Study On Capillary Electrophoresis-electrochemical Detection By Chemically Modified Electrode And Its Application On Neurotoxicity Of TBBPA

Brominated flame retardants(BFRs)have been widely used in plastics,textiles,electronic circuitry and other materials to prevent fires.They can accumulate in the environment and biont,and have been proved to be harmful to mammals,birds and fish.Because of their wide spread,stable structures and uncertain risk to human health,great concern has been aroused. Their effects on the hepatic enzyme activities,thyroid gland,nerve system,and immune system have been studied.Recent studies have shown that neonaltal exposure to some of the BFRs produce persisitent aberrations in spontaneous behaviour and affect learning and memory function in the adult mice.These findings suggest that several of the BFRs must be neurotoxic.In order to investigate the neurotoxicity of TBBPA on molecular level,monitoring monoamines,neuropeptide in brain,plasma,urine,and tissue is useful for studying the role of them in neurophysiology,behavioral effects,pathology,disease diagnosis and control.Since the concentrations of these molecules in brain are extremely low,it is important to develop highly sensitive analytical techniques with high resolution for their detections.Recently,capillary electrophoresis(CE)has exhibited powerful capability for the analysis of complex samples with many advantages,such as the high separation speed and efficiency,low volume sample for analysis,the relatively simple instrumentation and very low running costs.Amperometric detection is an important detection method due to its sensitivity.It has been successfully coupled to CE to detect electroactive analytes.Common amperometric detectors,based on glassy carbon or carbon paste electrodes,exhibit no response for many analytes because of a high overpotential.The benefits of using chemically modified electrodes(CMEs)in these systems include acceleration of electron transfer reactions,permselective transport,reduction in fouling,and preferential accumulation of analyte.In this dissertation,we laid our emphasis on the research of novel methods of sample analysis in neuroscience by CE-AD couple with chemically modified electrodes(CMEs).To our knowledge,it was the first time by using CE-AD couple with CMEs to determine the monoamine neurotransmitters,neuropeptides in rats' brain after exposure to TBBPA.The dissertation includes five chapters: Chapter 1.IntroductionIn this chapter,it introduced the characteristics of TBBPA,neurotransmitters and CE.The goal and significance of this dissertation are introduced too.Chapter 2.Determination of monoamine neurotransmitters in rats' brain after exposure to TBBPA by CE-AD with Platinum Nanoparticles(PtNPs)modified electrodeSeveral studies have shown that BFRs affect the dopaminergic system in CNS,especially the synaptosomal and vesicular uptake.It is suggested that such toxins may influence the level of monoamines neurotransmitters.In this chapter,the fabrication and application of CE-AD with the platinum nanoparticles modified electrode were described.The chemically modified electrode had a good catalytic effect on monoamines neurotransmitters.In CE-AD with dopamine(DA),epinephrine(E),norepinephrine(NE)and 5-hydroxytryptamine(5-HT) in rat brain were separated and determined effectively at CMEs.The electrophoresis conditions followed as running voltage was 15 kV,working potential was 1.0 V,running buffer was 160 mmol/L Na₂HPO₄～NaH₂PO₄(pH=5.8)and the injection time was 10 s.A good linear relation was observed in 0.01～10 nmol/L.The detection limits of them was 2.3,2.7,3.1 and 3.3 pmol/L(S/N=3),respectively.The method can be applied directly to the determination the four monoamines neurotransmitters in brain sample with satisfactory results.Chapter 3.Determination of TRH in rats' brain after exposure to TBBPA by CE-AD with Multiwalled Carbon Nanotubes modified electrodeStudies have shown that neonatal exposure to some brominated flame retardants(BFRs) produces neurobehavioral aberrations.In our study,thyrotropin-releasing hormone(TRH) was used to explore the neurotoxicity of BFRs.Tetrabromobiaphenol-A(TBBPA)was administered intraperitoneally(i.p.)to investigate its effect on TRH levels in rat brain.CE with electrochemical detection(ED)using a carbon nanotubes(CNTs)-modified carbon electrode in conjunction with on-capillary copper complexation was developed for the determination of TRH.TRH forms an electroactive Cu(â…¡)complex thus is detectable with the above method.Carbon nanotubes have excellent electrocatalytic activity,so the CNTs-modified carbon electrode was used to improve the sensitivity to at least 10-fold.The detection limit for TRH was 0.3 nmol/L(S/N=3)with the linear calibration curves ranging from 1～100 nmol/L,which enables the possibility for the determination of TRH in biological samples.An increase TRH concentration in different regions of rat brain was observed 24 h after administration of TBBPA in a single dose of 1000 mg/kg body weight.The highest TRH concentration was detected in hippocampus.It was also found that the higher dose of TBBPA affects the TRH level in brain much more.The present study shows that neuropeptides could be used as targets to evaluate the neurotoxicity of BFRs.This rapid,effective and sensitive method can be used to study the neurotoxicity of BFRs via detection of TRH in rat brain.Chapter 4.Determination of enkephalins in rats' brain after exposure to TBBPA by CE-AD with Platinum Nanoparticles(PtNPs)modified electrodeTo observe the changes of M-ENK and L-ENK concentration in the brain regions such as hypothalamus;hippocampus;cerebellum and cortex to explore the neurotoxicity of TBBPA, CE-AD applied with a platinum nanoparticles(PtNPs)modified electrode was developed. This work on M-ENK and L-ENK assays has been established based on the reversible electrochemistry of the Cu(â…¡)/Cu(â…¢)coupled with M-ENK and L-ENK.Under the optimum condition,M-ENK and L-ENK could be separated in 75 mmol/L Na₂B₄O₇～NaOH(pH=9.8) containing 1.0 mmol/L Cu²⁺.A good linear relation was observed in 0.1～50 nmol/L.The detection limits of them was 7 and 8 pmol/L(S/N=3),respectively.This rapid,effective and sensitive method can be used to study the neurotoxicity of BFRs via detection of enkephalines in rat brain.Chapter 5.Determination of GSH and GSSG in rats' brain after exposure to TBBPA by CE-AD with Multiwalled Carbon Nanotubes/Mv RuO-RuCN Films modified electrodeGlutathione(GSH)is often involved in protective and detoxifying functions of the cell. Glutathione disulfide(GSSG)is known to be formed in biological systems because of the function of GSH as antioxidant.Analysis of GSH and GSSG is of continuous interest because of their biological significance.To determine concentration of GSH and GSSG,a glassy carbon electrode doped multiwalled carbon nanotube was deposited from a solution containing micromolar concentrations of RuCl₃ and K₄Ru(CN)₆ to fabricate the GC/MWCNTs/mv RuO-RuCN as a working electrode.It was found that the CMEs exhibited efficiently electrocatalytic effect on GSH and GSSG.A good linear relation was observed in 0.01～10μmol/L.The detection limits of them was 2.8 and 1.2nmoI/L(S/N=3),respectively. This rapid,effective and sensitive method can be used to study the toxicity of BFRs via detection of GSH and GSSG in rat serum.