| Food safety incidents caused by biotoxins take place worldwide, which are not only a threat to human health, but more horrible, used by terrorists as a weapon in biological warfare due to their strong physiological toxicity. In order to fundamentally solve these problems, the each link of food supply chain, from fram to dinner table, must be controlled and monitored in the whole course. However, traditional detection methods of biotoxin contaminations are high performance liquid chromatography and mass spectrum and antibody-dependent immunoassay, which require expensive device and complex and time-consuming operations. Thus they are difficult in satisfaction of rapid and sensitive and convenient demands for current food safety detection. As a consequence, this dissertation was established in the research of food safety detection methods based on the aptamer and time-resolved fluorescence assay technology, to develop a series of progressive, rapid, accurate, sensitive and convenient determinations of Staphylococcal enterotoxins(SEs) through fluorescence resonance energy transfer platform. Besides, the related mechanism was investigated.On the one hand, amine-functionalized Fe3O4 magnetic nanoparticles(magnetic beads) were synthesized through a facile one-pot solvothermal route. The crystal composition, morphology and surface groups were respectively characterized by X-ray diffraction(XRD), transmission electron microscope(TEM) and fourier transform infrared(FT-IR), suggesting the eligibility for surface chemical modifications.At first, the selection of DNA aptamer specific against Staphylococcus enterotoxin A(SEA) was studied based on magnetic separation. After immobilization of SEA on magnetic beads, the SEA-coated magnetic beads were chose as screening target, bare beads as negative selection target and SEB-coated and SEC1-coated beads as counter selection target. SEA aptamer was selected through a modified fourteen-round Flu Mag-SELEX. The binding affinity and specificity of selected sequences recognizing SEA were tested and verified by fluorescence analysis. The results showed that the optimum aptamer owned a low dissociation constant(Kd) of 48.57 ± 6.52 nmol/L, and high selectivity to SEA among other structure-similar enterotoxins and potential interfere proteins existing in food matrices. The potential application of selected aptamer in quantification of SEA in milk was investigated, and with a limit of detection of 8.7 ng/m L.Secondly, based on the similar principle and method, the selection of DNA aptamer specific against Staphylococcus enterotoxin A(SEA) was studied. The SEC1-coated magnetic beads as screening target, bare beads as negative selection target and SEA-coated and SEB-coated beads as counter selection target, SEC1 aptamer was harvested through a modified fourteen-round Flu Mag-SELEX. Kd of the aptamer binding to SEC1 was measured to be 49.43 ± 11.76 nmol/L. The limit of detection of SEC1 was 6 ng/m L.On the other hand, through the one-pot solvothermal synthesis and optimization conditions, the multiple lanthanide-doped(Ln3+-doped) time-resolved fluorescence nanoparticles(NPs) were prepared, which exhibited long lifetimes, good fluorescent property and well-dispersed in aqueous solution. Besides, the modified NPs were available for bio-labeling as probes due to their biological functional groups. The synthesis and modified of nano-probes were characterized by TEM image, time-resolved fluorescence spectrum, XRD, FT-IR and ultraviolet absorption spectrum(UV).During the applications of new the Ln3+-doped NPs in the filed of time-resolved fluorescence assay, the reported ricin aptamer was firstly chose as recognition element to build the high-sensitive detection method. Eu3+-doped KGd F4NPs were applied as a time-resolved fluorescent probe in labeling recognition element. By employing good water dispersibility and high quenching efficiency of graphene oxide(GO), a aptamer-based time-resolved fluorescence assay was developed to detect ricin in the homogeneous portable water. Under the optimal conditions, the detection linear range of ricin covered from 0.05 to 50 ng/m L(R2 = 0.9975), and the limit of detection was 0.008 ng/m L(3S/N). The precision of this method was favorable and the recovery rates for ricin added in portable water(0.075 ~ 12.5 ng/m L) were found to be 89.42% ~ 107.1%. The samples were subjected to the aptamer-based method and commercial applicant ELISA kit, and a good linear correlation was found between the results of two detections. It was inferred that the established method could be feasible in real analysis.Based on successful research of detection for single target biotoxin, this aptamer based time-resolved fluorescent nano-probe technology was applied in multiplex biotoxins simultaneous high sensitive detection. The aptamers of SEA, SEB and SEC1 as recognition elements and multi-color lanthanide-doped NPs as detective signals and GO as high-efficient quencher for broad-wavelength fluorescence, a multilex TR-FRET-based assay was established for simultaneous determination of three SEs. Under the critical conditions of Eu3+: Tb3+: Dy3+ = 2: 1: 3.5 and 0.25 mg/m L GO solution, the linear ranges of detection for SEA, SEB and SEC1 were 0.08 ~ 10 ng/m L, 0.10 ~ 9 ng/m L, 0.05 ~ 5 ng/m L, respectively, and the limits of detection were 0.062 ng/m L, 0.069 ng/m L, 0.040 ng/m L(3S/N) in sequence. The SEs(0.5 ng/m L, 1.0 ng/m L, 5.0 ng/m L) added in milk sample without diluted were detected by the developed method, and recoveries were 92.76% ~ 114.58%. The results showed that this method exhibited features of high specificity, high sensitivity and high throughput, which was specially helpful to improve the analysis performance for multiplex determination in complicated food matrices. |
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