| Diarrhetic Shellfish Poisoning Toxins (DSP) are the most widely distributed marine biotoxins all around the world, with okadaic acid (OA) and its derivatives as the main components. The DSP toxins are accumulated in the bodies of shellfishes, clams and mussels when the toxic algae are consumed by those marine animals. The diarrhea shellfish poisoning symptoms, such as diarrhea, nausea and vomiting will be appeared if people consume such contaminated seafood. Because of its strong toxicity with obviously poisoning symptoms and strong thermal stability, it is difficult to remove DSP toxins by just boiling. So the development of sensitive, rapid and on-field detection assay is required to guarantee consumers’health and life. Recently, the lateral flow biosensor (LFB) has been widely exploited for fast screening of several biotoxins. Compared to other analytical assay, LFB shows some advantages: no complicated sample preparation procedure, visualized results, fast signal response and miniaturization of detection device. In this thesis, a nanocomposites-based lateral flow biosensor for the detection of DSP toxins was established by using DSP toxin antibodies as biological recognition elements as well as using signal-enhanced nano composites as labels. The competitive immune detection format was introduced for the recognition of diarrhea shellfish toxins (okadaic acid). The research of DSP contaminated shellfish in the seafood market was accomplished, and the research results were verified by traditional bioassay with good consistence.The main contents of this thesis include:Firstly, the study of the preparation of gold nanoparticles-silica nanorods (GNPs-SiNRs) material. Based on the comparison of GNPs coverage and loading amounts as well as the stability of GNPs-SiNRs in water solution (coagulate easily or not), the best GNPs-SiNRs material was selected as antibody labels. The antibodies were immobilized onto the surface of GNPs-SiNRs materials by physical adsorption method. The GNPs-SiNRs materials based LFB was built up, and the working conditions of the biosensor (such as the pH value of antibodies adsorbed onto the GNPs-SiNRs surface, the amount of antibodies for making conjugates and et al) were optimized in order to reduce non-specific adsorption, and to improve the detection sensitivity.Secondly, the study of the analytical performance of the biosensor. High sensitive detection of DSP toxins in shellfish samples were achieved. Under the optimal experimental conditions (the pH value of antibodies adsorbed onto the GNPs-SiNRs surface was 9.0, the amount of antibodies for making conjugates was 1 ug/mL and et al), a good linear relationship between signal intensity and standard DSP toxin concentrations was obtained at the range of 15.5-100 ng/mL, and the LOD was as low as 15.5 ng/mL. After one month storage at room temperature, the response signal changes of the same concentration of the target toxin was less than 10%, showing a good reproducibility and stability of the biosensor. Compared to the blank control signal, no significant signal response changes were observed when other non-target toxins (such as aflatoxin, saxitoxin) were tested, while the signal response was greatly decreased with target toxin demonstrating the good specificity of the biosensor. For the DSP toxin contaminated shellfish sample test, a good linear relationship between signal intensity and DSP toxin concentrations in complex matrix was obtained at the range of 16.8-100 ng/mL, and the LOD was as low as 16.8 ng/mL. The dynamic range and the LOD showed no much difference between the test in standard solution and in the real sample, meaning the applicability of the biosensor for the real sample detection. Finally the test results of LFB were verified by mouse bioassay and a good consistence was obtained. |
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