Dissertations / Theses on the topic 'Dopamine detection'

Colorimetric probes have become important tools in analysis and biomedical technology. This thesis reports on the development of such probes for the detection of dopamine (DA). Liquid and different solid state probes were developed utilizing un–functionalized gold nanoparticles (UF–AuNPs). The liquid state probe is based on the growth and aggregation of the UF–AuNPs in the presence of DA. Upon addition of the UF–AuNPs to various concentrations of DA, the shape, size and colour change results in spectral shifts from lower to higher wavelengths. The analyte can be easily monitored by the naked eye from 5.0 nM DA with a calculated limit of detection of 2.5 nM (3σ) under optimal pH. Ascorbic acid (AA) has a potential to interfere with DA detection in solution since it is often present in biological fluids, but in this case the interference was limited to solutions where its concentration was beyond 200 times greater than that of DA. Since most of the previously reported colorimetric probes, especially those for DA are solution based, the main focus of the thesis was in the development of a solid state based colorimetric probe in the form of nanofibre mats. To overcome the interference challenges experienced in the solution studies (the interference by high concentrations of AA), the suitability of molecularly imprinted polymers (MIPs) for the selective detection of DA was investigated. The results showed that the MIPs produced did not play a significant role in enhancing the selectivity towards DA. A probe composed of just the UF–AuNPs and Nylon–6 (UF–AuNPs + N6) was also developed. The UF–AuNPs were synthesized following an in situ reduction method. The probe was only selective to DA and insensitive to other catecholamines at physiological pH. Thus, the probe did not require any addition functionalities to achieve selectivity and sensitive to DA. The liquid state probe and the composite UF–AuNPs + N6 nanofibre probe were successfully applied to a whole blood sample and showed good selectivity towards DA. The simple, sensitive and selective probe could be an excellent alternative for on–site and immediate detection of DA without the use of instrumentation. For quantification of DA using the solid state probe, open–source software imageJ was used to assist in the analysis of the nanofibre colours. It was observed that the intensity of the colour increased with the increase in concentration of DA in a linear fashion. The use of imageJ can also be a great alternative where the colour changes are not so clear or for visually impaired people. The solid state probe developed can detect DA qualitatively and quantitatively. The work also forms a good foundation for development of such probes for other analyte.

The development of dopamine (DA) electrochemical sensors and biosensors is described in this thesis. The aim was to develop (bio)sensors that would continuously and specifically detect DA over a wide concentration range, with high sensitivity and low detection limits. The electrode surfaces used were platinum (Pt), boron-doped-diamond (BDD), and carbon-paste (CP). The first two were used to construct DA sensors, while Pt was shown to be superior for the construction of biosensors. The DA biosensor configurations were based on the use of a Pt electrode as a transducer surface, polyphenol oxidase (PPO) as an enzyme, ferrocene (Fc) as a mediator, and polypyrrole (PPY) and Nafion as membranes. A range of electrochemical techniques were employed in this research.
It was determined that the oxidation of DA on bare Pt is a surface-controlled reaction, occurring at low overpotentials. The reaction is electrochemically reversible, involving the spontaneous adsorption of DA on the electrode surface. The Pt and BDD sensors were efficiently used to determine DA in aqueous solutions. In order to increase their resistance to the ascorbic acid (AA) interference, the sensor surfaces were modified by a thin Nafion film. This configuration was shown to selectively detect DA even when AA was present with DA at a 1000-time larger concentration. The lowest DA detection limit was achieved using the unmodified BDD sensor, 50 nM. Nevertheless, both the unmodified and Nafion-modified Pt and BDD sensors were suitable for monitoring of DA at concentration levels typical for urine samples.
It was shown that the sensitivity and detection limit of the developed Pt-based biosensors depend on the amount of PPO and Fc incorporated into the PPY membrane, and also on their ratio. The modification of the biosensor by a Nafion membrane offered three benefits: an increase in sensitivity, an improvement in detection limit, and a significant minimization of the AA interference. An optimum biosensor architecture was made by polymerizing PPY for 40 minutes from a pyrrole solution containing 2,400 U mL-1 of PPO and 10 mM of Fc, on top of which a thin Nafion film was formed. Using chronoamperometry as a detection technique, this biosensor yielded a DA detection limit of 20 nM, which makes it suitable for monitoring DA levels in brain. Even a lower detection limit, 10 nM, and higher sensitivity were achieved by using electrochemical impedance spectroscopy (EIS) as a detection technique. Unfortunately, the developed biosensor lacked operational stability, predominately due to the leakage of PPO and Fc into the storage solution.

A controlled assembly of natural beta-cyclodextrin modified Au NPs mediated by dopamine is demonstrated. Furthermore, a simple and sensitive colorimetric detection for dopamine is established by the concentration-dependent assembly.

This thesis investigates quantitative methods for the characterisation of dopamine (DA) release in preclinical PET. The goal was to develop methodologies that improve quantification of the PET signal and test to what extent these techniques can reliably detect subtle DA fluctuations. Ultimately, reliable quantification could help to better understand the role of DA in human brain conditions. The focus of this work is kinetic modelling, whose accuracy depends on different factors, from the quality of the measured imaging data to the interpretation of parameter estimates. An investigation of the model, linear parametric neurotransmitter PET (lp-ntPET), that enables the description of time-varying DA changes, was performed in 3 contexts. First, its performance was tested in a Bayesian framework, the PET-ABC method. Second, it was evaluated in 1D-simulations by varying the activations and the priors, to study their effects on uncertainty in the estimates. Third, a [11C]raclopride study was performed in monkeys using the high sensitivity MiniExplorer, where different amphetamine doses were injected to induce variable DA releases. Finally, towards exploiting the synergies of PET/fMRI data in the future, a preclinical PET insert was evaluated in 3 configurations: as a standalone unit, in the MR bore and with the MR pulsing. In conclusion, the results of the studies showed that the lp-ntPET model used in conjunction with PET-ABC is a robust quantitative method that not only provides parameter estimates and their uncertainties but also statistically sound inferences about model preference. Further, PET-ABC is able to reliably estimate parameters describing transient DA release in preclinical studies. The PET insert presented good performance in all configurations, enabling future investigation of parallel analysis of fMRI and PET data for improved parameter estimation.

We have investigated electropolymerisation for fabrication of a chemically modified working electrode for the determination of dopamine and catechol neurotransmitters in the presence of ascorbic acid. A variety of film compositions were investigated that would allow discrimination of the neurotransmitters through a combination of electrostatic barrier and the film porosity. The films investigated were based on different compositions of () poly-o-toluidine-co-aniline (POT-co-PA), () poly-o-toluidine-co-o-anisidine (POT-co-POA) and () polyacriflavine (PAF). The POT-co-PA and POT-co-POA gave the most promising result although the POT-co-PA was preferred because of higher current enhancement and better separation of dopamine and catechol neurotransmitters in the presence of ascorbic acid. The uses of electropolymerisation make the investigated films attractive candidates for the fabrication of a chemically modified microelectrode with application in capillary electrophoresis separation with electrochemical detection. The active area of nano particle (Au, Pt and Ag) screen printed electrodes was determined using cyclic voltammogram with ferro/ferricyanide couple. The active surface of the nano particle coated electrode was found surprisingly to be 5% - 65% lower than that geometrically calculated surface area for the electrode. This is ascribed to the limitation of the screen printing approach that was used. A low cost high replication approach that would allow development of a capillary electrophoresis microfluidic chip with electrochemical detection (CE-ECD) on a polymer substrate was investigated. A fluidic top layer was fabricated using hot embossing and an electrode bottom layer by metal patterning on a polymer substrate using metallisation and photolithography.

An ideal biosensor is a compact and in-expensive device that is able to readily and rapidly detects different types of analytes with high sensitivity and specificity. The affectability of a biosensing methodology is subject to the limit of nanomaterials to transduce the target binding process to an improved perceptible signal, while the selectivity is accomplished by considering the binding and specificity of certain moieties to their targets. Keeping these requirements in mind we have chosen nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) that has catalytic properties combined with their size, shape and configuration dependent chemical and physical properties as essential precursors and signaling components for creation of biosensors with tremendous sensitivity. The primary goal of the research work described in this dissertation is to develop and evaluate novel methods to detect various analytes using nanomaterials, at the same time making an affordable architecture for point-of-care (POC) applications. We report here in chapter 3 a simple and new strategy for preparing disposable, paper-based, porous AuNP/M-SWCNT hybrid thin gold films with high conductivity, rapid electron transfer rates, and excellent electrocatalytic properties to achieve multiple analyte electrochemical detection with a resolution that greatly exceeds that of purchased flat gold slides. We further explored the use of nanomaterial-based paper films in more complex matrices to detect analytes such as NADH, which can act as a biomarker for certain cellular redox imbalances and disease conditions. Carbon nanotubes with their large activated surfaces and edge-plane sites (defects) that are ideal for performing NADH oxidation at low potentials without any help of redox mediators minimizing surface fouling in complex matrices is described in chapter 4. With an instrument-free approach in mind we further focused on a colorimetric platform using split cocaine aptamers and gold nanoparticles (AuNPs) to detect cocaine for on-site applications as described in chapter 5. In chapter 5, the split aptamer sequences were evaluated mainly on three basic criteria, the hybridization efficiency, specificity towards the analyte (cocaine), and the reaction time to observe a distinguishable color change from red to blue. The assay is an enzyme-assisted target recycling (EATR) strategy following the principle that nuclease enzyme recognizes probe–target complexes, cleaving only the probe strand releasing the target for recycling. We have also studied the effect of the number of binding domains with variable chain lengths on either side of the apurinic (AP) site. On the basis of our results, we finally shortlisted the sequence combination with maximum signal enhancement fold which is instrumental in development of colorimetric platform with faster, and specific reaction to observe a distinctive color change in the presence of cocaine.

Les signaux neuromodulateurs induisent une adaptation des fonctions neuronales par le biais de mécanismes d’intégration dynamiques complexes. Parmi les voies de signalisation intracellulaires, celle de l’AMPc/PKA joue un rôle essentiel dans la réponse cellulaire à la dopamine. Pour analyser ces processus d’intégration, nous combinons l’imagerie de biosenseurs dans des préparations ​ex vivo de tranches de cerveau de souris avec de la modélisation de la signalisation intracellulaire dans les neurones D1 et D2 striataux. Dans une première partie de mon travail de thèse, nous analysons la dynamique de la signalisation striatale en réponse à des stimulations dopaminergiques transitoires telles celles associées aux récompenses. Nous montrons par imagerie que, contrairement à ce qui est communément admis, les récepteurs D​2 à la dopamine permettent la détection de dopamine phasique au niveau de l’AMPc. De plus, les simulations suggèrent que les neurones D2 pourraient détecter une diminution du niveau de dopamine tonique, indicateur d’une situation aversive chez l’animal. Ce travail a fait l’objet d’une publication (​Yapo et al., ​J. Physiol 2017​). Dans une deuxième partie, nous avons analysé l’effet dans le noyau de ces stimulations dopaminergiques rapides. En comparaison avec les neurones du cortex, nous montrons que les neurones du striatum disposent d’un mécanisme de contrôle en-avant (“​feed forward​”) qui renforce les réponses PKA nucléaires. Cette situation originale, à l’opposé des rétrocontrôles homéostatiques habituels en biologie, amène à une réponse du noyau tout ou rien, extrêmement sensible. Nous pensons que ce mécanisme est impliqué dans la détection des signaux dopaminergiques transitoires. Ce travail a été publié dans un article (​Yapo et al., ​J Cell Science​ 2018​). Enfin une troisième partie, sous forme de résultats préliminaires, consistait à analyser l’adaptation des neurones du striatum à la perte des afférences dopaminergiques, caractéristique de la maladie de Parkinson. Nous avons observé l’hypersensibilité à la dopamine affectant les neurones D1, largement décrite dans la littérature. De plus, nous montrons que les neurones du striatum présentent une activité phosphodiestérase accrue. Une meilleure compréhension de ces adaptations pathologiques pourrait mener à de nouvelles stratégies thérapeutiques
Neuromodulatory signals trigger adaptations in neuronal functions via complex integrative properties. Among the various existing intracellular signaling pathways, the cAMP/PKA cascade plays a critical role in the cellular response to dopamine. To analyze these integrative processes, we combine biosensor imaging in mouse brain slices with in silico modelisation of the intracellular signaling in D1 and D2 medium-sized spiny neurons. In a first part of my thesis work, we analyze the dynamics of cAMP/PKA signaling in striatal neurons stimulated by transient dopaminergic signals, such as those associated with reward. With imaging we show that the dopamine D​2 receptors can sense phasic dopamine signals at the level of cAMP, a thought that has been argued for long. Moreover ​in silico simulations suggest that D2 spiny neurons could sense the interruptions in tonic dopamine levels associated with aversion in the animal. This work was published in (​Yapo et al., ​J Physiol 2017​). In a second part, we analyzed the effect of such brief dopaminergic signals on the nuclear PKA-dependent signaling. In comparison to cortical neurons, we show that the striatal neurons display a positive feedforward mechanism which strengthens the nuclear responses. This peculiar situation, which contrasts with the usual homeostatic feedback mechanisms found in biology, leads to all-or-nothing and extremely sensitive responses. We believe that this mechanism allows for the detection of transient dopaminergic signals. This work was published in (​Yapo et al., ​J Cell Science​ 2018​). Lastly a third part, that will be introduced as preliminary data, consisted in analyzing the adaptations of the striatal neurons following a dopamine depletion, such as the one found in Parkinson’s disease. We observed in our mouse model an hypersensitivity of the D1 spiny neurons to dopamine, already described by other groups. Additionally we show that striatal neurons display an increased phosphodiesterase activity. A better understanding of these pathological adaptations could lead to the emergence of new therapeutic strategies

Chemistry
Ph.D.
Biogenic monoamines such as dopamine play an important role as major neurotransmitters. Simultaneous determination of the concentration changes is thus crucial to understand brain function. Additionally, quantification of pharmaceutically active compounds (PhACs) and their metabolites in biological fluids is an important issue for forensic tests, clinical toxicology and pharmaceutical analysis. We have developed two postcolumn luminescence detection methods coupled to a 2-dimensional-solid phase extraction (2D-SPE) system. The postcolumn reaction methods used in this study are the redox-dependent photoluminescence-following electron-transfer (PFET) and Fenton-based chemiluminescence techniques, for the determination of certain neurotransmitter and nonsteroidal anti-inflammatory drugs (NSAIDs). A stable [Os(tmphen)3]3+ (tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline) reagent was prepared in neutral aqueous solution by oxidation of [Os(tmphen)3]2+ with lead(IV) oxide. [Os(tmphen)3]2+ and [Os(tmphen)3]3+ are characterized by absorption spectroscopy. [Os(tmphen)3]3+ stability is compared with [Ru(tmphen)3]3+ in the same pH 7 environment. The properties of Os(III) and Ru(III) complexes were investigated for use as the oxidant in a PFET system. Studies of photophysical and electrochemical properties, the stability of the Os(III) and Ru(III) complexes, and analytical application in PFET detection of oxidizable analytes are presented. The spectroscopic properties of the complexes were not very advantageous, but careful control of the detection system and reaction conditions enabled sensitive detection of the analytes. The method was fully validated and the optimized system was capable of detecting dopamine and acetaminophen at about 30.2 µg L-1 and 33.5 µg L-1, respectively. The limit of detection (LOD) was 1.5 µg L-1 for acetaminophen and 4.3 µg L-1 for dopamine. The accuracy and precision were within bioanalytical method validation limits (90.9 to 101.5 % and RSD < 12.0 %, respectively). Typical analysis time was less than 15 minutes. Two Fenton-based flow-injection chemiluminescence (CL) methods were developed and validated for the determination of naproxen. Under the optimal experimental conditions the proposed methods exhibited advantages in a larger linear range from 2,760 ng mL-1 to 207,000 ng mL-1 for the first CL method and 41.4 ng mL-1 to 700.0 ng mL-1 for the second CL method. The LOD was 13.8 ng mL-1 for naproxen. The CL mechanisms for the system, H2O2-FeIIEDTA-naproxen was further studied by batch experiments, chemiluminescence spectroscopy, fluorometry, high pressure liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR). The effects of various interferences commonly found in biological and wastewater systems on the chemiluminescence intensity were also investigated. We used these methods to determine NSAIDs in commercial pharmaceutical formulations. Another application of these method was for detecting NSAIDs in biological samples. A 2x-1-Dimensional Solid Phase Extraction (2x-1D SPE) method was developed for determination of acetaminophen and naproxen in urine. This method uses both the methanol concentration and the pH advantageously to preferentially isolate analytes of interest from complex sample matrix. These methods were fully validated and had sufficient sensitivity (limit of quantification: acetaminophen; 40.41 mg L-1 - 360.0 mg L-1 and naproxen; 23.03 mg L-1 - 214.8 mg L-1) for biological matrices and applications.
Temple University--Theses

The work presented in the thesis is about the evaluation of Organic Electrochemical Transistors (OECTs) for fast scan cyclic voltammetry (FSCV). FSCV is a method which has been used for real time dopamine sensing both in vivo and in vitro. The method is sensitive to noise and could therefore benefit from signal preamplification at the point of sensing, which could be achieved by incorporation of OECTs. In this study the OECTs are based on the conductive polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS). The gate consists of gold microelectrodes of different sizes to be used one at a time. When dopamine is reacted at the gate electrode, the redox state of the PEDOT:PSS OECT channel is modulated and the resulting change in drain current can be measured. The gate current, which contains the sensing information, is after filtering obtained by differentiating the channel potential with respect to time. The derived gate current is plotted in cyclic voltammogram for different dopamine concentrations and the amplitude of the oxidation/reduction peaks can be used to determine the dopamine concentration. In this thesis for the first time it is demonstrated that OECTs can be used for FSCV detection of dopamine. The results are discussed and an outlook on future work is given.

Most of the topics dealt with in this thesis belong to surface science. The starting point was the fundamental understanding of phenomena at the oxide-gas interface and the effect of its modification. Such knowhow was then used to solve (or, at least, to attempt to solve) issues of critical impact in everyday life: the increasing lifetime of building materials employed in low-impact smart houses; the fouling prevention in electroanalytical sensors for neurotransmitter detection; the unspecialized laboratories accessibility to microlithography, critical to device miniaturization. These challenges might seem not related, but they actually share deep scientific and technological foundations. The physicochemical modification of oxide surfaces, the creation of organic/inorganic hybrids and the exploiting / the enhancing of semiconductor peculiar properties allowed us, starting from the foundation, the realization of proof-of-concept protocols and devices, ready for the pre-commercial development. The Leitmotif of my research was the synthesis and the modification of titanium dioxide surfaces. TiO2 has been the main character in physico, physicochemical and material science researches of the last 50 years. Biocompatibility and low cost make it engaging for many applications. Its (near-UV active) semiconductor features, well known and abundantly investigated by the scientific community, are acquiring central interest also in many markets with the development of self cleaning coatings, windows and asphalts, anti-fogging mirrors and self-sterilizing surgery rooms and instrumentations. New generation batteries and solar cells are going to be developed as commercial prototypes. One of the biggest challenges in the titania fundamental research is the enhancement of activity in the solar spectrum. First, the most recent aspects in titania doping and promotion were touched. While, in the last twenty years, great effort has been made in the mono-atomic doping of titania and in the understanding of the influence of the dopant position in the titania lattice and its electronic behavior, the most recent literature describes the co-promotion of the material by two (or more) atoms doping. The metal/non-metal codoping seems especially promising; the synergetic effect of the two atoms in the TiO2 lattice was both theoretically and experimentally proved. In this contest, the N/Nb codoping was analyzed, investigating the effect of the atoms in the lattice from morphological (surface area, porosity and crystallographic structure) and electronic point of view (EXAFS, UV-Vis absorption and EPR analyses). N/Nb codoping was compared with N/Ta co-doped samples, synthesized by two different procedures. The photoactivity of the two sample families was tested by a model reaction (the degradation of ethanol, throughout acetaldehyde intermediate) both under UV and solar simulated irradiation. Then, a different approach in the modification of surfaces was tested. The assembly of organic/inorganic hybrids was tested; thanks to the formation of organic mono- or multi-layers at the surface,they can tune the chemistry, the polarity and the adhesion properties of the interface. Siloxanes were used as active agents, thanks to their compatibility with oxide materials and, especially, for the ability to self-assemble at the surface to form a monolayer. Siloxanes are able to react with the -OH groups at the surface, chemisorbing and polymerizing at the interface in such a way to form a monolayer with tunable functionalities. Many different silanes were tested and their dipole momenta were related to their wettability properties. Such siloxanes chemisorb strongly both from the gas phase and the liquid phase. Their reactivity, both on smooth and rough surfaces, was tested vs the temperature of functionalization in gas phase. Many characterization techniques were adopted to understand the behavior of such molecules from a molecular point of view: magnetic (solid state NMR), microscopic (SEM, TEM, AFM), optical and electrochemical (CV and EIS). The science of adhesion and wettability was also adopted for the development of superhydrophobic coatings. Titanium dioxide particles with engineered morphology were used as the best candidate to create superhydrophobic/superhydrophilic patch-wise surfaces, exploiting their photoactivity (photolithograpy). The core of the thesis was the synthesis, modification and application of transparent photoactive thin films. A procedure for the synthesis of smooth, transparent and photoactive TiO2 thin layers was developed, and used to produce highly applicative devices and protocols. Such synthetic strategy is highly tunable and reproducible; the obtained films are robust and active and, most of all, require simple instrumentation (sol-gel procedure), which is highly appealing for the market. The films were properly characterized both form the morphological/mechanical and photochemical point of view. Apart their transparency and their thickness, the films were highly crystalline (pure anatase phase). Such procedure was firstly designed as a proof-of-concept for self-cleaning windows, but, thanks to its versatility and the high activity of the films, it leads the path towards highly applicative procedures and devices. The smoothness and the photoactivity brought me to the field of photolithograpy, especially in the direction of microlithography. The high activity of the titania allowed the use of safe and low-energetic lamps. No collimation was required to obtain a resolution lower than 5 µm. First of all, I tested the lithography on siloxane monolayer films, as a proof-of-concept of resolution and efficiency. But siloxanes, as many other self-assembled monolayer molecules, can be the pillars for 3D fabrication. Such monolayers were used as polymerization initiators for polymer brushes. If the initiators of polymerization are patterned, patterned polymer brushes will be obtained. That was the first report of polymer-brushes lithography exploiting the photoactivity of TiO2. Remote photocatalytic lithography makes this procedure extremely versatile. Exploiting the remote photocatalysis, in principle, any material can be used as a support for patterned polymer brushes growth (provided that the initiator are able to graft the surface). The developed protocol for the synthesis of TiO2 thin films was also used to design and engineer complex electrodes for cyclovoltammetric analyses of biological samples. Electrochemistry seems to be the best candidate for the development of an analytical option with sensitivity comparable with present analytical procedures but reduced time-per-analysis and cost. Unfortunately, catecholamines chemisorb and polymerize on metal and oxide electrodes quickly, making the device useless. Covering the electrode by a homogeneous, nano-porous thin layer of titania makes the surface photoactive. That is the first example in literature of self-cleaning nano-engineered electrodes for cyclic voltammetry. After the detection, also in simulated human serum and liquor, a fast and simple irradiation of the device, under non-hazardous UV-A lamp, degrades all the fouling on the surface without altering its features. The sensor, after each UV treatment, recovers its pristine performances, with full recovery in terms of selectivity and sensitivity. Irradiation trials were also performed directly in the analytical mixture, as a proof of concept for on-site application. Modern era requires flexible and light materials for the building industry. Polymers are acquiring more and more interest thanks to their increasing performances and their smart properties. The drawbacks of such materials are connected to the low resistance to the UV light, the softness and the difficulties in cleaning procedure. The use of organic/inorganic hybrid, or better the coverage of plastic materials with an oxidic thin layer, can solve many of these problems, lengthening the lifetime of such materials. If the covering oxide is also photoactive, the material can be self-cleaned when exposed to solar light. That is a big chemical challenge, because of many synthetic problems. Two different approaches were tested to solve this relevant issue. On one side, the hydrophobicity of ionic liquid modified SPES (sulfonated polyether sulfone) was combined with designed morphological features to confer superhydrophobicity. On the other side, the polymeric surface was covered with a transparent titania layer active in the near UV-region, able to mineralize organic molecules chemisorbed at the surface. Eventually, a different approach to modify oxidic (and not only) surfaces is the creation of a homogeneous layer of Ag nanoparticles by an innovative microwave procedure. That simple and accessible strategy allowed us to produce plasmonic surfaces (thanks to the dimension and the homogeneity of the Ag particles) with countless applications. The layer was shown to be a very active substrate for surface enhancement Raman spectroscopy (SERS). Thanks to the versatility of the synthetic method, all shapes and dimensions can be covered. That makes it a perfect candidate for the production of new generation of SERS sensors. The sensitivity towards molecules of environmental and biomedical interest was proved.

碩士
國立暨南國際大學
應用化學系
95
Dopamine, the derivative of catechol, is an important neurotransmitter in human body. Dopamine can be oxidized to o-dopaminoquinone (DOQ) electrochemically. It was found that the nucleophilic attack of sodium sulfanilate (ABSA) to o-dopaminoquinone occurred and a new adduct was produced. The original reduction wave of dopamine at Ered = 0.3 V vs. Ag/AgCl is disappeared and a new reduction wave at Ered = 0.1 V vs. Ag/AgCl is appeared. The concentration of dopamine was determined in terms of the reduction current of the new adduct by using dual working electrode-FIA. The concentration of dopamine ranging from 0.4 to 20 μM gives a good linear relationship and the detection of limit is 0.048 μM. In the second part, the p-phenylenediamine (PPD) was used as an indicator for the determination of thiol. The oxidized form of PPD is PDI (p-phenylenediimine), which is attacked by thios nucleophiles. The increasing oxidation current after the addition of thiol into PDD solution was used to quantitative analysis of the concentration of thiols.

碩士
國立交通大學
應用化學系碩博士班
100
Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) existing super paramagnetic characteristic and can be easily manipulated by external magnetic field. After coating with gold shell, the functional magnetic composites can be widely used in biochemical or clinical related researches. Based on the specific interaction between 4-mercaptophenylboronic acid (4-MPBA) and dopamine, the functional magnetic composites were decorated with 4-MPBA on the surface via Au-S self-assembly mechanism to capture dopamine in the sample solution. By applying magnetic field, the dopamine in the sample solution which captured by magnetic nanocomposites can be gathered to the surface of the electrode. This procedure concentrated the analyte at the electrode surface, which enhanced the sensitivity of the measurement. The developed method had high sensitivity and good selectivity detecting dopamine. The linear range was from 10 pM to 1 μM with R2 equal to 0.9935 and the estimated limit of detection (LOD) was 0.16 pM (S/N = 3). The proposed method improved the sensitivity of detection and can be further applied to different biosensors or bioelectronics devices in various fields.

碩士
國立清華大學
電子工程研究所
97
本研究目的在於開發出感測濃度達到fM(10-15 M)等級的多巴胺感測元件，此感測元件由TSMC CMOS 0.35 �慆製程製作而成，我們把從製程廠製作回來的晶片，經由一連串的後製程，首先經由硫酸濕蝕刻掉金屬層，再經由KOH濕蝕刻掉多晶矽，以露出我們所需要的感測區域，而完成了感測元件OGFET(開閘極場效電晶體)。 此篇論文的特點在於將感測元件與感測電路整合在一顆晶片上，而感測電路方面則是4乘4的感測陣列，感測電路原理為將感測元件電流轉換至數位脈波輸出的感測機制，有鑑於以往傳統式ISFET感測電路為電阻式放大器，但輸出的變化量都在mv(毫伏)範圍；而我們所設計的電路正好彌補了此不足，其優點在於能夠將大範圍的電流感測出來並且以數位脈波輸出，而我們所設計的電路感測電流範圍在40 nA ~6 �嫀之間。 在整個晶片完成了硫酸濕蝕刻等後製程後，必須在其感測表面進行分子固定化，用意在於能夠抓取我們想要量測的生物分子(在此為多巴胺)，抓取到的多巴胺分子帶有負電，使得所量測的I-V曲線產生位移而能感測到fM(10-15 M)等級的多巴胺溶液。 我們在此篇論文中利用傳統CMOS製程開發出極靈敏的離子電晶體感測器，而能夠達到與奈米金線場效電晶體相同的感測靈敏度(NWFET)；本研究最重要的地方在於能夠將感測元件與電路整合在一個系統中，這對於低成本開發無疑是一個絕對的幫助，且因晶片面積小而能夠達到隨身攜帶的目的，對於病人的即時偵測非常的有用。

碩士
國立中興大學
精密工程學系所
106
In this study, poly(ethylenedioxythiophene)/carbon nanotubes (PEDOT/CNTs) composite materials were used to modify acupuncture needles for the detection of serotonin (5-HT) and dopamine (DA), simultaneously. Firstly, the gold film was applied to the surface of the acupuncture by physical vapor deposition to increase its conductivity, and then the PEDOT/CNTs composite was deposited on the gold film by electrochemical deposition. The PEDOT layer has a high affinity for biomolecules while the oxygen functional groups of CNTs contains a porous interface layer, so that it can provide many active sites and improve the sensitivity of electrochemical detection of 5-HT and DA. Furthermore, detecting 5-HT and DA with Au/PEDOT/CNTs acupuncture biosensors by cyclic voltammetry and differential pulse voltammetry analysis. The result shows that PEDOT/CNTs modified acupuncture has greater current response than Au and PEDOT modified acupuncture. In the process of detecting neurotransmitters, the oxidation potential of 5-HT is 0.38 V and DA is 0.18 V. Both of them have a good linear relationship between the concentration range of 1 to 100 μM. The linear correlation coefficient of 5-HT is R² = 0.9712 with the detection limit of 0.81 μM (S/N=3), the linear correlation coefficient of DA is R² = 0.9915, with the detection limit of 0.49 μM (S/N=3). When the two coexisting concentrations differ by a factor of 10, the acupuncture needles can still distinguish two different substances clearly and also detect the signal in a well reproducibility (RSD is lower than 7%, RSD is lower than 10%, n = 5). The acupuncture sensor has the advantages of easy preparation, high reproducibility, smaller volume than traditional electrodes and successfully detecting 5-HT and DA with are a good prospect in future research on acupoint detection.

碩士
國立中興大學
生醫工程研究所
104
Recently, graphene oxide nanoribbon (GONR) has attracted attention for applications in many fields, especially in biomedical research since it contains various oxygen groups and is easy to be functionalized and integrated into the fabrication of biosensors. However, GONR has been only used so far for biomolecular detection at low ionic strength solution and for monitoring the binding events occurring at the interface. This limits the sampling capability to be carried out at the patient site since physiologically relevant ionic strength is 100 mM. Therefore, it will be highly advantageous if these biosensors are able to detect not only at low concentration but also at high-salt concentration. Taking inspiration from impedance measurement and high-frequency technique, this study aims to develop a system based on the impedimetric of reduced GONR field-effect transistors (rGONR-FETs) for the sensing of dopamine (DA) in the presence of high concentration of ascorbic acid (AA) at high frequency. We successfully demonstrate that the fundamental ionic screening effect can be mitigated by operating rGONRs-FETs as high-frequency biosensors. This simple, label-free and sensitive method allows determination of DA concentration without using redox probe. 3-aminopropyl trimethoxysilane (APTMS) self-assembled monolayer was used to modify the specific opening areas on patterned-circuit silicon chips. After that, GONRs with oxygenated functional groups were assembled on APTMS modified surfaces through covalent amide bond. GONR-FETs were then reduced by hydrazine at 95°C for 1 h to gain semiconductive property of rGONR-FETs. Impedance measurements were performed in the frequency range of 20 Hz to 2 MHz using a precision LCR meter, which was connected to the source and drain terminal of rGONR FET. Moreover, the positive direct current (DC) was provided to the solution-gate, which can help to reduce the interference of the system and saturate solution. Measurement at high frequency using single-rGONR FET device showed a significant change in the impedance magnitude (|Z|) and impedance phase (θ) with increasing DA concentration. Herein, a wide linear range of DA in phosphate-buffered saline (from 20 nM to 100 μM) was observed at higher frequency region due to its molecular dipole field. Furthermore, detection of DA in the presence of 2.5 mM AA was also investigated. The recorded signals were further interpreted into either impedance magnitude and impedance phase or resistance and capacitance. Those recorded parameters were applied to probe the subtle changes between charged ions/ DA molecules and sensing surface of rGONR. These results confirm improved sensitivity in the detection of DA at high background salt concentrations at high frequency and provide a solution and platform to detect other physiological charged molecules.

碩士
國立清華大學
材料科學工程學系
104
Abnormal dopamine level in human body is related to some diseases. However, traditional dopamine sensing methods were confronted with problems such as interference and biofilm-fouling. This study adopted electrochemical method to detect dopamine, and the electrode we used was conductive nitrogen-incorporated ultrananocrystalline diamond (NUNCD) films which were prepared through the biased enhanced growth method. The synthesis conditions for NUNCD were 1400 W plasma power, -300 V applied bias at the substrate, 50 torr chamber pressure, and deposition for 1 hour. The best resistivity of the film was about 63.15 μΩ·cm. For the dopamine detection in the phosphate buffer solution, the detection limit was about 0.32 μM. In addition, detection of dopamine in the presence of ascorbic acid and uric acid were performed and it is found that the detection limit was not affected by the interference of biomolecules and the electrode showed good selectivity. In fetal bovine serum, the electrode also performed excellently with a detection limit of about 0.39 μM. We also built up calibration curves and equations to calculate the content of the dopamine in a blind sample. By substituted the current, obtained from the result of differential pulse voltammetry, into the equation and calculated the dopamine level, we found the dopamine concentration calculated from the equation has the same level with the value we added. Recovery of the electrode is about 90-120%, indicating that the NUNCD film has great potential to be used as an electrode for dopamine sensing.

碩士
國立清華大學
生醫工程與環境科學系
99
In this study, the dumbbell-like Pt-Fe3O4 nanoparticle was prepared by using Pt nanoparticle with different sizes as the seeding nanoparticles. The 3, 6, and 13 nm of Pt nanoparticles were obtained by using Pt(acac)2 and oleylamine as the precursor and reducing agent, respectively. In addition, the different sizes of PT nanoparticles can be obtained by controlling the amount of reducing agent and synthesisis temperature from 140-180ºC. Using different sizes of Pt nanoparticles in the presence of iron-oleate, the different sizes of dumbbell-like Pt-Fe3O4 nanoparticle were successfully fabricated by tuning the ratios of Pt and iron-oleate. The synthesized Pt and Pt-Fe3O4 were characterized by TEM, XRD, TGA, ICP-AES, SQUID. The Pt showed better electron transfer ability. Pt and Pt-Fe3O4 were dispersed on graphite electrode to fabricate the amperomertic biosensor for sensing dopamine，the Pt-Fe3O4 electrode is linearly dependence on dopamine concentration in the range of 20-850,15-850 and 35-850 ?嵱 receptively , and the Pt-Fe3O4 electrode were 15-850,25-850,10-850 ?嵱 receptively. The detection limits of Pt were 7.49,7.36 and 8 ?嵱 receptively, and Pt-Fe3O4 were 7.22,6.14 and 0.13 ?嵱 receptively. In addition, the Pt-Fe3O4 shows better current value of signal intensity , linear range and more lower detection limits when compared Pt nanoparticles, clearly showing the application potential on biosensing and catalytic activity.

碩士
國立清華大學
生物科技研究所
97
Dopamine is an important neurotransmitter that involved in many physiological activities such as movement, emotion, sensation and cognition. The imbalance of dopamine metabolism results several neurodegenerative disorders, including Parkinson’s disease and depression. Researches in experimental model organisms and clinical studies have shown that the degeneration of dopaminergic neurons in substantia nigra (SN) located at basal ganglia could lead to Parkinson’s disease. Because the dysfunction of dopamine underlying many brain disorders, understanding how dopamine being regulated in the nervous system is crucial for resolving the pathogenic mechanism of those diseases. Currently, detecting dopamine in vivo has not been possible due to its instability under physiological condition. Therefore, we develop fusion proteins composed of human monoamine oxidase B (hMAO B) and green fluorescent protein (GFP) as dopamine probes that can detect dopamine in living animal model. The MAO is a flavoenzyme that associates FAD by covalent band, and it can bind and oxidize dopamine specifically. Previous studies showed that human MAO presented spectral absorption from 400~500 nm, but this property can be reduced after the reduction by substrates binding. Our preliminary results show the MAO B-GFP fusion proteins can absorb the excitation wavelength of GFP and thus block its emission when MAO is in oxidized form. If the substrates like dopamine are present, the MAO is reduced after substrates binding and the excitation wavelength can trigger GFP emission, a phenomenon we called “shield effect”. Our probes may provide a novel approach for dopamine detection in vivo.

碩士
國立清華大學
電子工程研究所
99
In this study, high sensitivity UNCD (ultrananocrystalline diamond) micrielectrodes for real-time dopamine detection are investigated. It is believed that such a device could be used in the future for monitoring the change of dopamine concentration in Parkinson's disease and Alzheimer's Disease patients. High Pressure Liquid Chromatography (HPLC) has been widely used in hospitals due to its high sensitivity; however the expensive cost makes it inconvenient for general users. In order to improve this problem, a new dopamine sensor based on electrochemical detection is proposed. The dopamine sensor in this thesis includes N-doped UNCD interdigitated microelectrodes and CMOS (Complementary Metal Oxide Semiconductor) sensing circuit. First of all, by employing MEMS (Micro-Electro-Mechanical Systems) technology the sensor is fabricated on the silicon substrate coated with Si3N4. As the dopamine is detected by microelectrode, the sensor generates a current signal resulted from oxidation and reduction. Then, this current signal is converted to a voltage signal by the sensing circuit. Finally, the dopamine concentration can be calculated from the output data. Since the UNCD has flat surface, superior physical and chemical properties, it’s an excellent candidate material for microelectromechanical (MEMS) application. Its conductivity is close to that of an the insulator, so it has limited applications. By doping some elements to improve its conductivity, it can help the UNCD on the semiconductor and biomedical device applications.

Biogenic amines are neurotransmitters involved in several physiological processes. Changes and disturbs in their function are associated to several neurological disorders or diseases, as well as consumption of psychotherapeutic or psychotropic drugs. The detection of these compounds in biological fluids is therefore of clinical and neurochemical interest. The goal of this work was to develop and validate and analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in both urine and plasma samples. The internal standard used was 3,4-dihydroxybenzylamine (DHBA). The MEPS extraction procedure was optimized using the design of experiments (DOE) tool, and the final conditions of 8 strokes, no washing, and two elutions of 100 FL methanol were chosen. The method was fully validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50-1000 ng/mL for 5-HT and between 5-1000 ng/mL for DA and NE, with determination coefficients (R2) higher than 0.99 for all compounds. The limits of detection and quantification were respectively 20 and 50 ng/mL for 5-HT, and 2 and 5 ng/mL for DA and NE. Intra- and interday precision ranged from 1 to 10 %, while accuracy was within a ±15% interval for all compounds. Authentic urine and plasma samples were analysed by the validated method, and the three compounds were detectable and quantifiable in urine, while only 5-HT was detected and quantified in plasma. This is the first time that a commercially available MEPS column was used for the simultaneous detection of biogenic amines in urine, and also the first time that 5-HT was detected and quantified in plasma using MEPS. MEPS proved to be fast to perform with the use of less solvent volumes, saving time and money and being less laborious. The validated method is useful for the determination of biogenic amines in laboratorial routine.

博士
國立成功大學
化學工程學系
102
The three kinds of modified electrodes were prepared with carbon nano materials, 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and silver/silver sulfide for simultaneous determination of dopamine (DA), uric acid (UA) and ascorbic acid (AA). An ABTS-immobilized carbon nanotube (CNT) electrode was used to simultaneously detect DA and UA in the presence of ascorbic acid. DA and UA are oxidized to DAox and UAox, respectively, at the active sites of the CNT surface, and the released electrons are transported to the ITO electrode through the ABTS mediator. Ag/Ag2S was applied on an electrochemical system. The Ag/Ag2S nanoparticles were dispersed with CNT in Nafion to be an Ag/Ag2S-immobilized CNT electrode. Ag/Ag2S worked as an electrocatalyst for DA and AA, and promoted electron transfer on the modified electrode surface. Nitrogen doped graphene oxide (r-NGO) was prepared from graphite oxide and polyvinylpyrrolidone (PVP) mixture by rapid thermal treatment. The r-NGO modified electrode was applied to simultaneously determine the concentrations of DA, UA and AA. The result shows that the nitrogen-carbon bonds promoted electron transfer on the modified electrode surface and the r-NGO structural defect showed highly electrocatalytic activity towards the oxidation of DA, UA and AA.

碩士
國立中興大學
生醫工程研究所
104
Since graphene was successfully isolated in 2004, it has received much attention and proceed with many research application owing to its high charge mobility, low background noise, high surface area, and biocompatibility property. In this study, the patterned-circuit silicon chips was first modified with by (3-aminopropyl) trimethoxysilane (APTMS), forming self-assembled monolayer which containing with amine groups. Then, the inherited oxygenated functional groups on solution-based graphene oxide (GO) were activated by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide (EDC/NHS) and further covalently conjugated with amine-functionalized silicon chips through amide bonds (-CONH-). After that, chemical reduction using hydrazine was implemented to remove a variety of oxygenated groups from immobilized GO surface gaining reduced GO (RGO). Electron spectroscopy for chemical analysis (ESCA), contact angle, Raman spectroscopy, biocompatibility test and I-V curve measurement were used to characterize the chemical components, hydrophilic/hydrophobic properties, lattice structure, biological toxicity and electrical characteristics of different reduced time of RGO. Analysis of results indicated 60-min RGO featured of optimal material properties which was suitable for the base material of the biosensors. Further, using 60-min RGO-based biosensor in the detection of the concentration gradient of dopamine. Making use of phosphate buffered saline to prepare dopamine solution, simulating physiological environment in human body. The medium frequency range of electrical impedance were measured by using LCR meter (E4980A, Agilent Technologies). Since the dopamine concentration in our cerebrospinal is around 1.89~16.65 nM, our 60-min RGO-based biosensor could discriminate 1 fM~100 nM which proved that 60-min RGO biosensor could be as the effective sensing dopamine platform. Furthermore, through the change in the impedance value could observe proliferation condition of rat adrenal pheochromocytoma (PC12) cells, proved 60-min RGO biosensor also could be non-invasive monitoring of living cells platform. By adding nerve growth factor (NGF) to stimulate PC12 cells differentiation, along with synapses growth and exhibited neuron characteristics. With 100μM KCl stimulation, neurotransmitters would be released from synaptic vesicle in differentiated PC12 cells. Through 60-min RGO biosensor could measure the impedance change which resulted from attachment of differentiated PC12 cells or emancipation of dopamine. In this research, through LCR meter imposing a sinusoidal AC voltage to trigger disturbances of frequency, avoiding hydrazine-reduced 60-min RGO biosensor to be interfered by Debye length. According to impedance response of dopamine and cell physiology, 60-min RGO biosensor could be used to detect early symptoms of imbalance dopamine concentration which related to Parkinson''s disease or schizophrenia, showing promising application in detection and tracking of the lesion.

碩士
國立成功大學
化學工程學系
105
A low-cost and simple fabrication of phosphorus-doped reduced graphene oxide (PTRG) was performed by using triphenylphosphine (TPP) as phosphorus source and simultaneously phosphorus-doping and reducing the graphene oxide through thermal annealing. Compared with screen-printed carbon electrode (SPCE) and several carbon materials modified electrodes, the PTRG electrode had the highest oxidation peak current to background current ratio for dopamine (DA) detection. For simultaneous sensing of DA and uric acid (UA), two well-separated voltammetry peaks were obtained with the PTRG electrode in differential pulse voltammetry (DPV) measurements, and the oxidation peak between DA and UA was 146 mV. The linear response ranges for the determination of DA and UA were 1-25 μM and 5-1000 μM, respectively. The attractive features of the PTRG provided potential applications on the simultaneous determination of DA and UA.

博士
國立中山大學
機械與機電工程學系研究所
102
In the conventional sample extraction approach, to achieve efficient liquid-liquid phase separation for the sample analysis is an important issue. However, it is challenging to separate the immiscible liquid of low surface tension from water by using microfluidic device. Therefore, this study developed a microfluidic chip that composed of T-junction, reaction channel and a novel liquid-liquid phase separator for continuously synthesizing fine gold nanoparticles (AuNPs) in the organic solvent (toluene). The design of glass chip is capable for separating water (surface tension = 72.75 mN/m) and toluene (surface tension = 30.9 mN/m) with 92% separation efficiency, owing to design different depths of microchannel that creates large difference between liquid surface tension and capillary force. Furthermore, AuNPs that synthesized in the microdevice exhibits narrower size distribution and better dispersion in comparing to the typical vessel synthesis process. Besides, this study successfully developed a novel and high performance colorimetric probe for dopamine (DA) detection. Aqueous-phase AuNPs extracted via 4-(dimethylamino) pyridine (DMAP) from toluene were used as the reaction probes. Interestingly finding that the original diameter of AuNPs around 13 nm which separated into 2-5 nm size after adding DA. This exhibits change in the color of AuNPs colloid from red to blackish green. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) showed the AuNPs break into the smaller sizes right after addition of DA. The DA concentration is quantitatively monitored by using UV-Vis spectrometer with a limit of detection (LOD) as low as 5 nM. In addition, the developed DA detection approach appears no significant problems in detecting DA with present common interferents such as ascorbic acid (AA), homovanillic acid (HVA) and catechol (CA). However, many study reported that using microfluidic chip is capable to provide fast chemical reaction and rapid detection approach for biochemical analysis. This study developed a novel optical detection sensor by using the etched multi-mode optical fibers assembling in a droplet-based microfluidic system to achieve on-site absorbance measurement. Hence, the reaction of AuNPs detecting DA biosample was also capable to achieve rapid and continuously detection by using the microdevice. The proposed optical detection sensor composed by initially forming AuNPs droplet in segmented flow and measuring for sample absorbance in a 10 mm long of optical detection channel. Note that using the microdevice for absorbance measurement only required sample volume for 50 nL, which exhibits lower sample consumption in comparing to detect in the conventional cuvette system. Results indicated the developed microdevice capable for steady measuring sample absorbance with operating flow rate in the range from 5-25 μL/min. In addition, the detection approach shows faster reaction response for kinetic measurement of DA core etching AuNPs. Therefore, this study successfully developed microfluidic chip to provide efficient liquid-liquid phase separation, which benefit to use for the sample extraction and synthesizing AuNPs of uniform size distribution in toluene. In addition, assembling optical fibers on the microfluidic chip that have offers simple and high performance optical detection to the bioanalysis. In this regard, the proposed microdevice with using AuNPs probes shows great potential to achieve high sensitivity detection for the future applying to such as biology, medical and clinic diagnostic applications.

碩士
國立臺灣師範大學
化學系
105
Dopamine, a catecholamine neurotransmitter, plays an important role in mammalian central nervous system. Abnormal concentration of dopamine in biological fluids causes several diseases such as Parkinson’s and Huntington’s disease. Therefore, it is an important research topic to develop a quantitative method to accurately estimate the level of dopamine. In this study, we designed a whole-cell biosensor for dopamine detection using monoamine regulon in Escherichia coli and RFP (red fluorescence protein) as a signal output. The detection of limit was 1.43M. Afterward, we replaced RFP with MjDOD (4,5-DOPA extradiol dioxygenase from Mirabilis Jalapa) . L-DOPA can be converted into betalamic acid (a precursor of betaxanthin and absorb at 432nm) by MjDOD. We proposed that dopamine can be converted into 6-decarboxylated betalamic acid by MjDOD. This biosensor could detect dopamine and L-DOPA with relatively high selectivity and without the interferences of phenethylamine and phenylacetaldehyde. Finally, phenethylamine, dopamine, L-DOPA, and (-)-Epinephrine can be distinguished with as-developed dual-signal biosensor carrying RFP and MjDOD simultaneously and each analogue has a unique fingerprint profile. With feature, this biosensor could potentially improve the accuracy and specificity in the diagnosis of dopamine.

碩士
國立中興大學
化學系所
99
The electrochemical detection of dopamine (DA) at conventional solid electrodes was interfered by the coexisted ascorbic acid (AA). In this study, we develop the nanoporous gold electrode to circumvent this problem. The nanoporous gold electrode exhibited excellent electrocatalytic activity towards the oxidations of DA and AA in 0.1 M phosphate buffer solution (pH 6.0).The results also indicated that the nanoporous gold electrode exhibited substantial enhancement in electrochemical sensitivity and selectivity for DA due to its large surface area. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used to investigate the electrochemical behavior of AA, DA and mixture. By CV and SWV, the separation of the oxidation peak potentials for dopamine–ascorbic acid were about 0.210 V and 0.232 V, respectively. The calibration curve for DA was obtained in the range of 0.1－10 μM. The detection limit (S/N = 3) was 8.5 nM. In the presence of 0.5 mM AA, the calibration curve for DA was obtained in the range of 0.1－10 μM and the detection limit was 13 nM.

碩士
國立成功大學
材料科學及工程學系
103
In this research, we aim to understand the influence of nanostructures and surface charge on the detection of dopamine. We synthesize several functionalized poly(3,4-ethylenedioxythiophene) (PEDOT), including carboxylic acid, ethylene glycol, and phosphocholine functional groups. The results showed the nanostructured PEDOT with carboxylic acid groups provides best detection limit mainly contributed by electrostatic interaction between carboxylic acid and dopamine. Furthermore, we evaluate the performance of our electrodes in the presence of proteins. The electrodes are immersed into solutions containing BSA, lysozyme and fibrinogen for 6 hours before test. The results show the non-specific binding of protein lower the detection sensitivity. However the electrode presents a similar detection ability from 1 to 50 μM of domamine concentration, and achieves a lower detection limit about 1 μM. These show the electropolymerized functionalized PEDOT having the properties of light stability, consistency under protein influence and mass production.

碩士
國立臺灣師範大學
化學系
93
An unequal electric field assisted sweeping-MEKC (UEFA/sweeping-MEKC) technique is proposed, for the first time, and compared to the normal sweeping-MEKC with reference to sensitivity and separation efficiency. NDA (naphthalene-2,3-dicarboxaldehyde) derivatized-dopamine and -norepinephrine, were selected for use as model compounds. In the case of normal sweeping-MEKC, the findings show that, when a portion (30 cm, ~1/3 of the total length of the capillary) of sample matrix was injected into the capillary, the separation resolution was only 1.5. However, this was dramatically improved to 9.2 when the UEFA/sweeping-MEKC technique was applied. In the view of sensitivity, when a violet-LED (~ 2mW) was used as the fluorescence excitation source, the limit of detection for NDA-labeled dopamine was determined to ~10-9 M, the similar to that for normal sweeping-MEKC, since the injected sample volume is same. In addition, this method also provides sufficient sensitivity and separation efficiency for the detection of low concentrations of dopamine in urine.

ABSTRACT

Phenols are ubiquitous in our surroundings including biological molecules such as L-Dopa metabolites, food components, such as whiskey and liquid smoke, etc. This dissertation describes a new method for detecting phenols, by reaction with Gibbs reagent to form indophenols, followed by mass spectrometric detection. Unlike the standard Gibbs reaction which uses a colorimetric approach, the use of mass spectrometry allows for simultaneous detection of differently substituted phenols. The procedure is demonstrated to work for a large variety of phenols without para‐substitution. With para‐substituted phenols, Gibbs products are still often observed, but the specific product depends on the substituent. For para groups with high electronegativity, such as methoxy or halogens, the reaction proceeds by displacement of the substituent. For groups with lower electronegativity, such as amino or alkyl groups, Gibbs products are observed that retain the substituent, indicating that the reaction occurs at the ortho or meta position. In mixtures of phenols, the relative intensities of the Gibbs products are proportional to the relative concentrations, and concentrations as low as 1 μmol/L can be detected. The method is applied to the qualitative analysis of commercial liquid smoke, and it is found that hickory and mesquite flavors have significantly different phenolic composition.

In the course of this study, we used this technique to quantify major phenol derivatives in commercial products such as liquid smoke (catechol, guaiacol and syringol) and whiskey (o-cresol, guaiacol and syringol) as the phenol derivatives are a significant part of the aroma of foodstuffs and alcoholic beverages. For instance, phenolic compounds are partly responsible for the taste, aroma and the smokiness in Liquid Smokes and Scotch whiskies.

In the analysis of Liquid Smokes, we have carried out an analysis of phenols in commercial liquid smoke by using the reaction with Gibbs reagent followed by analysis using electrospray ionization mass spectrometry (ESI-MS). This analysis technique allows us to avoid any separation and/or solvent extraction steps before MS analysis. With this analysis, we are able to determine and compare the phenolic compositions of hickory, mesquite, pecan and apple wood flavors of liquid smoke.

In the analysis of phenols in whiskey, we describe the detection of the Gibbs products from the phenols in four different commercial Scotch whiskies by using simple ESI-MS. In addition, by addition of an internal standard, 5,6,7,8-tetrahydro-1-napthol (THN), concentrations of the major phenols in the whiskies are readily obtained. With this analysis we are able to determine and compare the composition of phenols in them and their contribution in the taste, smokey, and aroma to the whiskies.

Another important class of phenols are found in biological samples, such as L-Dopa and its metabolites, which are neurotransmitters and play important roles in living systems. In this work, we describe the detection of Gibbs products formed from these neurotransmitters after reaction with Gibbs reagent and analysis by using simple ESI‐MS. This technique would be an alternative method for the detection and simultaneous quantification of these neurotransmitters.

Finally, in the course of this work, we found that the positive Gibbs tests are obtained for a wide range of para-substituted phenols, and that, in most cases, substitution occurs by displacement of the para-substituent. In addition, there is generally an additional unique second-phenol-addition product, which conveniently can be used from an analytical perspective to distinguish para-substituted phenols from the unsubstituted versions. In addition to using the methodology for phenol analysis, we are examining the mechanism of indophenol formation, particularly with the para-substituted phenols.

The importance of peptides to the scientific world is enormous and, therefore, their structures, properties, and reactivity are exceptionally well-characterized by mass spectrometry and electrospray ionization. In the dipeptide work, we have used mass spectrometry to examine the dissociation of dipeptides of phenylalanine (Phe), containing sulfonated tag as a charge carrier (Phe*), proline (Pro) to investigate their gas phase dissociation. The presence of sulfonated tag (SO₃^-) on the Phe amino acid serves as the charge carrier such that the dipeptide backbone has a canonical structure and is not protonated. Phe-Pro dipeptide and their derivatives were synthesized and analyzed by LCQ-Deca mass spectroscopy to get the fragmentation mechanism. To confirm that fragmentation path, we also synthesized dikitopeparazines and oxazolines from all combinations of the dipeptides. All these analyses were confirmed by isotopic labeling experiments and determination and optimization of structures were carried out using theoretical calculation. We have found that the fragmentation of Phe*Pro and ProPhe* dipeptides form sequence specific b₂ ions. In addition, not only is the ‘mobile proton’ involved in the dissociation process, but also is the ‘backbone hydrogen’ is involved in forming b₂ ions.

碩士
國立清華大學
生物科技研究所
103
Dopamine circuit has been shown to associate with locomotion, sleep and arousal, decision making, and associative learning in mammals and fly. To understand the mechanism of dopaminergic-associated behaviors and disorders, we aim to visualize dopamine release in vivo. Currently, monitoring the dynamics and distribution of dopamine released in live is unattainable; the traditional ways to measure dopamine level in neurons is cumbersome and lacks of temporal and spatial resolutions. Our goal is to develop a probe that can switch fluorescence upon detecting dopamine release from dopaminergic neurons in vivo. By modifying our previously established cytosolic probe MMG1, in which we used intrinsic spectral properties of MAO B to switch GFP emission upon dopamine binding (we have coined this phenomena as “shield effect”) as an intracellular dopamine sensor, I have developed different membrane dopamine sensors that could potentially use for detecting dopamine release. Current results show that two full-length constructs, MSG11 (MAO B-ste2p-sfGFP11) and MSG (MAO B-ste2p-GFP), and two MAO B C-terminal truncated probes could be embedded on the cell membrane with expected topology. Additional tests are currently ongoing to validate the shield effect and enzyme activity of these sensors. My ultimate goal is to make the transgenic flies and test the probes in the brain. With this type of molecular probe on hand, we may have a better way to determine how brain process information in dopamine circuits under different environmental stimulations and behavioral responses.

The thesis deals with the testing and optimization of hydrodynamic conditions of the electrochemical detector, which consists of a flow cell containing the electrochemical sensor with an array of electrodes. The flow cell includes a rotating disc-shaped component, which allows radial flow of analytes along the sensor electrodes. This principle imitates the rotating disk electrode, with the difference that here the electrodes are immobile. Hydrodynamic phenomena in this case are similar to RDE. Therefore, the theoretical part of my work deals with hydrodynamics and mass transfer for the well-described case of rotating disk electrode. It was found that in the current set-up under certain conditions, an analyte flowed back from the output cell channel to the sensor, even at the laminar flow. This causes a nonlinear response of the electrodes and their low reproducibility. The solution is to reduce the distance between the rotation component and the electrodes surface and reduce flow speed of analyzed liquid towards the sensor. A novel type of sensor with annular electrodes was designed and tested. It showed four times higher conversion rate of the analyte compared with the originally used AC9 electrochemical sensor having electrodes in the form of full circles lying on a circumference of a common circle. Based on calculations, conversion of the analyte on the plane electrode can be increased up to 100% when using this annular sensor, adjusting flow cell and reducing of the input flow rate. The detector was connected to the liquid chromatograph and its function was tested on two substances - ascorbic acid and dopamine. For the current detector these parameters were set: dynamic range, linearity, noise, detection limit, time constant and the temperature range at which the detector can be operated.

Research Doctorate - Doctor of Philosophy (PhD)
Schizophrenia is a debilitating disorder of neurodevelopmental origins that likely stems from the cumulative action of a range of genetic and environmental factors. Epidemiological evidence has identified maternal infection during gestation as one significant environmental risk factor for the development of the disorder. Evidence from animal models has further validated the link between maternal immune activation (MIA) in the absence of an active infection and the later life development of schizophrenia-like pathology in the offspring. In particular, work in mouse models has suggested that the gestational time at which MIA occurs can alter the behavioural and neurobiological phenotype displayed. Specifically, that MIA in late gestation is involved in schizophrenia-relevant cognitive dysfunction and altered NMDA receptor expression, whereas MIA in early gestation is more closely associated with behavioural deficits reminiscent of positive symptomology and dopaminergic neurotransmission. The aim of the current thesis was to extend the mouse findings to another species, the rat, and further explore the effects of MIA. In addition to producing a reliable rat model of schizophrenia where distinct behavioural and neurological phenotypes associated with schizophrenia are produced following MIA at either early or late gestational time-points (gestational day 10 or 19, respectively), the current thesis extends on previous work by examining the schizophrenia biomarker of mismatch negativity and assessing the neuroinflammatory state of offspring. Behavioural assessments revealed that MIA in either early or late gestation produced transient impairments in working memory and reductions in PPI. In these behavioural studies, there was no clear distinction between a dopamine and glutamate-related behavioural phenotype based on the gestational timing of exposure. However, early but not late gestation MIA did produce alterations in the dopaminergic system of males, as indicated by increased dopamine 1 receptor mRNA in the nucleus accumbens. EEG experiments demonstrated that although the male rat brain is able to generate human-like (adaptation-independent) mismatch responses (MMRs), and although MIA (regardless of gestational timing) does alter MMRs, it does not do so in a manner comparable with schizophrenia. Immunohistochemical techniques revealed that MIA does result in subtle neuro-immune changes in adult offspring, with an increase in microglial immunoreactivity identified in the frontal white matter of late, but not early, gestation MIA animals. Furthermore, a strong trend towards increased astrocyte immunoreactivity that approached significance was identified in the prefrontal cortex of late, but not early MIA offspring. The combined results have demonstrated that MIA during the chosen gestational time-points are sufficient to disrupt neurodevelopmental processes producing long-term alterations in behavioural and neuropathological measures relevant to schizophrenia. However, the phenotype characterised here deviates slightly from previous findings from mouse models indicating potential differences in the critical periods of neurodevelopmental susceptibility to MIA exposure between the rat and mouse. Importantly this research has provided insights into the underlying neuro-immune changes which may contribute to the behavioural abnormalities seen in adult MIA offspring and has provided evidence that MIA in rats can alter the prominent schizophrenia relevant electrophysiological biomarker of adaptation-independent MMRs, providing a basis to further investigate these measures and their underlying mechanisms.

碩士
國立高雄大學
化學工程及材料工程學系碩士班
101
In this study, thiol-capped CdSe, graphene oxide (GO), chemically reduced graphene oxide (rGOc), and carbon nanotube (CNT) were dispersed in chitosan (CS) aqueous solution followed by casting films on glassy carbon electrodes to investigate the electrocatalytic activities of the films by cyclic voltammetry for developments of electrochemical sensors for dopamine (DA), uric acid (UA), and ascorbic acid (AA). CV curves revealed that CdSe/rGOc/CS exhibited high electrocatalytic activity and selective detection ability for DA, UA, and AA compared with bare GCE and rGOc/CS. The CdSe/rGOc/CS composite film had enhanced porosity after adding CdSe to rGOc/CS and exhibited specific interactions with DA, UA, or AA. The composite films of CNT/rGOc/CS and CNT/GO/CS exhibited also high electrocatalytic activities. The sulfonated chitosan (sCS) in the CNT/GO/sCS had enhanced the electrocatalytic activities much higher toward the oxidation of DA, UA and AA. This could be attributed to the swelling of sCS in aqueous solutions leading to enhanced porosity in the CNT/GO/sCS film. The CNT/GO/sCS- and CNT/GO/CS-modified electrodes were used for simultaneous and quantitative determinations of DA, UA, and AA. For the CNT/GO/CS, the linear ranges for detections of DA, UA, and AA were 1.25

碩士
國立暨南國際大學
應用化學系
98
Dopamine（3-hydroxytyramine , DA）and serotonin（5-hydroxytryptamine, 5-HT）are neurotransmitters widely distributed in the central and peripheral nervous systems of both mammals and insects. Without them, lots of disease ensues, including sleep disorder, Parkinson’s disease and Schizophrenia. Therefore, it is important to determine the amount of neurotransmitters in biological and medical studies. Recent researches reported fruit flies have short lifetime and most important of all genome of fruit flies was similar to that of human about 66%. We choose fruit flies as animal model and made use of high performance liquid chromatography coupled with fluorescent detector（HPLC-FLD）to quantitatively determine dopamine and serotonin in head of fruit flies. In this study, we used Canton-S（2U）wild-type and mutant strain. Besides, we control the amount of dopamine and serotonin by knocking down dopa decarboxylase and tryptophan hydroxylase with UAS-dsRNAiddc or UAS-dsRNAitph, respectively, by means of Actin-Geneswitch. The method（HPLC-FLD） we demonstrated for detection of changes of amount of dopamine and serotonin in heads of fruit flies provides a new choice for further research in the field chemical analysis.