Studies On Novel Electrochemical Biosensor And Its Application On The Detection Of Environmental Poison And Cancer Biomarker

With the rapid development of industry and agriculture, a large number of pollutant emission has caused serious environmental pollution and ecological resource destruction, which threatens the human survival environment and health. On the other hand, with the improvement of human living standard, people’s health consciousness gradually strengthens. Disease is one of the major threats to human health. Early diagnosis of diseases can greatly shorten the treatment time and improve the cure rate of the disease. Environmental pollution and medical level, two factors that closely related to human health, have become the focus on priority in People’s Daily life. Thus, the development of non-destructive, rapid, high sensitive analysis and detection technology for environmental toxins and disease markers have raised more and more researchers’ attention.Electrochemical biosensor is one of the hot spot of current biosensor development, It adopts immobilized biomolecules or organisms as the sensitive element and electrodes as transducer. Through the detection of electric signal change induced by the reaction between target analytes and sensitive element, the analysis and detection of chemical target can be achieved. Electrochemical biosensor combines powerful analysis functions of electrochemical methods and the specificity of biometrics identification process. Electrochemical biosensor has been widely used in environmental monitoring, food safety, biological medicine, military and other fields for it owns the advantage of good selectivity, high sensitivity, portable instrument, and low cost. This paper focus on development of novel electrochemical biosensors for environmental toxin analysis and testing and detection of cancer markers.In this paper, the main contents and innovative work are as follows:1. A label-free, real-time and high-throughput cell-based impedance sensor for heavy metal toxicity detectionWe utilized self-developed high-throughput cell-based impedance sensor and impedance detection instrument for dynamic monitoring the hepatotoxicity of heavy metal ions Hg2+, Cd2+ and Pb2+ and the inhibition of Zn2+ on Cd2+-induced cytotoxicity. The experimental results reveals the toxic level and interaction rate of three different heavy metal ions and cell’s response pattern to heavy metal ions. The results also revealed that the inhibition of Zn2+ on Cd2+-induced cell apoptosis is dose-dependent. Certain dose of Zn2+ may be beneficial for preventing Cd2+-induced hepatotoxicity. However, its excessive intake may intensify the hepatotoxicity. The protective effects of Zn2+ were based on the anti-oxidant effects of Zn2+ and the occupation of Cd2+’s binding sites on cells. The results may provide some reference for Cd2+ poisoning treatment. In conclusion, we have provided a high-throughput chemical toxicity screening platform, which has broad application prospects in environmental poison evaluation and drug screening.2. An electrochemical DNA sensor for Hg2+ detection based on T-Hg2+-T coordination chemistryThis paper firstly employed AuNPs based colorimetric assay for the detection of Hg2+, which verified T-Hg2+-T mispairing and its selectivity. Then the electrochemical DNA sensor was proposed. The probe DNA which contain several thymidine was firstly immobilized on electrode through Au-S bond. In the presence of Hg2+, the detection DNA could hybridize with the probe DNA, causing ferrocene groups which were tagged on detection DNA being introduced into the electrochemical detection system. By detecting the electrochemical oxidation current of ferrocene, the sensor could be used to detect Hg2+with high sensitivity. The results reveals that the sensor showed a sensitive response to Hg2+ in a concentration range from 5nM to 200 nM, with a detection limit of 2.7nM. In addition, this method afforded well selectivity and reproducibility to Hg2+ detection.3. A carbon nanotubes modified screen print electrode (SPE)-based electrochemical enzyme sensor for rapid detection of okadiac acid (OA).We firstly utilized electrochemical polymerized method for the fabrication of poly(o-aminophenol)_carbon nanotubes (PoAP_CNTs) composite film modified SPE, then we immobilized PP2A on PoAP_CNTs/SPE. The inhibition effect of okadiac acid on PP2A was evaluated by detecting the oxidation current of enzymatic reaction product, p-nitrophenol (p-NP). Owing to the excellent physiochemical characteristic of CNTs, the electrochemical performance of electrode was enhanced, as well as the enzyme loading capacity of electrode. The detection limit of enzyme sensor was 0.67μg/L, which was one order of magnitude lower than that of PP2A colorimetric inhibition assay and PP2A/SPE. The results of spiked sample tests showed acceptable recovery rate and low RSD, demonstrating the applicability of this method for OA assessment in the quality and toxicity monitoring of seafood.4. A novel magnetic beads (MBs)-based electrochemical immunosensor for sensitive detection of okadiac acid (OA)We utilized magnetic beads as carriers of OA-BSA composite to develop a electrochemical competitive immunoassay. OA in sample and OA on MBs competed to react with the HRP-labeled anti-OA antibody, inducing the amount of HRP-labeled anti-OA antibody bound on the MBs inversely proportional to the concentration of OA in solution. The MBs complex was then magnetically immobilized on SPE. By applying the catalysis of HRP, the amount of OA was determined by differential pulse voltammetry. The experimental results indicated that the detection limit of this method is 0.039μg/L and the detection range is 0.05μg/L-50μg/L. By using magnetic beads, the complexity of operation has been significantly reduced. And the time needed for analysis has reduced to 40min. Moreover, the interference of other substances in samples and nonspecific binding could be effectively reduced. The results of OA determination in real samples demonstrate the applicability of this method for field OA monitoring in the quality and toxicity monitoring of seafood.5. An Au nanoparticles (AuNPs) and staphylococcal protein A (SPA) based electrochemical immunosensor for high-sensitive detection of carcino-embryonic antigen (CEA)We utilized AuNPs and SPA as the dual-amplication matrix for antibody immobilization. Due to the good biocompatibility of AuNPs and SPA’s strong affinity to Au atom, more SPA was absorbed on electrode. SPA bind with the Fc portion of antibody, leaving the antigen binding region Fab portion arranged in the lateral surface of the electrode to bind their target antigens. The electron-transfer of redox was hindered by the immune complex. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were employed in the determination of electrode responses and applicability. The immunosensor displays a linear response to CEA with a range from 1pg/mL to 100ng/mL and a detection limit of O.1pg/mL. Furthermore, samples of 1～50ng/mL CEA in rat serum was detected using the immunosensor and the immunosensor showed acceptable recoveries. It can be concluded that the immunosensor provides a promising ultrasensitive immunology strategy for clinical diagnosis.