Study On Novel Hyphenated Techniques Based On Electrochemistry And Their Applications In Neuroscience

Isolation and analysis of the complex system has become an important research direction of current analytical chemistry in the development of biological sciences.To obtain comprehensive and accurate data about complex samples,the analytical process includes trace analysis,dynamic analysis and nondestructive analysis. Accordingly,the analytical methods must be accurate,sensitive and selective to realize the in vivo and on-line analysis.It is apparent that a single analytical technique is no longer qualified for the analysis of complex biological samples.The hyphenated technique is one of the trends in the development of analytical chemistry in the future.In this dissertation,we laid our emphasis on the establishment of hyphenated techniques based on electrochemistry and their applications in neuroscience.The electrochemical determination was coupled with high performance liquid chromatography for the separation and analysis of complex biological samples.The introduction of microdialysis sampling technique carried out the on-line determination. On the other hand,we combined electrochemistry with photochemistry and developed a novel semiconductor nanomaterial photoelectrochemical system for determination of enzyme inhibition.This dissertation includes five chapters:Chapter 1.Liquid chromatography-electrochemical detection for studying the effects of tetrahydrobiopterin on monoamine neurotransmitters in rat striatum Tetrahydrobiopterin(BH₄)is an essential co-factor in the biosynthesis of monoamine neurotransmitters.A nano-Pt/Pd modified electrode as the electrochemical detector for high-performance liquid chromatography coupled with microdialysis sampling technique,is used to explore the effect of administration of BH₄ on the monoamine neurotransmitters in the rat striatum.The researches demonstrate that the contents of dopamine,5-hydroxytryptamine, 5-hydroxyindoleacetic acid and homovanillic acid increase significantly with the administration of BH₄.The pharmaceutical kinetics is carried out to research into the time course of BH₄ effect on the concentration of monoamine neurotransmitters in rat striatum,which provides reliable data for pathology and pharmacology research on neuroscience.Chapter 2.Studying the effects of endogenous neurotoxins on the monoamine neurotoxin in rat striatum with liquid chromatography-electrochemical detectionEndogenous neurotoxins(R)-salsolinol and N-methyl-(R)-salsolinol have been reported to be involved in the pathogenesis of Parkinson's disease.In this chapter a novel electrochemical detector with the acetylcholine film for high performance liquid chromatography was described.It was found that the chemically modified electrode exhibited efficiently electrocatalytic effect on the current responses of(R)-salsolinol, N-methyl-(R)-salsolinol and monoamine neurotransmitters.Combined with microdialysis sampling,the method was successfully applied to study the effect of endogenous neurotoxins on the concentrations of monoamine neurotransmitters in rat brain.Chapter 3.Effect of endogenous neurotoxins(R)-salsolinol and N-methyl-(R)-salsolinol on the balance impairment between dopamine and acetylcholine in rat brainThe endogenous neurotoxins such as(R)-salsolinol and N-methyl-(R)-salsolinol have long been thought to play a major role in Parkinson's disease.Much attention has been paid on the degeneration of dopamine neurons induced by these neurotoxins. However,in this chapter we reported a novel effect of endogenous neurotoxins on the balance between dopamine and acetylcholine by impairing cholinergic system as well as dopaminergic system.We took advantage of high performance liquid chromatography coupled with electrochemical detection to detect the concentrations of dopamine and its metabolites.Meanwhile,we assessed the influence of neurotoxins on acetylcholinesterase activity and developed a novel microdialysis-electrochemical device to measure acetylcholine concentrations with enzyme-modified electrodes.The results demonstrate that endogenous neurotoxins(R)-salsolinol and N-methyl-(R)-salsolinol not only affect the dopaminergic system by inhibiting the related enzymes in the synthesis or metabolism of dopamine,but also impair the cholinergic system by inactivating acetylcholinesterase.Both phenomena would lead to the disruption of balance between dopamine and acetylcholine and contribute to the development of Parkinson's disease.Chapter 4.A new microdialysis-electrochemical device for in vivo simultaneous determination of acetylcholine and choline in rat brain treated with N-methyl-(R)-salsolinolAcetylcholine(ACh)and choline(Ch)play a critical role in cholinergic neurotransmission and the abnormalities in their concentrations are related to several neural diseases.Therefore,the in vivo determination of ACh and Ch is important to the research on neurodegenerative disorders.In this work,electrochemical biosensors based on poly(m-(1,3)-phenylenediamine)(pmPD)and polytyramine(PTy)modified enzyme electrodes were fabricated.The electropolymerized pmPD polymer was used to exclude interfering substances and the PTy layer facilitated the immobilization of acetylcholinesterase(AChE)and choline oxidase(ChOx).Then,ACh/Ch sensor and Ch sensor were coupled with microdialysis to produce a novel device,which provides a sensitive and selective method for simultaneous determination of ACh and Ch.The integrated device was successfully applied to assessing the impact of endogenous neurotoxin N-methyl-(R)-salsolinol on ACh and Ch concentration,which is of great benefit to understand the pathogenesis of Parkinson's disease.Chapter 5.Study on acetylcholinesterase inhibition induced by endogenous neurotoxin with enzyme-semiconductor photoelectrochemical systemThe inhibition of acetylcholinesterase(AChE)induced by N-Methyl-(R)-salsolinol, a dopamine-derived endogenous neurotoxin,has been considered to participate in the pathogenesis of Parkinson's disease.Therefore,a sensitive and simple method for the evaluation of AChE inhibition is urgently needed.In this chapter,the well-ordered and uniform TiO₂ nanotubes are fabricated by electrochemical anodic oxidation and modified with Au nanoparticles by a novel photoelectrochemical deposition.Then,the integration of semiconductor TiO₂ nanotubes with biomolecule AChE yields a novel AChE-Au-TiO₂ hybrid system,which provides a new,valid and rapid photoelectrochemical approach to the determination of AChE inhibition induced by endogenous neurotoxin.Due to the separation of excitation source and detection signal,the photoelectrochemical method has been proved to be not only simple but also quite sensitive.Furthermore,the semiconductor TiO₂ nanotubes can be also coupled with other enzymes to design diverse photoelectrochemical sensors for the inhibition of enzymes.