Rozprawy doktorskie na temat „Electrophoresis microchip devices”

Laboratory test results are important in making decisions regarding a patient's diagnosis and response to treatment. These tests often measure the biomarkers found in biological fluids such blood, urine, and saliva. Immunoassay is one type of laboratory test used to measure the level of biomarkers using specific antibodies. Microfluidics offer several advantages such as speed, small sample volume requirement, portability, integration, and automation. These advantages are motivating to develop microfluidic platforms of conventional laboratory tests. I have fabricated polymer microfluidic devices and developed immunoassays on-chip for potential cancer markers. Silicon template devices were fabricated using standard photolithographic techniques. The template design was transferred to a poly(methyl methacrylate) (PMMA) piece by hot embossing and subsequently bonded to another PMMA piece with holes for reservoirs. I used these devices to perform microchip immunoaffinity electrophoresis to detect purified recombinant thymidine kinase 1 (TK1). Buffer with 1% methylcellulose acted as a dynamic coating that minimized nonspecific adsorption of protein and as sieving matrix that enabled separation of free antibody from antibody-TK1 complexes. Using this technique, I was able to detect TK1 concentration >80 nM and obtained separation results within 1 minute using a 5 mm effective separation length. Detection of endogenous TK1 in serum is difficult because TK1 is present at the pM range. I compared three different depletion methods to eliminate high abundance immunoglobulin and human serum albumin. Cibacron blue columns depleted abundant protein but also nonspecifically bound TK1. I found that ammonium sulfate precipitation and IgG/albumin immunoaffinity columns effectively depleted high abundance proteins. TK1 was salted out of the serum with saturated ammonium sulfate and still maintained activity. To integrate affinity columns in microfluidic devices, I have developed a fast and easy strategy for initial optimization of monolith affinity columns using bulk polymerization of multiple monolith solutions. The morphology, surface area, and porosity, were qualitatively assessed using scanning electron microscopy. This method decreased the time, effort, and resources compared to in situ optimization of monoliths in microfluidic devices. This strategy could be used when designing novel formulations of monolith columns. I have also integrated poly(ethylene glycol dimethacrylate-glycidyl methacrylate) monolith affinity columns in polymer microfluidic devices to demonstrate the feasibility of extracting human interleukin 8 (IL8), a cancer biomarker, from saliva. Initial results have shown that the affinity column (~3 mm) was successfully integrated into the devices without prior surface modification. Furthermore, anti-IL8 was immobilized on the surface of the monolith. Electrochromatograms showed that 1 ng/mL of IL8 can be detected when in buffer while 10 ng/mL was detected when IL8 was spiked in saliva. Overall, these findings can be used to further develop immunoassays in microfluidic platforms, especially for analyzing biological fluids.

This work presents the development of 3D printed microfluidic devices and their application to microchip analysis. Initial work was focused on the development of the printer resin as well as the development of the general rules for resolution that can be achieved with stereolithographic 3D printing. The next stage of this work involved the characterization of the printer with a variety of interior and exterior resolution features. I found that the minimum positive and negative feature sizes were about 20 μm in either case. Additionally, micropillar arrays were printed with pillar diameters as small as 16 μm. To demonstrate one possible application of these small resolution features I created microfluidic bead traps capable of capturing 25 μm polystyrene particles as a step toward capturing cells. A second application which I pioneered was the creation of devices for microchip electrophoresis. I separated 3 preterm birth biomarkers with good resolution (2.1) and efficiency (3600 plates), comparable to what has been achieved in conventionally fabricated devices. Lastly, I have applied some of the unique capabilities of our 3D printer to a variety of other device applications through collaborative projects. I have created microchips with a natural masking monolith polymerization window, spiral electrodes for capacitively coupled contactless conductivity detection, and a removable electrode insert chip. This work demonstrates the ability to 3D print microfluidic structures and their application to a variety of analyses.

Master of Science
Department of Chemistry
Christopher T. Culbertson
Chemical separation involves selective movement of a component out of a region shared by multiple components into a region where it is the major occupant. The history of the field of chemical separations as a concept can be dated back to ancient times when people started improving the quality of life by separation of good materials from bad ones. Since then the field of chemical separation has become one of the most continually evolving branches of chemical science and encompasses numerous different techniques and principles. An analytical chemist’s quest for a better way of selective identification and quantification of a component by separating it from its mixture is the cause for these ever evolving techniques. As a result, today there are numerous varieties of analytical techniques for the separation of complex mixtures. High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Capillary Electrophoresis (CE) and Gel Electrophoresis are a few out of a long list. Each these techniques manipulates the different physical and chemical properties of an analyte to achieve a useful separation and thus certain techniques will be suited for certain molecules. This work primarily focuses on the use of Capillary Electrophoresis as a separation technique. The mechanism of separation in Capillary Zone Electrophoresis and principles of UV detection will discussed in chapter one. Chapter two contains a discussion about the application of Capillary Electrophoresis (CE) on microfluidc devices. This will include sections on: microfabrication techniques of PDMS and photosensitized PDMS (photoPDMS), a UV detector for microfluidic devices and its application for the detection of wheat proteins. In Chapter three we report the experimental part of this project which includes; investigations on the effect of UV exposure time and thermal curing time on feature dimensions of photoPDMS microfluidic device, investigations on the injection and separation performances of the device, characterization of a UV detector set up and its application for the separation and detection of wheat gliadin proteins. The results of these investigations are presented in chapter four.

The electrochemical detector provides a promising detection mode for capillary electrophoresis (CE) due to its excellent sensitivity, good portability, high selectivity, easy miniaturization, low capital and running cost. To widen its scope for determining trace analytes in complex samples, three dual-electrode detectors were fabricated to enable the determination of electro-inactive analytes, to assess co-eluted peaks and to give a large enhancement of the detection sensitivity by modifying electrode surface using multi-walled carbon nanotubes (MWNTs). To determine trace non-electroactive amino acids present in human tears, a serial dual-electrode detector was developed using an upstream on-capillary Pt film electrode to oxidize bromide to bromine at +1.0 V and a downstream Pt disk electrode to detect the residual bromine at +0.2 V after their reaction with amino acids eluted out from the separation capillary. The bromide reagent was introduced after CE separation by a newly designed coaxial post-column reactor fabricated onto the PMMA chip. Using optimized CE buffer containing 20 mM borate, 20 mM SDS at pH 9.8, L-glutamine, L-alanine and taurine were baseline separated with detection limits ranging from 0.56-0.65 μM and a working range of 2-200 μM for L-glutamine and of 2-300 μM for both L-alanine and taurine. Method reliability was established by close to 100% recoveries for spiked amino acids and good agreement between the measured and the literature reported amino acid concentrations in tears. For the determination of polyphenols in wine, a microchip-CE device was fabricated with a dual-opposite carbon fiber microelectrode operated in a parallel mode to assess peak purity. Under optimized conditions, (+)-catechin, trans-resveratrol, quercetin, (-)-epicatechin and gallic acid were baseline separated within 16 min with detection limits ranging from 0.031- 0.21 mg/L and repeatability of 2.0-3.3 % (n=5). The use of an opposite dual-electrode enables the simultaneous determination of peaks and measurement of their current ratios at +0.8 V and +1.0 V vs Ag/AgCl. The capability of using current ratio to identify the presence of co-migrating impurities was demonstrated in a mixed standard solution with overlapping (+)-catechin and (-)-epicatechin peaks and in a commercial red wine with interfering impurities. Matching of both the migration time and the current ratio reduce false positive and validate polyphenol quantitation in red wine. Lastly, a dual-opposite MWNTs modified carbon fiber microelectrode (CFME) was developed to determine the biomarkers (4-nitrophenol, 4-nitrophenyl-glucuronide and 4-nitrophenyl-sulfate) needed to assess exposure to methyl parathion. Use of the MWNTs modified CFME showed a much higher sensitivity than bare CFME, with a detection limit of 0.46 μM for 4-nitrophenol. Baseline separation of all three biomarkers was obtained within 31 min by a 45 cm long capillary under 12 kV in a 20 mM phosphate buffer at pH 7.0. The method developed was successfully utilized to determine low levels of biomarkers in human urine without using complex pretreatment steps and delivered recoveries ranging from 95.3 - 97.3% and RSDs within 5.8% (n=3). Using a parallel dual-electrode detector was shown to deliver reliable results with matching current ratios and comparable migration time to those obtained from biomarker standards.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

My dissertation work integrates the techniques of microfabrication, micro/nanofluidics, and bioanalytical chemistry to develop miniaturized devices for healthcare applications. Semiconductor processing techniques including photolithography, physical and chemical vapor deposition, and wet etching are used to build these devices in silicon and polymeric materials. On-chip micro-/nanochannels, pumps, and valves are used to manipulate the flow of fluid in these devices. Analytical techniques such as size-based filtration, solid-phase extraction (SPE), sample enrichment, on-chip labeling, microchip electrophoresis (µCE), and laser induced fluorescence (LIF) are utilized to analyze biomolecules. Such miniaturized devices offer the advantages of rapid analysis, low cost, and lab-on-a-chip scale integration that can potentially be used for point-of-care applications.The first project involves construction of sieving devices on a silicon substrate, which can separate sub-100-nm biostructures based on their size. Devices consist of an array of 200 parallel nanochannels with a height step in each channel, an injection reservoir, and a waste reservoir. Height steps are used to sieve the protein mixture based on size as the protein solution flows through channels via capillary action. Proteins smaller than the height step reach the end of the channels while larger proteins stop at the height step, resulting in separation. A process is optimized to fabricate 10-100 nm tall channels with improved reliability and shorter fabrication time. Furthermore, a protocol is developed to reduce the electrostatic interaction between proteins and channel walls, which allows the study of size-selective trapping of five proteins in this system. The effects of protein size and concentration on protein trapping behavior are evaluated. A model is also developed to predict the trapping behavior of different size proteins in these devices. Additionally, the influence of buffer ionic strength, which can change the effective cross-sectional area of nanochannels and trapping of proteins at height steps, is explored in nanochannels. The ionic strength inversely correlates with electric double layer thickness. Overall, this work lays a foundation for developing nanofluidic-based sieving systems with potential applications in lipoprotein fractionation, protein aggregate studies in biopharmaceuticals, and protein preconcentration. The second project focuses on designing and developing a microfluidic-based platform for preterm birth (PTB) diagnosis. PTB is a pregnancy complication that involves delivery before 37 weeks of gestation, and causes many newborn deaths and illnesses worldwide. Several serum PTB biomarkers have recently been identified, including three peptides and six proteins. To provide rapid analysis of these PTB biomarkers, an integrated SPE and µCE device is assembled that provides sample enrichment, on-chip labeling, and separation. The integrated device is a multi-layer structure consisting of polydimethylsiloxane valves with a peristaltic pump, and a porous polymer monolith in a thermoplastic layer. The valves and pump are fabricated using soft lithography to enable pressure-based sample actuation, as an alternative to electrokinetic operation. Porous monolithic columns are synthesized in the SPE unit using UV photopolymerization of a mixture consisting of monomer, cross-linker, photoinitiator, and various porogens. The hydrophobic surface and porous structure of the monolith allow both protein retention and easy flow. I have optimized the conditions for ferritin retention, on-chip labelling, elution, and µCE in a pressure-actuated device. Overall functionality of the integrated device in terms of pressure-controlled flow, protein retention/elution, and on-chip labelling and separation is demonstrated using a PTB biomarker (ferritin). Moreover, I have developed a µCE protocol to separate four PTB biomarkers, including three peptides and one protein. In the future, an immunoaffinity extraction unit will be integrated with SPE and µCE to enable rapid, on-chip analysis of PTB biomarkers. This integrated system can be used to analyze other disease biomarkers as well.

Microfluidics is a vibrant and expanding field that has the potential for solving many analytical challenges. Microfluidics shows promise to provide rapid, inexpensive, efficient, and portable diagnostic solutions that can be used in resource-limited settings. Microfluidic devices have gained immense interest as diagnostic tools for various diseases through biomarker analysis. My dissertation work focuses on developing electrokinetically operated integrated microfluidic devices for the analysis of biomarkers indicative of preterm birth risk. Preterm birth (PTB), a birth prior to 37 weeks of gestation, is the most common complication of pregnancy and the leading cause of neonatal deaths and newborn illnesses. In this dissertation, I have designed, fabricated and developed several microfluidic devices that integrate various sample preparation processes like immunoaffinity extraction, preconcentration, fluorescent labeling, and electrophoretic separation of biomarkers indicative of PTB risk. I developed microchip electrophoresis devices for separation of selected PTB biomarkers. I further optimized multiple reversed-phase porous polymer monoliths UV-polymerized in microfluidic device channels for selective retention and elution of fluorescent dyes and PTB biomarkers to facilitate on-chip labeling. Successful on-chip fluorescent labeling of multiple PTB biomarkers was reported using these microfluidic devices. These devices were further developed using a pH-mediated approach for solid-phase extraction, resulting in a ~50 fold enrichment of a PTB biomarker. Additionally, this approach was integrated with microchip electrophoresis to develop a combined enrichment and separation device that yielded 15-fold preconcentration for a PTB peptide. I also developed an immunoaffinity extraction device for analyzing PTB biomarkers directly from a human serum matrix. A glycidyl methacrylate monolith was characterized within microfluidic channels for immobilization of antibodies to PTB biomarkers. Antibody immobilization and captured analyte elution protocols were optimized for these monoliths, and two PTB biomarker proteins were successfully extracted using these devices. This approach was also integrated with microchip electrophoresis for combined extraction and separation of two PTB biomarkers in spiked human serum in <30 min. In the future, these optimized microfluidic components can be integrated into a single platform for automated immunoaffinity extraction, preconcentration, fluorescent labeling, and separation of PTB biomarkers. This integrated microfluidic platform could significantly improve human health by providing early diagnosis of PTBs.

Within one tumor, cancer cells exist as different sub-populations due to the variations in expression of crucial bio-markers. The prevalence of even minor cell sub-populations can determine overall cancer progression and treatment response. Single-cell protein analysis is a way to identify these cell sub-populations; therefore we developed a microfluidic platform with ultrahigh-sensitivity for single-cell protein analysis. As the key step to develop such a platform, protein migration under the application of an electric field has to be understood. COMSOL multi-physics software is used as a tool to understand the protein migration in microfluidic channels, which contain ion-selective hydrogels as the separation matrix. The objective of this thesis work, is to minimize the protein losses to diffusion and to maximize the fluorescent signal in order to quantify the protein expression in single cells. The novelty of this work lies in the use of ion-selective hydrogels to eliminate the diffusional losses and separate the proteins based on their mass and charge. This thesis project has been performed thanks to an Erasmus fellowship at MCS Department of the University of Twente.

A eletroforese capilar em microchips (µCE ou MCE) é uma forma diferente de eletroforese capilar que tem se desenvolvido muito nos últimos anos. Essa técnica usa microdispositivos feitos em placas que podem ser de vidro ou polímero contendo canais de dimensões micrométricas ao invés de um capilar de sílica. Ganhos significativos têm sido obtidos em termos de tempo de análise, volume manipulado, dimensões físicas, consumo energético e integrabilidade com outros sistemas. Neste trabalho foi empregada uma técnica diferente de microfabricação, usando toner de impressora laser e folhas de transparência para a construção de dispositivos para microfluídica. A técnica se mostra simples, rápida e excelente para prototipagem. Visando a aplicação desses microchips de toner-poliéster, esse trabalho teve como objetivo o desenvolvimento de instrumentação para separações químicas usando microdispositivos de toner-poliéster. Fontes de alta-tensão e de corrente foram desenvolvidas usando módulos conversores de baixa para alta-tensão elétrica. As programações das fontes foram feitas usando tensões elétricas geradas por uma placa de aquisição de dados ou por um conversor digital-analógico (DAC) com uma interface de comunicação I2C. Todo o controle foi desenvolvido em sistema GNU/Linux. Um sistema de injeção hidrodinâmico também foi desenvolvido usando um compressor de ar de diafragma juntamente com um sistema de amortecimento pneumático de pulsações e tendo sua pressão interna estabilizada por uma coluna d\'água. Um medidor de pressão eletrônico foi desenvolvido, usando um sensor de pressão, e calibrado com um manômetro de coluna d\'água. Registros de pressão de -10, -1, +1 e +10cm de coluna d\'água em função de diferentes tempos de injeção foram feitos usando um software controlando o acionamento do injetor hidrodinâmico e efetuando leituras do medidor de pressão eletrônico. Os dados mostram que colunas d\'água de 10cm e tempos de injeção maiores que 3 segundos exibem um desvio padrão relativo (RSD) de aproximadamente 0,5% em módulo. uUma proposta diferente de construção de reservatórios é apresentada. Tal proposta usa mantas de silicone e um bloco de acrílico para a definição dos reservatórios. Observou-se que essa configuração promove o estrangulamento dos canais nas microestruturas de toner-poliéster. Assim, a configuração de colagem de reservatórios por pedaços de tubos, mostrou-se melhor para uso com dispositivos de toner-poliéster descartáveis. Uma nova forma de confecção de eletrodos para detecção condutométrica sem contato acoplada capacitivamente (C4D) foi desenvolvida usando placas de circuito impresso (PCB). Após a confecção dos eletrodos pelo processo de corrosão de PCB a placa foi recoberta com uma resina para que os espaços entre os eletrodos ficassem da mesma altura da camada de cobre. Essa configuração é simples e permite uma maior integração de circuitos eletrônicos. Testes de separação eletroforética foram feitos usando a instrumentação desenvolvida neste trabalho. Soluções de 100µM dos cloretos de K+, Na+ e Li+ dissolvidos em tampão HLac/His 2mM foram usadas para os testes. Essas espécies foram injetadas eletrocineticamente e separadas usando tampão HLac/His 20mM. A quantificação não foi possível por apresentar irreprodutibilidade no processo de injeção devido ao uso de espécies de elevada mobilidade, juntamente com longos canais de injeção. Também foram realizados testes com amostras de sangue permitindo a separação de K+ e Na+ sem pré-tratamento. Separações isotacoforéticas de 1mM dos cloretos de K+, Na+ e Li+ e 1mM de HCl, como eletrólito líder, e 1mM de cloreto de tetrametilamônio, como terminador, foram realizadas para demonstrar a funcionalidade do sistema em sistemas isotacoforéticos.
The Microchip Capillary Electrophoresis (µCE or MCE) is a different kind of capillary electrophoresis that has been growing. This technique uses devices made with small plates of glass or polymer with a microchannel instead of a silica capilar. Improvements in time analysis, sample volumes, physical dimensions, power consume, and integrability with diferent systems have been archieved. A diferent microfabrication technique using laser printer toner and polyester sheets was used to build devices for microfluidic devices. This tecnique is simple, fast and suitable for prototyping. In this work were developed instruments for use with these toner-polyester microdevices. High-voltage and current sources were developed using high-voltage conversors (DC/HVDC). The programming was obtained by electric voltages from a data acquisition board and a digital-analogic conversor (DAC) with a I2C interface communication. Its control was made in a GNU/Linux System. An hidrodynamic injector was developed using an air compressor with a pulse dumper. The internal pressure was regulated by water column. An electronic manometer was built and calibrated with a water manometer. Recording of pressure using -10, -1, +1, and +10cm water column using different injection times were acquired with a data acquisition system. The data show that when water columns of ca. 10cm and injection times greater than 3 seconds are used, the relative standard deviation (RSD) is about 0.5% in modulus. A different way to build vials is presented. This method uses a silicone mantle and plastic glass block with holes. As a result, channels are stragled due to the poliester sheets. A new way to build electrodes for capacitively coupled contactless conductivity detection (C4D) using printed circuit boards (PCB) is shown. After the corrosion of the copper board, varnish is applied on the board to planify its surface. This configuration is simple and allows good integrability with the electronic circuit. Electrophoretic tests using the instrumentation developed was performed by separation of 100µM K+, Na+ and Li+ solutions in 2mM HLac/His buffer. This solutions were injected by electrokinetic method and separated using 20mM HLac/His buffer under high-voltage. The three species were detected but not quantified due to irreprodutibilities of the electrokinetic injection with high mobility ions. Demonstrative separations of K+ and Na+ were made with the same chemical system and blood samples without pretreatment. Isotacophoretic separations of 1mM K+, Na+, and Li+ in 1mM HCl (leader electrolyte) and 1mM tetramethylammonium (terminate electrolyte) were carried outto demonstrate the system functionality.

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed.

Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues.

Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.

博士
國立交通大學
應用化學系所
93
Polymers are the most promising materials for fabrication of microfluidic devices since they are applicable for conventional mass replication technologies such as hot embossing, casting, and molding. In this paper, we demonstrate several polymer microfabrication technologies for fabricating microfluidic devices for microchip capillary electrophoresis and microreactor applications. First, a molding process using silicone mold and polymer resins for the casting and duplication of microchannels from a master template to plastic substrates is described. These silicone molds can be used repeatedly to replicate inexpensive and disposable polymeric microfluidic devices. Second, we describe an effective method for controlling the surface properties and performance of polymeric microchips by using a bulk copolymerization approach during the fabrication process. The loading of a hydrophilic modifier (2-hydroxyethyl methacrylate) has a dramatic effect on the contact angle and electroosmotic mobility (μeo) of the modified copolymer chips. This method is a simple and potentially useful approach toward preparing plastic chips that have different intrinsic bulk properties and electroosmotic flows in their microchannels. In addition, we demonstrate a simple, efficient, economical and environmentally friendly continuous method for preparing polyaniline in a water medium within a flexible dry-film-photoresist-based polymeric microreactor. Finally, we present a new approach for integration of electrophoresis microchips with electrochemical detector using dry film photoresist in conjunction with photolithographic and lamination techniques. Rapid and efficient separation of dopamine, catechol, and uric acid was achieved within 50 s at 200 V/cm in microchip capillary electrophoresis. Combined with this easily performed fabrication procedure, dry film photoresist can be considered as promising alternative materials for constructing microfluidic devices. This approach for miniaturized microchip with electrochemical detector is time- and cost-effective, which suggests that it has great potential for use in prototyping of disposable microscale analytical system.

The introduction of the “microscale total analysis system (μTAS)” concept in the late 80’s triggered the evolution of microfluidic devices that cover a vast range of applications. Automation, integration of multiple processes, and near zero dead volume for separation techniques are some benefits. Closing the gap between research and commercialization in a resource-limited environment is the main aim of this research. This project feeds into two main streams. The first is to integrate on-chip sample preparation for biological applications, like therapeutic drug monitoring (TDM) and diagnostics, using nanojunctions created by controlled dielectric breakdown (Chapters One - Five). The second part focuses on fast prototyping of microfluidic devices with multiple integrated functionalities using a consumer-based 3D-printer (Chapters Six & Seven). These two approaches were tailored to solve specific problems inherent to each sample type and application. Chapter One starts with a general introduction to the unique ion transport phenomena associated with nanojunctions. Many factors act together to determine whether a certain ion will be blocked or preferentially transported through the nanojunction. I developed controlled dielectric breakdown as a cost-effective alternative to conventional nanolithography methods. Pore size control was achieved by tuning the breakdown voltage in response to the feedback current measured through the formed nanojunction. Higher pre-set current limits result in larger pore size and hence the nanojunction will be permeable to larger molecules. I demonstrate the use of single nanojunction for simple extraction and the use of two nanojunctions acting together to form a size/mobility trap (SMT) for the simultaneous extraction, concentration, and desalting. In the SMT format, the second nanojunction was introduced on the other side of the separation channel and offset by a 500 μm. While the role of the first junction remains the same, extraction, the second junction made with smaller pore size blocks the analyte but permits smaller ions. The two nanojunctions work together as a trap that concentrates the injected plug and simultaneously desalt it. This approach is very flexible and can be tuned for different applications as demonstrated in the following chapters. Chapter Two is an introduction to microfluidic systems used for analysis of small molecules, especially pharmaceuticals, in biological samples. The methods were reviewed regarding the hardware and fluid handling processes. The chapter concludes by discussing the requirements for point-of-care devices and decision making based on the results obtained. There are still many challenges and issues that need to be addressed before the wide spread use of these devices becomes a reality. In Chapter Three, the pore size of the nanojunctions was optimized for the analysis of small molecules in blood. First, a single nanojunction was integrated between the sample compartment and the separation channel of the microfluidic device. The nanojunction will permit the analyte of interest and small ions but block blood cells and other macromolecules. Isotachophoresis (ITP) and blue light emitting diodes (LEDs) were employed for the determination of small organic acids in blood with indirect fluorescence detection. The acids chosen in this study were pyruvate, lactate, and 3-hydroxy butyrate due to their significance as biomarkers for diabetes and ketoacidosis. The single nanojunction allowed for the extraction of acids directly from whole blood within 60 s without interference from other macromolecules. The limit of detection (LOD) was 12.5 mM and can be further improved by changing the microchannel geometry near the detection point. The need for point-of-care devices for TDM was addressed through two examples: quinine (an example for positively charged drug) and ampicillin (an example for negatively charged drug). Quinine is a counter-ion at the experimental conditions employed, which is also the case for many pharmaceuticals like antidepressants, and hence its transport is favoured through the negatively charged nanojunction. A single nanojunction was integrated between the sample compartment and the separation channel of the microfluidic device for extraction. Peak mode ITP was employed to concentrate the injected plug and achieve a linear response that covers the therapeutically relevant range. Direct fluorescence detection was feasible due to the native fluorescence of quinine. Finally, SMTs were employed for TDM of ampicillin. This eliminated the need to use other preconcentrating techniques like ITP. The electroosmotic flow (EOF) can be tuned in relation to the electrophoretic mobility by carefully selecting the buffers in the separation channel and the waste/desalting channel. This enables trapping of ions within a certain size/mobility range. Ampicillin is one of the front line antibiotics used for managing sepsis, a critical condition with 30-50% mortality rate. The device may facilitate accurate dose adjustment and improve the survival of septic patients. Chapter Four is a general introduction to different electrokinetic methods for biological sample pretreatment with an interest in biopolymers like proteins and DNA. A special attention was given to devices that incorporate nanojunctions as they exhibit unique behaviour and have already being demonstrated for DNA manipulation, protein concentration, and single molecule detection. Their use was highlighted for sample pretreatment processes like purification, extraction, and concentration. Chapter Five demonstrates the use of the developed nanojunction methods for biopolymer applications. The single nanojunction format was employed to concentrate sodium dodecyl sulphate (SDS)-protein complexes from high ionic strength buffers. Enhancement factors up to 80-fold were achieved within 200 s. The above mentioned SMTs were employed for the direct extraction of short single strand DNA (ssDNA), 20 bases, from blood. As examined with small molecules, DNA molecules were extracted into the separation channel while cells and proteins were blocked. The second nanojunction trapped the DNA in the separation channel leading to simultaneous concentration and desalting. The LOD achieved for fluorescein labelled DNA was 12.5 nM. Chapter Six is an introduction to 3D-printing. Different modes were discussed and compared regarding their capabilities and suitability for microfluidic applications. This was followed by brief discussion of the recent portable systems reported for environmental analysis and design requirements in comparison to biological samples. Chapter Seven explores the microfabrication capabilities of a desktop 3D-printer based on stereolithography (SL). The printer employed for this work is a commercially available low-cost printer that photocures a clear resin that resembles polymers commonly used for large-scale manufacturing. A wide range of microfluidic processes was demonstrated like mixing, gradient generation, droplet extraction and ITP. Multiple functionalities were integrated into one device for nitrate analysis in water. The final design features standard addition at five levels to correct for the matrix effect, passive mixers to shorten reaction time, and detection at different path lengths to extend the linear response range and accommodate samples regardless of their initial concentration. Development and refining of the design was accelerated by the short turn-around times as 3D objects were printed at 2 cm/h speed, in height regardless of xy dimensions. The low price of the printer makes it a very accessible tool for small research laboratories. In Chapter Eight, I summarise the findings of this project and suggest future directions. The outcomes of this research provide valuable solutions for multiple process integration for on-site analysis. Whether it is dielectric breakdown for controlled integration of nanojunctions or fast prototyping of complex devices, both approaches are simple and low-cost. They are suitable for disposable devices and onsite analysis and there is still a great opportunity for improvement in this area.

碩士
國立中興大學
精密工程研究所
92
In this study, we integrate a three-electrode electrochemical detector in a microchip electrophoresis device and find the best fabrication process of different manufactures. In electrodes, the patterns are drawn by AutoCAD and manufactured by screen-print with 250 mesh/inch2 and 10 μm thickness. These high voltage electrode, low voltage electrode, work electrode, counter electrode and reference electrode were screen-printed into 5 cm × 10 cm PMMA substrates using carbon and silver inks. For microchannel, we printed electrodes into PMMA, then coated JSR photoresistor on PMMA and to make the microchannel structures. The final device was bonded in a PMMA/JSR/PMMA sandwich configuration. It is found that combination of photolithography and bonding techniques is the most suitable process during the hot-embossing, pour molding, laser photolithography and photolithography techniques. The microchannel has smooth and shape geometry, and the process is easily control. This microchip is used to detect and separat the standard uric acid and L-ascorbic acid samples. The working voltage is +0.7 V (reference electrode Ag/AgCl), and a duration of 33 seconds at 200 V/cm, 48 seconds at 100 V/cm and 65 seconds at 50 V/cm. These conditions can get the signification signal. In human urine sample, we choose 100 V/cm and it takes 44 seconds. This study presents a low cost, simple and fast process to develop a microchip electrophoresis device. The screen-print process, PMMA substrates, JSR photoresistor and bonding process are successfully integrated to the application of electrochemical detection. Furthermore it can be applied to the other analyses in various biological for medical and clinical diagnosis.