donors (UCNPs) were attached to the3’end of the MB. The good resultof detection was benefited from UCNPs labeling, aptamer affinity and magnetic concentration.The detection limit was0.01ng·mL-1. Furthermore, the new FRET system based ondual-colour UCNPs as fluorescent energy donors was explored. Graphene oxide (GO) werefound as the entire and effective acceptor because of a good overlap between the fluorescenceemission of dual-colour UCNPs and the absorption spectrum of GO. Hence, a multiplexedFRET system has been first presented and applied on the basis of multiplexed energy donorsto the entire energy acceptor. We have constructed a novel sensor for the simultaneousdetermination of ochratoxin A(OTA) and fumonisin B1(FB1) using a multiplexed FRET fromaptamers modified dual-colour UCNPs on the surface of graphene oxide, and the detectionlimit was0.1ng·mL-1and0.02ng·mL-1, respectively. Our study has paved the way to widenthe applications of FRET systems: the multiplexed FRET-based detection can be used forvarious targets with fluorescence nanoparticles of which emission spectra can bedistinguished as donors.Fifthly, the signal amplification of UCNPs by laser induced fluorescence method wasresearched in this section. Enzyme-catalysed target recycling strategy was an effectiveamplification approach except for magnetic separation and concentration. Against to thecomplex of aptamer and target, exonuclease I, an exonuclease specific to single-strandedDNA, was used to amplify the signal of UCNPs and improve the sensitivity of detection byselectively digesting a particular DNA for analyte recycling. An ultrasensitive bioassay wasdeveloped to detect staphylococcal enterotoxin B (SEB) using UCNPs labeling and anexcellent limit of detection of0.3pg·mL-1was obtained by exonuclease-catalysed targetrecycling strategy. The sensitivity of LIF method was high because of the advanced propertyof UCNPs, and even higher sensitivity was displayed because of exonuclease-catalysed targetrecycling strategy. It was a significant method to solve the problem of ultratrace toxins hard todetect in foodstuffs.In conclusion, lanthanide doped upconversion nanoparticles were synthesized asfluorescent labels in this study. Based on its unique luminescent properties, a series of novel,sensitive, specific, stable and practical assays were developed for the determination of microbial toxins in food coupled with laser induced fluorescence, FRET, aptamer recognition,immnuo-recognition, DNA hybridization, MNPs separation and concentration, andexonuclease-catalysed target recycling strategys. Moreover, multicolor UCNPs fluorescentlabels were successfully applied in food safety analysis and detection, which provide atechnical support for guaranteeing food safety.