Research, New Organic Phosphorus Detection Method Based On Microplate

Organophosphate pesticides are toxic compounds, which have resulted in many ecological problems and damages. These compounds are inhibitors of acetylcholinesterase (AChE), which catalyses the hydrolysis of neurotransmitter acetylcholine. These pesticide residues are intended to kill living organisms, and a potential dose-related acute and chronic toxicity exists in humans. Traditional methods for insecticide detection are based on gas chromatography (GC) or high performance liquid chromatography (HPLC) coupled with mass selective detectors (MSD), but these are expensive, time-consuming and involved in complicated sample treatment and currently not suitable for field use. Consequently, there is a growing interest in fast and more sensitive detection systems.Biosensor array is widely used as class of biological sensors. It has been applied to medical diagnosis and environmental monitoring because of its characteristics of high-throughput, miniaturization, portability. Biosensor array for detection of organophosphates is promising, especially in screening samples.This thesis includes three chapters as followings. The first chapter introduced the current research activities of biosensor array. Polystyrene microplate was used as support to construct of biosensor array.In Chapter 2, a protocol for enzyme covalent immobilization on polystyrene microplate was developed, employingβ-glucosidase as model enzymes. Recommended conditions for the developed method are as followings. Wells are activated with 2.5% glutaraldehyde for about 15 min at room temperature. Then wells are incubated in 1% chitosan in HAc solution (1 vol. conc. HAc+99 vols. H2O) to provide more potential binding site. Non-specific binding site is blocked in 2% bovine serum albumin. For stabilization of schiff's base intermediate, reduction with 0.1 M NaBH4 in 0.1 M Na phosphate pH 8.0 buffer is included in step. Using this method,β-glucosidase was immobilized on amino-microplate 24 fold greater than no spacer molecular. The storage stability of immobilizedβ-glucosidase retained about 78.9% activity after 60 days at 4℃in pH 5.0 buffers. The developed glutaraldehyde protocol was compared with the manufacturer's recommendation BS3 protocol to assess its practicality and reliability. Glutaraldehyde protocol gave the similar values in 0.7 Uβ-glucosidase solution for 2 h incubation comparing to BS3 protocol, which utilized very expensive BS3 as activation reagent. As for in 1.1 Uβ-glucosidase solution, BS3 protocol resulted in only a slightly higher amount of immobilized enzyme. Thus, the developed immobilization method requires much less cost and also achieves a high efficiency of immobilization to amino-microplate.In chapter 3, biosensor array employed AChE as molecular recognization element was constructed using the above developed method. Under the optimized conditions, the response of the biosensor fabricated was related linearly to concentration of paraoxon in the range of 5.0×10-7-5.0×10-5 g·L-1 and with the detection limit of 2.1×10-7g·L-1.The fabricated biosensor array can be used for high-throughput screening of organophosphate. The performance of biosensor array will be tested and the detection procedure for real samples will be also optimized in the further work..