Numerical Simulations of Flow and Mass Transport in Micro-Fluidic Components for Modular Bio-Analytic Chip Applications

Microfluidics has received a great deal of attention in the past decade. The ability of modular microfluidic chips to miniaturize integrate chemical and biological systems (µTAS) can be greatly productive in terms of cost and efficiency. During the design of these modular devices, misalignment of materials, geometrical or both is one of the most common problems. These misalignments can have adverse effect in both pressure driven and electrokinetically driven flows. In the present work, Numerical Simulations have been performed to study the effect of material and geometrical mismatch on the flow behavior and species progression in microfluidic interconnects. In the case of electrokinetic flows, simulations were performed for 13%, 50%, 58% and 75% reduction in the available flow area at the mismatch plane. Correlations were developed to predict the flow rate reduction due to the geometrical mismatch in electrokinetic flows. A 13% flow area reduction was found to be insignificant and did not cause an appreciable sample loss. As the amount of geometrical mismatch increases (i.e. area reduction is more than 40%), it can have a significant effect on the sample resolution and on the flow behavior. In the case of pressure driven flows, Numerical Simulations have been performed for three types of interconnection methods: End-to-End, Channel Overlap, and Tube-in- Reservoir interconnection. The effects of geometrical misalignments in these three interconnection methods have been investigated and the results were interpreted in terms of the pressure drop and equivalent length. The amount of misalignment was varied by changing the available flow area ratios. All the configurations were simulated for practically relevant Reynolds numbers ranging from 0.075 to 75. Correlations were developed to predict the pressure drop for any given misalignment area ratio. It was found that for the misalignment area ratio of 2:1 or more, the increase in pressure drop can be drastic. Numerical simulations of Injection and separation were also performed to study the effect of curvatures on the elongation of generated plugs. These end curvatures are commonly encountered during high precision micromilling process as a method to fabricate polymer microfluidic devices. The effect of pinching and pullback voltages on the generation of the sample plugs was investigated and optimum conditions to minimize plug dispersion were found

Development of an autonomous lab-on-a-chip system with ion separation and conductivity detection for river water quality monitoring

This thesis discusses the development of a lab on a chip (LOC) ion separation for river water quality monitoring using a capacitively coupled conductivity detector (C⁴D) with a novel baseline suppression technique.Our first interest was to be able to integrate such a detector in a LOC. Different designs (On-capillary design and on-chip design) have been evaluated for their feasibility and their performances. The most suitable design integrated the electrode close to the channel for an enhanced coupling while having the measurement electronics as close as possible to reduce noise. The final chip design used copper tracks from a printed circuit board (PCB) as electrodes, covered by a thin Polydimethylsiloxane (PDMS) layer to act as electrical insulation. The layer containing the channel was made using casting and bonded to the PCB using oxygen plasma. Flow experiments have been conduced to test this design as a detection cell for capacitively coupled contactless conductivity detection (C⁴D).The baseline signal from the system was reduced using a novel baseline suppression technique. Decrease in the background signal increased the dynamic range of the concentration to be measured before saturation occurs. The sensitivity of the detection system was also improved when using the baseline suppression technique. Use of high excitation voltages has proven to increase the sensitivity leading to an estimated limit of detection of 0.0715 μM for NaCl (0.0041 mg/L).The project also required the production of an autonomous system capable of operating for an extensive period of time without human intervention. Designing such a system involved the investigation of faults which can occur in autonomous system for the in-situ monitoring of water quality. Identification of possible faults (Bubble, pump failure, etc.) and detection methods have been investigated. In-depth details are given on the software and hardware architecture constituting this autonomous system and its controlling software

Modular integration and on-chip sensing approaches for tunable fluid control polymer microdevices

228 p.Doktore tesi honetan mikroemariak kontrolatzeko elementuak diseinatu eta garatuko dira, mikrobalbula eta mikrosentsore bat zehazki. Ondoren, gailu horiek batera integratuko dira likido emari kontrolatzaile bat sortzeko asmotan. Helburu nagusia gailuen fabrikazio arkitektura modular bat frogatzea da, non Lab-on-a-Chip prototipoak garatzeko beharrezko fase guztiak harmonizatuz, Cyclic-Olefin-Polymer termoplastikozko mikrogailu merkeak pausu gutxi batzuetan garatuko diren, hauen kalitate industriala bermatuz. Ildo horretan, mikrogailuak prototipotik produkturako trantsizio azkar, erraz, errentagarri eta arriskurik gabeen bidez lortu daitezkeenetz frogatuko da

Fabrication of Receptor-Modified Microfluidic Surfaces for Applications in Glycoprotein Screening

Glycoproteins have long been identified to have a profound association with human pathological processes, and they are much sought after as potential biomarkers to aid in the early diagnosis and clinical prognosis of cancers and diseases. There is currently high demand for high-throughput and low–limit–of–detection techniques that can afford profiling of the glycoproteome. Micro-total analysis systems (µTAS) based on microfluidics have the potential to fulfill these requirements, but in order to reduce the complexity of the protein pool, the µTAS devices must contain a pre-isolation and enrichment component. The research project undertaken here involved derivatization of microfluidic surfaces with ligands to allow for capture and isolation of glycoproteins in solution. It is envisioned that a microfluidic device operating in a serial affinity mode can be fabricated whereby a large set of glycoproteins are captured by a global capture element, followed by further fractionation of the previously captured glycoprotein pool into unique glycoproteins by capture elements specific to each unique protein. To that end, the research here involved (1) modification of poly(methyl methacrylate) surfaces with a boronic acid derivative as the global glycoprotein receptor and (2) investigation of a surface-amenable synthetic route for the creation of a thermoresponsive scaffold with immobilized lectin, as the specific glycoprotein receptor, and its complementary eluting sugar. Creation of these surfaces is the first step toward realizing a µTAS for glycoprotein analysis. The novel boronic acid derivative 4-[(2-aminoethyl)carbamoyl]phenylboronic acid was immobilized on carboxymethyl dextran surfaces, and its protein interaction analysis was investigated by surface plasmon resonance spectroscopy. Poly(methyl methacrylate) microfluidic surfaces were then functionalized with the novel boronic acid derivative to yield a first-generation global capture modality. Glycoprotein binding to and elution from the global capture surface was afforded using glycine- and Tris-binding buffer systems and borate-eluting buffer systems, respectively, with the aid of Tween 20. A thermoresponsive terpolymer poly(N-isopropylacrylamide–lactose–RCA120), with the lectin Ricinus communis agglutinin (RCA120) as the specific capture element, was successfully prepared by surface-amenable synthetic protocols. The synthetic strategy proposed in this work can be easily adapted in the creation of microfluidic devices that can afford the capture of specific glycoproteins

In vitro metabolic pathway construction in an immobilised enzyme microreactor (IEMR)

The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilised enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesising a chiral molecule. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesised from simple achiral substrates using two enzymes, transketolase (TK) and ω-transaminase (TAm). This project involves the design and the development of an IEMR using His6-tagged TK and TAm immobilised onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The IEMR was first characterised based on the operational and storage stability. Furthermore, kinetic parameters of both enzymes were determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. For the multi-step enzyme reaction, two model systems were investigated. The first model investigated was the dual TK (pQR 791)-TAm (pQR 801) reaction. With initial 60 mM (HPA and GA each) and 6 mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 minutes at the lowest flow rate. On the other hand, the second model reaction comprises of three sequential enzyme reaction, TAm (pQR 1021)-TK (pQR 791)-TAm (pQR 1021). A 6% yield of ABT was produced from initial substrate mixture of 100 mM serine and 40 mM GA at flow rate of 0.5 μL/min. Further considerations to improve the system would allow for better yield of the target product and potentially make this IEMR system a powerful tool for construction and evaluation of de novo pathways as well as for rapid determination of various enzymes kinetics

MICROFLUIDICS INTERFACING TO MASS SPECTROMETRY

Polymer-based microfluidic systems have received considerable attention for high throughput chemical analysis. Recently, the ongoing development of microfluidics interfacing to high-accuracy mass spectrometry to identify large molecules had an important impact on biochemistry. A primary goal of this dissertation is the development of a microfluidic apparatus for performing microscale gel electrophoresis, coupled with integrated electrospray tips for either direct interfacing to mass spectrometry through ESI-MS, or coupling to MALDI-MS through the deposition of separated analyte onto a MALDI target for offline analysis. In this dissertation, microfabrication techniques for polymer-based microchip are developed. A novel electrospray interface is demonstrated with good performance. The optimization of multi-channel electrospray tips for multiplexed analysis from a single microfluidic chip was demonstrated. Gas-phased electrophoretic protein/peptide concentration on a pre-structured MALDI target was further demonstrated via theoretical and experimental analysis. The results for developing μGE-ES using linear polymer gel validate the underlined principles and specify challenges involved in coupling μGE to MS. Finally, cross-linked polyacrylamide gel was explored and characterized using in-situ photo- polymerization method in microchannels

Design of microfluidic multiplex cartridge for point of care diagnostics

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonA 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

Doctor of Philosophy

dissertationAnalyte-detecting sensors have been developed in many fields. Chemical sensors, and especially biomedical sensors, deserve special attention because they can simplify time-consuming, costly and site-limited medical procedures. Sensor efficiency depends on its analyte sensing material, signal transducing device and data processing system. The biggest barrier to devise biomedical sensors is the development of analyte sensing material with a high selectivity for target molecules. The motivation of this research was to develop biomolecule-sensitive polymers that can be used in biomedical sensors. Thus, this thesis covers all stages of chemical sensor development, from developing target analyte sensitive materials to merging the developed materials with a signal transducing system. First, the potential application of a zwitterionic glucose-responsive hydrogel as a body implantable continuous glucose monitoring system was examined. After using thermodynamics to confirm the glucose sensing mechanism, synthesis of the hydrogels was optimized and analyzed using statistical methods (design of experiments (DOE)). Thermodynamics study showed that mixing contribution was an important factor to glucose selectivity as well as elastic contribution. By the DOE study, we confirmed that sensitivity of the hydrogels was determined by the molar ratio of cationic and anionic functional groups, and response time depended on the amount of cross-linker. A hydrogel degradation study was also performed to determine the effect of gamma ray sterilization and neutron irradiations on the hydrogel cross-linking network for biomedical applications. Results showed that gamma ray affected cross-linking networks of UV cured hydrogels. However, the neutron irradiation effect was not considerable. In addition, ferromagnetic particles-embedded, zwitterionic glucose-responsive hydrogels were developed to enable the response processing by a magnetoresistive transducer. The hydrogel with horizontally aligned ferromagnetic particles showed good sensitivity in the physiological glucose range (~10mM). Moreover, response time was reduced by almost seven-fold with twice thicker samples (800 um) than samples (400 um) with a pressure sensor measurement. A second project optimized the synthesis of a glutathione (GSH)-sensitive polymer. The selectivity of the polymer for GSH was improved by synthesizing a GSHimprinted polymer and adopting a cobalt ion-mediated chelating binding structure as analyte binding sites