Study On Cell And Molecule-based Biosensors And Their Application In Marine Toxins Detection

Marine toxins, produced by algae, are easily accumulated to a high level in filter-feeding fish or shellfish. People get poisoned commonly by eating the contaminated seafood, and even to death. In recently, there are several human poisoning reported every year with the increasingly severe environmental pollution. Current detection methods of marine toxins include the mouse bioassay (MBA), enzyme-linked immunoabsorbent assay (ELISA), high performance liquid chromate-graphy (HPLC) and liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Nevertheless, each of these methods has their own drawbacks, such as poor repeatability, expensive equipment and ethical concerns. As an emerging technology, biosensor employed biomolecules as the sensing element and combined the second sensor of physics or chemistry to realize the detection of various compounds. Biosensor has the advantages of easy to operate, fast analysis speed, high sensitivity, miniaturization and easy to realize automation. Therefore, biosensor has wide application in cell research, drug screening and food detection.In this paper, we study the potential of different biosensors to detect marine toxins on cell and molecular level.The major contents and contribution of this thesis are as follows:1.Detection of diarrhetic shellfish poisoning (DSP) toxins using high sensit-ivity human cell-based impedance biosensorA human cell-based impedance biosensor are introduced in marine DSP toxin-Okadaic acid (OA) detection. The cell impedance is linearly related to the concentration of OA in the range of 10-100 μg/L. From the results, this method has a good specificity and excellent repetition presents a good correlation with mouse bioassay in OA detection. In summary, all the results indicate that the cell impedance biosensor had great potential to be an effective complement in DSP toxins detection.2. Propose an improved sensitive assay for the detection of paralytic shellfish poison (PSP) toxins with Neuroblastoma cell-based impedance biosensorPSP toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of Neurological disorders symptoms. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na+. Under the treatment of ouabain and veratridine, Neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na+ and ouabain inhibits the activity of Na+/K+-ATPases. Based on that mechanism, we development a novel sensitive assay to detect PSP toxins-saxitoxin (STX) using a cell impedance biosensor. The proposed approach could allow for the determination of STX in the concentration range of 0.1-1000nM. Furthermore, this method shows high sensitivity and good specificity for STX detection in real sample.3. An novel piezoelectric method for the detection of OA based on the indirect competitive immunoassay and combined with gold nanoparticle amplif-ication has been developedIndirect competitive immunoassay was selected in this assay for molecular weight of OA is small. Gold staining solution is introduced and deposits onto nanogold, which results in amplification of gravimetrical signals. And the results show that the sensor signals was linear to the concentration of OA in range of 10-150 ng/mL with a detection limit of 5.45 ng/mL. The accuracy of this method was compared with the ELISA assay, and the result showed that this immunosensors is accurate and reliable in the analysis of practical samples.4. Novel SAW sensor system for OA detection with a lower detect limit and low costWe design a design a kind of sandwich structure of nucleic acid adapted to detect OA molecules in the sample based on the specific binding capacity of aptamer and the characteristics of complementary double-stranded DNA hybridization. The phase of sensor will decreased after OA aptamer shed from the surface of sensor in the presence of OA. The phase shift was linearly related to the concentration of OA in the range of 1-100 ng/mL with a detection limit of 0.3 ng/mL. This method offer a new dimension for toxins detection and the "sandwich construction" has a universal and practical in toxins detection. Furthermore, the application of aptamer not only reduce costs but also shorten the measurement time.