| Neurotransmitters are important molecules in the brain for the transmission of information in the central nervous system, can act on receptors on the cell membrane of neurons, or the dominant effect, thereby completing the information transfer function. Neurotransmitter’s dysfunction, such as conduction disorders and balance broken is one of the important causes of a variety of central nervous system diseases. Comprehensive, real-time understanding the mutual relation between the change of neurotransmitter levels in the central nervous system and dysfunction and disease, it’s contribute to the study the relation of central nervous system disorders, disease pathology and molecular mechanisms. It will make the possible that doctor cure the mental illness completely. Therefore, online sensitive monitoring the level of nervous system neurotransmitter release and study its mechanism of action has become one of the hot point in life sciences, chemistry, clinical medicine, and other fields.Currently, Real time, in vivo, online methods for the neurotransmitter release detection are mainly including vivo microdialysis sampling and analysis techniques and in vivo fast cyclic voltammetry. In vivo microdialysis sampling can be dynamically trace vivo biochemistry from the biological sample, it is little damage on the tissue, sampling less and can be effective in combination with HPLC, capillary electrophoresis separation technology, has become an important research tools for the detection of neurotransmitter releasing. It has play a unique role in neuroscience, pharmacokinetics, biological and behavioral sciences and other areas of drug development, thus it gradually attract researchers’ broad attention in biomedical and analytical chemistry community. In vivo microdialysis sampling technique rapidly developed as a new and sustainable compelling field into the study of life. It can be applied to all tissues and organs of experimental animals, especially the research and application to the detection of neurotransmitters in the nervous system, and the results have been proved very good. It has laid a favorable theory foundation and provides effective research methods to investigate the mechanism of diseases related to the nervous system. In vivo fast cyclic voltammetry is a cutting-edge research in the development of electrochemistry and electroanalytical chemistry field late in1970s. Due to the microelectrode with small size, high spatial and temporal resolution, diffusion efficiency and fast response etc., it has been widely used in the in vivo biological field analysis and single cell analysis.However, in vivo cyclic voltammetry and microdialysis sampling method just study neurotransmitters of quickly spreading to the extracellular fluid in the body, but has not been systematically explored the true level of neurotransmitter instantaneous releasing as the body is stimulated. Since the synaptic gap distance is small less than100nm, to direct detection the release level of neurotransmitter molecules and their activities mechanism in the synaptic gap by electrochemical methods, small size, high sensitivity, high spatial and temporal resolution in diameter less than100nm nanoelectrodes are needed. The purpose of this paper, we designed a simple, green method for preparing a controllable size gold electrode which can be flexibly adjusted the size to meet the requirements according to the subsequent experiments needed. Then the size controllable nanoelectrodes combined with chemical sensing technology, through specific molecular assembly, functional nano-sensors with good selectivity and good stability, sensitivity and biocompatibility can be developed to direct detection neurotransmitters in synaptic gap with high selectivity. The full text is divided into five parts, as follows:Chapter1:OverviewThis chapter mainly introduces the research background, the significance, the classification, the preparation methods, the applications in biochemistry of nano-electrodes. We focus on the preparation and characterization of nano-electrodes and the nano electrode’s applications. Chapter2:Preparation of controllable disk gold nanoelectrodes by electrochemical etching and Hybrid insulationIn this work, we have developed a simple and green method preparing newly, dimension controllable disk gold nano electrode. The gold electrode tip was characterized by SEM, TEM and steady-state cyclic voltammetry. SEM, TEM results showed that the etching potential control at1.7V,φ=25μm gold wire tip can be etched into a tip with radius of～6nm; the hybrid insulation film of the external electrode was uniform and thin about～30nm, so after insulating, the overall size of the electrode tip can keep in nanosized level; After further etching, the electrode tip’s morphology was approximately disklike. The Electrodes were steady cyclic voltammetry scanning in three standard solution (K3Fe(CN)6, Fc, K3IrCl6), all of the cyclic voltammetry have a good "S" shape. The fabrication of nano disk gold nanoelectrode in this work has good reproducibility, the effective radius of the electrode is controllable, and the surface of the insulating layer of the electrode both in the aqueous phase and the organic phase solution are not easy to peel off showing good stability. This part of the work provides a favorable experimental tool for the subsequent calculation of kinetic parameters and biological application.Chapter3:The electrochemical behavior of the disk gold nanoelectrode and its application for detection in a single cellIn this work, different effective radius Electrodes were steady cyclic voltammetry scanning in three standard solution (K3Fe(CN)6, Fc, K3IrCl6) to study the kinetics of the disk gold nano electrode. The transfer coefficients (α) and heterogeneous electron transfer rate constant (k°) of the electrode of the three standard systems were obtained by analysis the statistical data:k°=8.67±3.34cm s-1, α=0.73±0.03for Fc, k°=6.20±4.03cm s-1, α=0.66±0.09for K3IrCl6, k°=4.69±3.26cm s-1, α=0.74±0.06for K3Fe(CN)6, respectively. These experimental results are consistent with the literature, indicating that the mass transmit rate was quickly on the surface of the disk gold nanoelectrode with fast heterogeneous electron transfer rate constant. So the disk gold nano electrode with superior electrochemical performance is conducive to use in biological. After modified by nafion, the electrode has good selectivity, high sensitivity, low detection limits and wide linear range and so on for dopamine detection in0.2M PBS(pH=7.4). PC12(adrenal pheochromocytoma) cells was selected for the Single-cell experiments, nafion modified disk nano-electrodes was successful used to monitor a single vesicle exocytosis release from a single PC12cells. It was also proven that the main components of vesicles was dopamine; most sites on a single cell surface are inactive, only active sites have vesicle release; Furthermore, the distribution of release point on the cell have no obvious rule and not uniform; within the release sites, the number of vesicles release was different in each vesicle release sites and no significant law on the release; if in the same active release point can be distinguished with high resolution using the nanoelectrodes. Disk gold nanoelectrode makes feasibility of direct detection neurotransmitter molecule in the synapse which was between the nerve cells.Chapter4:Preparation of gold nanoelectrodes based on chemical deposition of gold nanoparticles on quartz nanocapillary tip and its application for the detection and analysis of dopamine in rat brainThis work was mainly discusses how to fabricate gold nanoelectrodes with the quartz glass tapered tip which attached a layer of gold particles on the surface of it and the gold nano-electrode applied in vivo analysis after modified by nafion. The nanoelectrodes of this work were size controllable by controlling the electrophoretic time, the potential of electrophoresis, electrophoresis number and deposition time of the gold particles.First, adjusting the laser Pullers’(P-2000, Sutter Corporation) five parameters to drawn a capillary into tapered tips with nanometer sizes; then have the tapered tip hydroxylated, functionalization by modified it in silane coupling agent, adhesion gold nanoparticle as the growth factor, the surface gold nanoparticles film is formed by dipping it into the chloroauric acid and hydroxylamine hydrochloride growth solution; finally the tapered tip of attached gold nanoparticles was connected to copper wire by a silver conductive adhesive and carefully placed into a cylindrical glass tube to get the gold cylindrical electrodes. Gold cylindrical electrode was insulation packaged by cathodic electrophoretic paint to get a gold nanoelectrode. SEM results show that gold electrodes with hemispherical tip. The electrochemical results show:a good "S" shape voltammogram was obtain when it cycle voltammetry scanning in a standard solution such as the ferricyanide, ruthenium hexamine trichloride. The radius of electrodes are calculated that its size achieve a nanometer size. After nafion modified, gold nano electrodes shows a good selectivity, wide linear range (5.6×10-5～2×10-8M), good stepped response, high sensitivity, low detection limit of1×10M for dopamine in2M PBS (pH=7.4). Gold electrode size is small and successful implanted it into the striatum of anesthetized SD rat’s brain and online current-potential curve was obtained. The experimental results indicate that releasing levels of dopamine in the rat’s striatal can be affected by external drug stimuli. The concentration of dopamine was up to49nM when stimulated the anesthetized rat with20mg kg-1of nomifensine by intraperitoneal injection.Chapter5:The electrochemical behavior of the gold nanoelectrode with the surface deposited different morphologies of gold nanomaterialIn this work, we have successfully prepared gold nano electrode by laser puller and finely polished with controllable size and good stability. Then the gold nanoelectrodes were acted as a base attached gold nanoparticles with different morphologies, and the effects of different morphologies gold nanoparticles response to dopamine were studied. The electrodes’ surface morphology was characterized by optical microscopy, SEM, TEM. The smallest effective radius of the electrode is6.82nm. Under+200mV, gold electrode tip attached with tapered gold nanoparticles modified by nafion shows a best response to dopamine, it was because the tapered gold particles have good self-purification capacity and good stability by itself. After calibration, the limit of detection of dopamine (S/N=3) were:cone gold nanoparticles (AuNP):～5.2×10-9 M, rod-shaped gold nanoparticles (AuNR):～1.71×10-8M, spherical gold nanoparticles (AuNS):～3.2×10-8M respectively. The linear range of conical gold nanoparticles to dopamine was1×10-8～2.55×10M. The gold nanoelectrodes deposited with tapered gold nanoparticles was implanted into the stratum of the anesthetized rats brain working as the working electrode to in vivo detection of the release level of dopamine in the rat brain. Subsequent experiments will take this as a base for exploring the nanoelectrodes’ modification or as a probe applying in electrochemical scanning microscopy technique to study the electrode surface behavior, and further applied it to detect the biological signaling molecules. |
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