Study And Application Of Microdialysis Sampling-electrochemical On-line Analytical System In In Vivo Measurement Of Brain Chemicals

Life science is a subject which focuses upon the researches of life process and the mutual relationships of life organisms and its neighboring environment. Nowadays, a series of vital problems we pay attention to, such as, human survive, health, population explosion, food safety, etc, have been demonstrated to be tightly associated with the development of life science. Meanwhile, the rapid progress in life science has also boosted the intersection of some related disciplines, including mathematics, chemistry and physics. What's more important, the successive technology breakthroughs in life science area like the discovery of DNA helical structure in the1950s, the innovation of PCR technique and the asexual reproduction in the90s, further confirms the leading position of life science which will definitely become the most important part in the future frontier research fields.All brain functions, including memory, movement, emotion, etc, have always involved the participation of various chemicals. The existence of the small molecular biological active substances (neurotransmitters and neuromodulators) provides the essential preconditions for these mentioned brain functions. Neurotransmitters are one of these chemicals that are secreted by neurons and relay messages to the target cells. The brain contains a vast network of neurons that are connected with each other at specialized junctions called synapses. Chemicals that are released into the synaptic gap interact with its corresponding receptors, which then lead to the intracellular changes in the postsynaptic neuron, manifested by the altered membrane potential or gene expression. The chemical signal is terminated by transport proteins that transfer transmitter molecules across the membrane to the intracellular space. This is just how the intact network operates and how the behaviors happen.On-line analytical techniques, consisting of microdialysis sampling and direct on-line detector, without any sample separation or pretreatment, are becoming increasingly common for continuous measurement of glucose and glutamate etc. in biological samples. Due to its short analytical time, high sensitivity and specificity to provide near real-time measurements, microdialysis sampling can provide both the temporal and chemical information that we need to fully elucidate the biochemical processes. Compared with various detection techniques coupled with microdialysis, electrochemical biosensors have been proved more efficient in view of its high time and spatial revolution, easy-to-prepare, and excellent selectivity and sensitivity. With these advantages, microdialysis-electrochemical biosensor has become a facile and reliable tool for the real-time determination of neurotransmitters in vivo.The main work of this paper focuses upon novel analytical methods and technique design that is suitable for life science. A series of promising nanomaterials based on metal nanoparticles and carbon materials are prepared and employed in the construction of enzyme biosensors. Combined with microdialysis sampling technique, the effective on-line detection system is developed in our group and applied for the real-time, continuous on-line measurement of physiologically important species such as glutamate, glucose and lactate. The method demonstrated here has been proved to be sensitive and reproducible in this paper, which enables its promising application in physiology and pathology. The primary research work is as followed:Chapter1OverviewIn chapter1, we mainly elaborated the development of microdialysis sampling technique and its combination with electrochemical biosensor, which is then applied for the real-time on-line determination of neurotransmitters in vivo. Moreover, we also pay more attention to the introduction of novel analytical principle and method into biology and chemistry based on the integration of functionalized nanomaterials with electrochemical biosensors. In the end, we also emphatically pointed out the purpose and significance of our research, as well as the innovation spot and contents.Chapter2Construction of on-line microdialysis-electrochemical biosensor systemFor the purpose of real-time, on-line, continuous determination of neurotransmitters in vivo, an effective on-line microdialysis-electrochemical biosensor system was constructed in our work. This system consisted of two parts:microdialysis sampling and electrochemical detection. The microdialysis sampling was powered by a microdialysis pump, and a perfusion fluid could be pumped into the rat brain through a microdialysis probe at a very low perfusion rate, which was then detected by the electrochemical biosensor. It was performed under a totally sealed condition, and both the sampling and detection procedure was continuously performed, so every time point change of the extracellular concentrations of neurotransmitters in brain can be detected in real time. For the preparation of electrochemical biosensors, various namomaterials including metal nanoparticles and carbon materials have been involved, which achieved a high sensitivity and selectivity. Through the integration of microdialysis with electrochemical biosensors, the on-line, real-time measurement of some physiologically important species such as, glutamate, glucose and lactate have been realized in vivo.Chapter3Morphology-tunable TiO2Nanostructure self-assembled on Graphene nanosheet for the Preparation of Palladium Nanoparticle and their Electrochemical application in Hydrogen Peroxide ReductionMorphology-tunable TiO2nanostructure synthesis using graphite oxide and TiCl3as the starting materials is demonstrated for the first time. Our results elucidate that both weight deposition ratio and morphology-controllable TiO2nanostructures can be facilely achieved on graphene nanosheet by simply manipulating the feeding volume of TiCl3, from0.6,1.2,1.8,2.4to3.0ml (i.e., TiCl3/GO weight ratio=9.4,18.8,28.2,37.6,47.0), forming five GTx nanocomposites (x=0.6,1.2,1.8,2.4to3.0). The nanostructures of TiO2are crystalline and vary from spherical to needle-like with the increase of TiCl3dosage. Moreover, these tunable GTx nanocomposites are further successfully employed as supporting materials for the reduction and dispersion of Pd nanoparticles (NPs). The photogenerated electrons from UV-irradiated TiO2are transported across the GTx composites to stepwise reduce Pd2+into Pd NPs. The diameters of these as-prepared Pd NPs show a similar GTx-dependent behavior. The five GTx-Pd hybrid nanocomposites are also observed to play a key role in deciding the electrochemical performance of H2O2, thereby tuning their electrocatalytic activities. Hence, this study demonstrates us a novel and facile method for constructing high-quantity graphene/metal oxide semiconductor/metallic NP hybrids with tunable morphology, size as well as electrocatalytic activity via an effective and low-cost method and their potential application in biocatalysis.Chapter4Development of On-line Microdialysis System with Poly(amidoamine)-encapsulated Pt Nanoparticles Biosensor for Glutamate Sensing in VivoIn this work, on-line microdialysis system with amperometric detection was constructed for in vivo glutamate measurement in rat's brain successfully. Firstly, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt) was synthesized and its electrochemical behaviors were investigated by linear sweep voltammetry (LSV) and amperometric i-t curve. Combined with MWCNTs fabrication, MWCNTs/PAMAM/Pt/Nafion modified electrode was prepared. It demonstrated excellent electrocatalytic responses to the reduction of H2O2at a potential of-200mV without HRP participation. Then, glutamate oxidase (GlutaOx) was immobilized on the MWCNTs/PAMAM/Pt films for glutamate measurement with on-line microdialysis system, which was powered by a microdialysis pump. The glutamate biosensor in the on-line microdialysis system showed good linear range from1.0μM to50.0μM with the detection limit of0.5μM (S/N=3). Under the above optimal conditions, the content of glutamate in the striatum of rat was detected in vivo continuously with this on-line system by implanting of microdialysis probe. The concentration of glutamate was5.80±0.12μM (mean±s.d., n=3) in the striatum of rats. It was proved to be sensitive and reproducible with wider linear range. These properties enabled its promising application in physiology and pathology.Chapter5Size-controllable Gold-Platinum Alloy Nanoparticles on Nine Functionalized Ionic Liquid Surfaces and Their Application as Electrocatalysts for Hydrogen Peroxide ReductionA series of room temperature ionic liquids (RTILs) containing different functional groups, such as hydroxyl, nitrile, carboxyl, and thiol attached to imidazolium cations, combined with various anions, e.g., chloride [Cl], tetrafluoroborate [BF4], hexafluorophosphate [PF6], bis[(trifluoromethyl)sulfonyl]imide [Tf2N], are successfully synthesized. Dissolved in chitosan (Chi), the RTILs are employed as flexible templates for the preparation of Au/Pt nanostructures. These Au/Pt nanostructures can be facilely in situ deposited on the surface RTILs through electrodeposition. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrate that the alloy size is significantly dependent on the structure of RTILs, with their sizes ranging from2.8nm to84.7nm. Based upon the functionalized RTILs, we fabricate nine RTIL-Au/Pt biosensors and the size-dependent electrochemistry of RTIL-Au/Pt is firstly investigated using potassium ferricyanide as the probe. Reversible electron transfer of Fe(CN)63-/4-redox couple is realized at nine biosensors while the peak current, as well as the peak-to-peak separation (⊿Ep) and electron transfer rate, differs greatly from each other, which is mainly caused by the diversity of RTILs. Further electrochemical researches reveal that the functional groups of these RTILs exert an obvious influence on the reduction behavior of H2O2, which in turn illustrates that the electrocatalytical activity of RTIL-Au/Pt nanocomposites can be tuned by means of employing RTILs with different functional groups and an appropriate combination of cations and anions could produce a higher activity. The facilitated electron transfer and the intrinsic catalytical activity of Au/Pt NPs provide us a facile way to construct a third generation H2O2biosensor with high sensitivity, lower detection limit, quick response time and excellent selectivity.Chapter6[C3(OH)2mim][BF4]-Au/Pt Biosensor for Glutamate Sensing in Vivo Integrated with On-line Microdialysis SystemA new type of hydroxyl functionalized room temperature ionic liquid (RTIL),[C3(OH)2mim][BF4], was synthesized herein and a novel H2O2biosensor was fabricated with [C3(OH)2mim][BF4] as the substrate and ultrasonically electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C3(OH)2mim][BF4] film. It was found that the functionalization of RTIL with hydroxyl groups provides an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs, of which the average diameter was2.5run. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic responses to glutamate at a potential of-200mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was successively set up and used for the detection of glutamate in the striatum of rats. The glutamate biosensor in the on-line microdialysis system showed good linear range from0.5μM to20.0μM with the detection limit of0.17μM (S/N=3). The application of the GlutaOx-[C3(OH)2mim][BF4]-Au/Pt-Nafion electrode is demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of100mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. The method demonstrated here was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.Chapter7Size-Tunable Pt nanoparticles assembled on ordered mesoporous carbons for the Simultaneous and On-line detection of Glucose and Lactate in Brain microdialysateThis study presents a facile electrochemical method for simultaneous and selective on-line detection of glucose and lactate in the striatum of anaesthetic rats through the integration of selective electrochemical detection with in vivo microdialysis system. A positively-charged polyelectrolyte,(diallyldimethylammonium chloride)(PDDA), was attached onto carbon mesoporous material (CMM) through non-covalent interaction, which provides an ideal environment for the dispersion of nanoparticle electrocatalyst. Platinum nanoparticles with uniform distribution and wide Pt loadings from5to50wt%are successfully self-assembled on PDDA-functionalized CMM via electrostatic interaction. TEM results show that with the increase in the Pt loadings on CMM, both the size and interconnectivity between particles increase, with Pt sizes ranging from3.2±0.4to6.8±1.4nm and interconnectivity from0.5～5.6. Moreover, the electrocatalytic activities of the as-prepared six Pt/PDDA-CMM hybrid nanocomposites are also observed to show an inverted V-shaped profile as a function of loading amount of Pt NPs. Integrated with glucose oxidase (GOx), lactate oxidase (LOD) and the in vivo microdialysis system, the constructed dual Oxidase/PDDA-CMM/Pt biosensors were successfully applied for the simultaneous and on-line detection of glucose and lactate. After post-calibration, the basal level of glucose and lactate in the striatum of anaesthetic rats was calculated to be0.27±0.03and0.71±0.05mM (mean±s.d., n=3), respectively. What's more important, the dual oxidase biosensors almost suffer from little cross-talk, which is characteristic of an excellent sensor with high performance. This property, along with the good linearity and a high stability toward glucose and lactate substantially enables this method elaborated in this work promising application in physiology and pathology.