A área de microfabricação de dispositivos de interesse em química analítica tem se expandido muito ao longo dos últimos anos. Uma série de produtos e processos tem sido proposta, tendo como base as tecnologias da área de microeletrônica. Muito destes processos são bastante sofisticados, estando além das necessidades para produção de alguns dispositivos relativamente simples e que são bastante úteis para a química analítica. Este é o caso, por exemplo, dos microcanais para implementação de sistemas eletroforéticos ou micro sistemas em fluxo. Neste contexto, surge a proposta deste trabalho, qual seja desenvolver processos e produtos de interesse nesta área. Esse objetivo foi alcançado pelo desenvolvimento de dois processos: um para produção de microcanais em resina de poliéster através de moldagem e outro de corrosão de vidro utilizando toner de impressora laser como resiste. O primeiro partiu de fotolito para produção de molde em silício através de processo de corrosão por plasma de SF6. Peças de resina de poliéster isoftálica são produzidas por polimerização sobre este molde. Para garantir a desmoldagem não traumática e boa reprodução de detalhes, foi incorporado óleo de silicone durante a preparação da resina. Com este procedimento, foi possível obter canais com 14,0 µm de profundidade e irregularidades superficiais de 1,4 µm para um molde com 15,3 µm de elevação e 0,5 µm de irregularidades superficiais. Com o uso de uma manta flexível de silicone como contraparte, foi possível gerar microcanais cuja altura foi avaliada como sendo da ordem de 5 a 7 µm. Esta avaliação foi conseguida através de medida de condutância após o preenchimento do microcanal com solução de KCl. No segundo processo, toner de impressora laser foi utilizado como resiste para corrosão de vidrO. O layout era diretamente impresso sobre papel aditivado com maltodextrina ou papel utilizado como suporte para etiquetas autocolantes através de uma impressora HP LaserJet 6L com resolução de 600 dpi. Após a transferência térmica da imagem para lâminas de vidro alcalino de 1,0 mm de espessura, a corrosão em ácido fluorídrico permitiu obter canais com 7,1 µm de profundidade e irregularidades de 1,0 µm. Embora este segundo processo apresente desvantagens com relação à resolução tanto no plano da lâmina como na profundidade do canal, quando comparado ao primeiro, deve-se ressaltar a extrema simplicidade, rapidez e baixo custo do processo que deve ser interessante para a produção de protótipos. Já para o primeiro processo, destaca-se a adequação à produção em pequena escala de dispositivos microcanais de baixo custo. / Several processes and products have been proposed to build and use microstructures for chemical purposes. Most of these processes were adapted from microelectronic technologies, which resulted in products with excellent resolution and quality. However, there are some devices that could be generated by simpler and rougher processes. In this work, two processes were developed in order to allow producing simple devices based on microchannels. The first process is a method to produce polyester based devices. A conventional microelectronic process was used to produce a silicon matrix. This matrix was used to produce blocks of isophthalic resin by in situ polymerization. The best results were obtained by adding 1 % (w/w) silicone oil during the polyester resin preparation. This additive improves the mold relief and the smoothness of the device surface. Channels 14.0-µm depth and roughness of 1.4 µm were obtained with a mold with structure height of 15.3 µm and roughness of 0.5 µm. A flexible sheet of silicone allows forming enclosed microchannels with depth of 5-7 µm. This dimension was evaluated by conductance measurement after filling the channel with KCl solution. A process for glass corrosion, using laser printer toner as resist, was proposed. In this method, the layout is printed over a special sheet of paper using a HP LaserJet 6L laser printer. The paper is used to transfer the toner to a soda-lime glass lamina by a thermic process. Hydrofluoric acid solution was used to promote the selective glass corrosion. Channels 7.1-µm depth and roughness of 1.0 µm were obtained. Although this second method does not give the saroe resolution and aspect ratio as the first one, it is suitable to easy and fast prototyping. Gn the other hand, the first method is suitable for low-cost production of devices in small scale.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text

Attachment of circulating tumor cells in microfluidic devices functionalized with proper antibodies was studied. Under static experimental conditions, microchambers were utilized to study the parameters such as cell suspension concentration, incubation time or ambient temperature that may affect the binding of cell to the functionalized surfaces. Specific capture of cells from suspensions increases exponentially with incubation time and linearly with concentration within the tested range. Functionalizing a surface with counter-receptors enables capture of almost 100% of cells within 15 minutes incubation time at ambient temperature higher than 25°C. Suspending cells with different receptors, changing the counter receptors immobilized on the surface, or incubation the cell suspension at low ambient temperature result in a poor capture ratio. To illustrate the specific binding of target cells, various binary mixtures of target cancer and blood cells were incubated in the microchambers. The microsystem sensitivity, specificity and accuracy were determined as a function of the incubated cell concentrations. In general, the system specificity increases while the sensitivity decreases with increasing cell concentration; the accuracy of the system depends weakly on cell concentration within the tested range. The cell attachment dynamics in shear flow was studied by driving the MDA-MB- 231 or BT-20 cells through microchannels functionalized with EpCAM antibodies. The cell attachment ratio was experimentally determined at different flow rates. A modeling system based on Stokesian as well as cell-adhesive dynamics is adopted to analyze the cell motion. The cell motion is modeled as a rigid sphere, with receptors on its surface, moving under shear flow above a surface immobilized with ligands. The system is described mathematically by the Langevin equation, in which the receptor-ligand bonds are modeled as linear springs. Primarily depending on the applied flow rate, three distinct dynamic states of cell motion have been observed: free motion, rolling adhesion, and firm adhesion. The fraction of cells captured due to firm adhesion, defined as attachment ratio, depends on the applied flow rate with a characteristic value that increases with either cellreceptor or surface-ligand density. Utilizing this characteristic flow rate as a scaling parameter, all measured and calculated attachment ratios for different receptor and ligand densities collapse onto a single exponential curve. Binary mixtures of target MDA-MB-231 cells and non-target BT-20 cells were driven through anti-cadherin-11 functionalized microchannels to study the selective isoaltion of target cells from binary mixtures. The system sensitivity is very high, above 0.95, while the specificity is only moderately high, about 0.85, essentially independent of the relative concentration of the target and non-target cells in the binary mixture. An attachment/detachment flow field pattern is proposed to enhance the system specificity. Utilizing this flow pattern with a 1:1,000 MDA-MB-231:BT-20 binary cell mixture, the microfluidic system specificity increased to about 0.95 while the sensitivity remained above 0.95. In order to obtain high experimental throughput allowing lower relative concentration of target cells, a microchannel array which enables processing samples containing about 510⁵ cells with a minimum target cell concentration ratio of 1/100,000 was designed and fabricated. To demonstrate selective isolation of target cells, binary mixtures of BT-20 cells and MIA PaCa-2 cells were driven through microchannel arrays functionalized with EpCAM antibodies; the EpCAM positive BT-20 cells served as target cells and the EpCAM negative MIA PaCa-2 cells as non-target cells. The relative concentration ratio of target/non-target cells varied from 1:1 to 1:100,000. The sensitivity was close to 1.0 while the specificity was also high, about 0.95. The additional detachment step, with a faster flow rate, enhanced the specificity to about 0.985. Initial results of two sets of experiments are reported as preliminary studies for future work. In the first set of experiments, whole blood samples from healthy donors were spiked with a known number of BT-20 cells at a concentration of 500 CTCs per milliliter blood or 50 CTCs per milliliter blood. After a pretreatment to enrich the CTCs, the samples were driven through microchannel arrays functionalized with anti-EpCAM. For both samples, around 55% of the target CTCs were captured in the microchannel arrays. The second set of experiments was dedicated to characterization of target cells exposed to applied shear stress. BT-20 or MDA-MB-231 cells were driven through microchannels functionalized with EpCAM antibodies to allow target cell attachment; then, a high flow rate was applied to detach the captured cells. The detached cells were collected and cultured in an incubator to test their viability. For both cell lines, the majority of the captured CTCs collected from the microchannels were viable. The images taken after three and seven days of culture demonstrate continuous cell growth and division.

This research demonstrates technology for detection of pathogens and environmental monitoring using a handheld optofluidic immunosensor and CFD simulation. The current methods such as ELISA and PCR require few hours for identification which means it is unavailable for early-monitoring. The use of a near-real-time, handheld biosensor device in a real animal/human environment is the key to monitoring the spread of dangerous pathogens. A 3-D computational fluid dynamics (CFD) simulation is needed to track the pathogens within an environment.This dissertation has four papers that demonstrate technologies for the detection and monitoring of pathogens and the miniaturization of these detection systems for in field applications with a handheld immunosensor and CFD simulation.In the first paper, an environmental prediction model was developed for optimal ventilation in a mushroom house by using sensible heat balance and 3-D CFD method. It is shown that the models can be used for farmers to predict the environmental conditions over different locations in a mushroom house.In the second paper, a field lab-on-a-chip system was constructed to detect mouse immunoglobulin G and Escherichia coli by using light scattering detection of particle immunoagglutination. Antibody-conjugated particles were able to be stored in a 4°C refrigerator for at least 4 weeks and to be lyophilized as a powder form for the storage in room temperature.In the third paper, rapid monitoring of the spreads of porcine reproductive and respiratory syndrome virus (PRRSV) was attempted using samples collected from nasal swabs of pigs and air samplers within an experimental swine building. An optofluidic device containing liquid-core waveguides was used to detect. It is shown that the developed optofluidic device and 3-D CFD model can serve as a good model for monitoring the spread of airborne viruses within animal and human environments.In the fourth paper, a handheld optofluidic immunosensor was developed for rapid detection of H1N1/2009 virus inside a 1:10 scale mock classroom. Both miniature spectrometer and cell phone camera were used as detector. A 3-D computational fluid dynamics (CFD) model was developed to track the transport/distribution of H1N1/2009 viruses, and corresponded very well with immunosensor readings.

The field of lipidomics encompasses the study of pathways, networks, and functionality of cellular lipids in biological systems. The lipid subclass, primary fatty acid amides, are crucial to nervous system signaling, receptor function, and numerous other physiological roles. Chapter 1 details these bioactive properties of several well-studied primary fatty acid amides as well as their biosynthesis, degradation, and most common analysis techniques. As these bioactive lipids are endogenously present in trace and ultra-trace abundancies, the field of microfluidics presents an attractive alternative analysis system to incorporate minimization of sample and reagent usage, analysis cost reduction, highly sensitive detection pairing, and decreased analysis time, all while limiting sample handling. Chapter 2 provides a microfluidics-based review of common device fabrication techniques, droplet microfluidics, and detection systems. Current primary fatty acid amide analysis techniques have detection limits on the periphery of endogenous concentrations, presenting the need for a more sensitive detection system, such as fluorescence. Chapter 3 serves as the foundation in developing methodology to analyze these amides and their conjugate fluorescently-tagged primary amines. Chapter 4 focuses on the development of a microfluidic platform capable of efficient on-chip fluorescent tagging reactions and the coupling of a highly sensitive laser induced fluorescence detection system capable of detection limits several orders of magnitude lower than currently employed mass spectrometry techniques. In addition, the appendix details the method development for the quantitative analysis of the anti-inflammatory and anti-cancer drug, celecoxib, uptake into novel drug delivery vehicles. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD / Dissertation;

A magnetic bead-based DNA extraction and purification device has been designed to be used for extraction of the target DNA molecules from whole blood sample. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in the cell lysis buffer in a circular chamber that is sandwiched between two electromagnets. Non-uniform nature of the magnetic field causes temporal and spatial distribution of the beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom electrode. DNA molecules are extracted from the magnetic beads by washing and re-suspension processes. The numerical simulation approach has been adopted in order to design the magnetic field source. The performance of the magnetic field source has been investigated against different physical and geometrical parameters and optimised dimensions are obtained with two different magnetic field sources; integrated internal source and external source. A new magnetic field pattern has been introduced in order to efficiently control the bulk of magnetic beads inside the mixing chamber by dynamic shifting of magnetic field regions from the centre of the coils to the outer edge of the coils and vice versa. A Matlab code has been developed to simulate beads trajectories inside the designed extraction chip in order to investigate the efficiency of the magnetic mixing. A preliminary target molecule capturing simulation has also been performed using the simulated bead trajectories to evaluate the DNA-capturing efficiency of the designed extraction chip. The performance of the designed extraction chip has been tested by conducting a series of biological experiments. Different magnetic bead-based extraction kits have been used in a series of preliminary experiments in order to extract a more automation friendly extraction protocol. The efficiency of the designed device has been evaluated using the spiked bacterial DNA and non-pathogenic bacterial cell cultures (B. subtilis, Gram positive bacteria and E. coli, Gram negative bacteria) into the blood sample. Excellent DNA yields and recovery rates are obtained with the designed extraction chip through a simple and fast extraction protocol.

This thesis presents a detailed study of complementary metal-oxide-semiconductor (CMOS) compatible silicon waveguide and ring resonator technologies. The project specifically focuses on a range of slotted ring resonator configurations comprised of rib-style waveguides. Single ring resonators and Mach-Zehnder interferometers with double rings and central drop port channels have been successfully characterised. Thermal tuning techniques using on-chip heaters were used to determine their sensitivities. A stringent signal cleaning method was also developed to remove systematic background noise. Analysing the transmission signals produced by the Mach-Zehnder interferometers with double rings and a central drop port, it was revealed that coupled resonator induced transparency (CRIT) is created along with Fano-type resonances when the resonant peaks of the two ring resonators are tuned to overlap. The tuning of these features revealed a 2.7 and 2-fold improvement in device sensitivity. A 3x3 transfer matrix model has been developed to simulate the behaviour of light travelling through this configuration. Modelling suggests that effective refractive index and relative phase are the key factors in determining this behaviour. When tuned to close proximity, a resonant ‘superstate’ is achieved in which a modified model is required. Applying the single ring resonators to biosensing applications, basic refractive index testing and a glucose sensing calibration were conducted. A polydimethylsiloxane (PDMS) based microfluidics system was also developed to improve the reliability of sensing and enable automation. Using silicon nitride ring resonators with inkjet-printed upconverting nanoparticles, it was found that the evanescent field of the rings could stimulate the upconversion process revealing visible spectrum emission around the rings.

A simple, but innovative microfluidic Lab-on-a-chip (LOC) device which is broadly applicable in point of care diagnostics of biological pathogens was designed, fabricated and assembled utilising explicit microfluidic techniques. The purpose of this design was to develop a cartridge with the capability to perform multiplex DNA amplification reactions on a single device. To achieve this outcome, conventional laboratory protocols for sample preparation; involving DNA extraction, purification and elution were miniaturized to suit this lab-on-a-chip device of 75mm X 50mm cross-sectional area. The extraction process was carried out in a uniquely designed microchamber embedded with chitosan membrane that binds DNA at pH 5.0 and elutes when a different solution at pH 9.0 flows through. Likewise, purification protocol that occurs in the designed waste reservoir is very significant in biomedical field because it is concerned with waste treatment and cartridge disposability, was performed with a super absorbent powder that converts liquid to a gel like substance. This powder is known as sodium polyacrylate, which is also they treated with anti-bacterial chemicals to prevent environmental contamination. Furthermore, this process also employed the use of a passive valve for a precise fluid handling operation involving flow regulation from extraction to waste reservoir. In order to achieve the intended multiplexing function a multiplexer was created to distribute flow simultaneously through a bifurcated network of channels connected to six similar amplification microchambers. Prior to fabrication, computational fluid dynamics (CFD) simulation was utilized at flowrates less than 10μL/s as the means to test the effectiveness of each design components and also to specifically deduct empirical values that can be analyzed to improve or understand the relationship between the fluid and geometrical constraints of the microfluidic modular elements. The device produced was a hybrid cartridge composed of PDMS and glass which is the most widely used materials microfluidics research due to their low cost and simplicity of fabrication by soft lithography technique. The choice of material also took into account the various physical and chemical properties advantages and disadvantages in their bio-medical applications. Such properties include but not limited to surface energy that determines the wetting fluid characteristics, biocompatibility, optical transparency. Subsequently, after a prototype cartridge was developed fluid flow experimentation using liquid coloured dye was used on the fully fabricated cartridge to test the efficacy of its microfluidic functionalities before expensive DNA amplification reagents were utilised at similar flowrates to the CFD simulations. This gave rise to comparison between similar and dissimilar flow Peculiarities in the microfluidic circuit of both experiments. The final experiment was performed with the aid of a recent molecular technique in DNA amplification known as of RPA kit (recombinase polymerase amplification reaction). It involved performing two main reaction experiments; first, was the positive experiment that bears the sample DNA and the latter, negative that served as the control without DNA. In the end, quantitative analysis of results was done using an agarose gel that showed 143 base pairs, for the positive samples, thus validating the amplification experiment.

This thesis describes the design, fabrication, and testing process for a microfluidic phosphate colorimeter utilized for water quality analysis. The device can be powered by, and interfaced for data collection with, a common cell phone or laptop to dramatically reduce costs. Unlike commercially available colorimeters, this device does not require the user to measure or mix sample and reagent. A disposable poly(dimethylsiloxane) (PDMS) microfluid chip, powered by an absorption pumping mechanism, was used to draw water samples, mix the sample at a specific ratio with a molybdovanadate reagent, and load both fluids into an onboard cuvette for colorimetric analysis. A series of capillary retention valves, channels, and diffusion pumping surfaces passively controls the microfluidic chip so that no user input is required. The microfluidic chip was fabricated using a modified SU-8 soft lithography process to produce a 1.67mm light absorbance pathlength for optimal Beer Lambert Law color absorbance. Preliminary calibration curves for the device produced from standard phosphate solutions indicate a range of detection between 5 to 30mg/L for reactive orthophosphate with a linearity of R²=91.3% and precision of 2.6ppm. The performance of the PDMS absorption driven pumping process was investigated using flow image analysis and indicates an effective pumping rate up to approximately 7µL/min to load a 36µL sample.

The aim to provide preventive healthcare and high quality medical diagnostics and treatment to an increasingly ageing population caused a rapidly increasing demand for point-of-care diagnostic devices. Disposables have an advantage over re-usable units as cross-contamination is avoided, no cleaning and sterilising of equipment is required and devices can be used out of centralised laboratories. To remain cost-effective, costs for disposables should be kept low. This makes polymer materials an obvious choice. One method for the realisation of fluidic micro devices is the stacking of several layers of microstructured polymer films. Reel-to-reel manufacturing is a promising technique for high-volume manufacturing of disposable polymer bio-analytical devices. Polyethylene terephthalate (PET) and cycloolefin copolymer (COC) were selected as suitable polymer substrate materials and polydimethyl siloxane (PDMS) as membrane layer. Bonding of polymer films with the help of adhesives carries the risk of channel blocking. Despite this drawback, no other method of bonding PDMS to a structural layer could be identified. Bonding with solvents avoids channel blocking issues, but adversely affects biocompatibility. Thermal diffusion processes enable bonding of COC and PET without the use of any auxiliary material. The extensive process times requires for thermal diffusion bonding can be considerably shortened by pre-treating the material with plasma or UV exposure. Welding with the use of a laser energy absorbing dye was demonstrated to be particularly suitable for selective bonding around channels and reservoirs. None of the assessed bonding methods provide a generic solution to all bonding applications. Instead, the selection of an appropriate technique depends on the intended application and the required level of biocompatibility. Since this selection has implications on the feasibility and reliability of microfluidic structures on the device, design rules which ensure design for production have to be established and followed.