Enzymatic Organophosphorus Pesticides Biosensors Based On Plant-esterase And Organophosphate Hydrolase Modified Electrodes

In modern agricultural, the widely use of pesticides in agriculture plays an irreplaceable important role due to it can effectively improve the crop and create a huge agricultural yields. Although the use of pesticides can enhance the yield of agricultural industry, they also pose huge threats to environment, public healthy and food safety. Highly neurotoxic organophosphorus pesticides?OPs? irreversibly inhibit the activity of acetylcholinesterase?AChE?, which interferes the function of fatal organs and eventually cause respiratory paralysis and death [1-3]. Up to date, the pesticide detection methods mainly rely on large test instrument, these traditional analytical technologies offer highly sensitive and accurate assays of OPs, but also suffer from many disadvantages such as time-consuming procedures, the use of bulk and requirement of skilled personnel. Therefore, they are not suitable for in-field application.In this study, two cost-effective types enzyme electrochemical biosensors based on the plant esterase?inhibition?/organophosphorus hydrolase?catalytic? were successfully constructed for sensitive detection of OPs. The surface morphology and crystal structure of the prepare biosensors were systematically characterized using UV–vis, SEM, TEM, FTIR, XRD, EDX and Raman. The electrochemical property and mechanism of the biosensor for OPs detection were thoroughly investigated by cyclic voltammetry, differential pulse voltammetry, chronoamperometry and electrochemical impedance spectroscopy. In this dissertation, we focused on following researchs:?1? Highly pure plant-esterase was prepared according to a two-step extraction method reported elsewhere [4], and the surface location of PLaE fusion was confirmed by SDS-PAGE analysis. The Citric acid stabilized gold nanoparticles?AuNPs? was firstly synthesized, we prepared AuNPs-GNs composite which has excellent conductivity and high surface areas through simple mixture treatment. Then the immobilization of PLaE by using chitosan?CS? which has good biocompatibility. Under the optimized operating conditions, the PLaE-CS/AuNPs-GNs composites based biosensor measured as low as 50 ppt?0.19 nM? of methyl parathion and 0.5 ppb?1.51 nM? of malathion?S/N = 3? with a calibration curve up to 200 ppb?760 nM? and 500 ppb?1513.5 nM? for methyl parathion and malathion, respectively. There is also no interference observed from most of common species such as inorganic ions, glucose and citric acid. In addition, its applicability to OPs-contaminated real samples?carrot and apple? was also demonstrated with excellent response recovery. Compared with AChE biosensors which share similar inhibition mechanism to OP pesticides, PLaE can be extracted from a number of plants and easy obtainment, with low-cost, thus holding great potential in development of enzyme electrochemical biosensor for OPs detection.?2? The elastin-like polypeptide-organophosphate hydrolase?ELP-OPH? was purified from cell culture according to a phase-transition method reported elsewhere [5]. Highly pure ELP-OPH was purified from a genetically engineered Escherichia coli?E. coli? which based on the unique phase transition of ELP by a simple and rapid thermal-responsive method, and the surface location of ELP-OPH fusion was confirmed by SDS-PAGE analysis. Titanium dioxide nanofiberswere prepared using electrospinning followed by calcination. Abundant free OP compounds were selectively adsorbed to the TiO₂ NFs surface due to the strong affinity with phosphoric group of TiO₂ NFs, while BSA was used to stabilize OPH activity in the nanocomposite. Under optimized conditions, The ELP-OPH/BSA/TiO₂NFs/AuNPs/GCE biosensor can detect OPs with a fast responsed?less than 5s?, a wide detection ranges?up to 46.4 ?M?, a good sensitivity of 926 ?A.cm-2.mM-1 and the limits of detection?S/N = 3? as low as 26 nM for methyl parathion. Its application for determination of OPs compounds in lake water sample and acceptable stability was also demonstrated.?3? Compared with AuNPs, c-MWCNTs was used to enhance the electron transfer in the amperometric detection as well as for covalent immobilization of ELP-OPH. The as-prepared TiO₂NFs/c-MWCNTs electrodes were immersed into freshly prepared N-hydroxysuccinimide?NHS? and 1-ethyl-3-?3-dimethylaminopropyl??EDC? co-addition solution to active the carboxylic groups on c-MWCNTs surface. Under the optimized operating conditions, the ELP-OPH/BSA/TiO₂NFs/MWCNTs based biosensor for OPs shows a wide linear range, a fast response?less than 5 s? and limits of detection?S/N = 3? as low as 12 nM and 10 nM for methyl parathion and parathion, respectively. Such excellent sensing performance can be attributed to the synergistic effects of the individual components in the nanocomposite. Its further application for selectively monitoring OPs compounds spiked in lake water sample was also demonstrated with good accuracy. These features indicate that the developed nanocomposite offers an excellent bisoensing platform for rapid, sensitive and selective detection of organophosphates compounds.