Studies On Enzyme Electrochemical Biosensors Associated With Neurotransmitters Based On Porous Electrodes

Electrochemical biosensor is defined by the biological material as asensitive element, the electrode as a conversion element for detectingelectrical signals in bioassay, which are based on potential, current orcapacitance for feature detection signal of the biosensors. Based on theinnovative of the porous electrode preparation, in this paper, the porousworking electrodes were fabricated, the enzymes were immobilized, theexperimental conditions were optimized and the electrochemicalproperties of the fabricated biosensors were investigated. The mainresearch contents are as follows:1. Preparation and characterization of polystyrene microspheres inthe presence of β-cyclodextrin. Using styrene as raw material, potassiumpersulfate as an initiator, β-cyclodextrin as a stabilizer, polystyrene（PS）microspheres were prepared by soap-free emulsion polymerization. Theresults show that after optimized the applied amount of stabilizer, themonomer concentration and the dosage of initiator, polystyrenemicrospheres with finely tailored size can be obtained.2. Preparation and electrochemical behavior of nano-porous pseudocarbon paste electrode（nano-PPCPE）. By mixing graphite powders,polystyrene microspheres and pyrrole in proportion to form a paste,packing the formed paste into the glass tube, and then inserting a copperwire into the paste in the glass tube, inducing polymerization of the pasteand extracting the template polystyrene microspheres, the nano-PPCPEwas fabricated. Experimental results showed that when the applied PSand graphite powder at mass ratio of2:1, the PS with smaller particle size,the obtained nano-PPCPE possess larger specific surface area which iseven up to332.09m²/g. In comparison with the traditional carbon pasteelectrode, the nano-PPCPE has good electrochemical behavior, highsensitivity, good stability and long service life, etc., demonstratingimportant significance in electrochemical detection and analysis.3. Preparation and electrochemical behavior of L-glutamate enzymeelectrochemical biosensor. Using the homemade nano-PPCPE as theworking electrode, mixing L-glutamate oxidase, catalase and bovine serum albumin with phosphate buffer, followed by cross-linking withglutaraldehyde, a novel L-glutamate enzyme electrochemical biosensorwas formed. It was demonstrated that this biosensor has large surfacearea, high selectivity, strong current intensity, excellent selectivity, goodstability, nice reproducibility, easiness to make and use, and has lowpollution in comparison with the traditional carbon paste electrode.Ranging from5×10^-7M to1×10^-5M, the current signal is linear with theconcentration of glutamate, the regression equation is y=2.09x-1.55, andthe regression coeffient of the linear curve is0.995, with the detectionlimit as low as2.5×10^-7M（S/N=3）.4. Preparation and electrochemical behavior of acetylcholine enzymeelectrochemical biosensor. Immobilized acetylcholinesterase to thehomemade nano-PPCPE as working electrode, then the electrochemicalbehavior of the as-prepared biosensor was studied through theacetylthiocholine iodide（ATChI） as an enzyme substrate. Ranging from5×10^-6M to2×10^-4M, the current intensity is linear with theconcentration of ATChI, the regression equation is y=0.59x+9.92, and theregression coeffient of the linear curve is0.991, with the detection limitas low as1.5×10^-7M（S/N=3）.5. Fabrication of an enzyme electrochemical biosensor array forsimultaneous detection of L-glutamate and acetylcholine. Using twohomemade nano-PPCPEs as the working electrodes（modified glutamateoxidase and acetylcholinesterase respectively）, a saturated calomelelectrode as the reference electrode, and a Pt electrode as the counterelectrode to form a four-electrode system, a novel type ofglutamate-acetylcholine enzyme electrochemical biosensor array wasfabricated. It was shown that the simultaneous determination of glutamateand acetylthiocholine iodide enzyme substrate is feasible and almost nodifference is found in comparison with the three-electrode system.