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.

There is great interest in miniaturized analytical systems for life science research, the clinical environment, drug discovery, biotechnology, quality control, and environmental monitoring and numerous articles have been written which predict the success of microfluidic based systems. It was demonstrated in this work that a microfluidic flow system could be quickly and easily manufactured in a research lab environment without the need for clean room facilities. The microfluidic device was created using polymethylmethacrylate, a CO2 laser and a standard oven. The device was designed, manufactured and ready for use within three hours. This work also investigated a chemiluminescent system which was intended for use in protease assays in the microfluidic device. This work also focused on the use of photoinitiated polymer monoliths, with immobilized tannic acid, as protein preconcentrators. The function of the monolithic devices was demonstrated by pumping low concentration solutions of BSA BODIPY® FL through the monolith. Both loading and elution were done using pressure. It was shown that BSA could be concentrated on and successfully eluted from the monolith. The elution volume for a 125 nl monolith was found to be 4 μl. Therefore an injection of a 60 μl sample of 1 x 10⁻⁹M BSA BODIPY ® FL gave rise to a concentration factor of 15. The pH optimum for the binding of BSA BODIPY ® FL was found to be pH 8.0 and the loading capacity of the tannic acid monolith was found to be 0.6 mg.ml⁻¹.

The Life Marker Chip instrument, which has been selected to fly as part of the 2018 ExoMars rover mission payload, aims to detect up to 25 organic molecules in martian rocks and regolith, as markers of extant life, extinct life, meteoritic in-fall and spacecraft contamination. Martian samples will be extracted with a solvent and the resulting liquid extracts will be analysed using multiplexed microarray-format immunoassays. The LMC is under development by an international consortium led by the University of Leicester and the work described within this thesis was carried out at Cranfield University as part of the consortium’s broader program of work preparing the LMC instrument for flight in 2018. Within this thesis four specific areas of LMC instrument development are addressed: the investigation of immunoassay compatible liquid extraction solvents, the study of likely interactions of martian sample matrix with immunoassays, the development of antibodies for the detection of markers of extinct life and demonstration of solvent extraction and immunoassay detection in a flight representative format. Cont/d.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to non-invasively map the physical and chemical environment within microfluidic devices. In this work FLIM has been used in conjunction with a variety of other techniques to provide a greater insight into flow behaviour and fluid properties at the microscale. The pH-sensitive fluorescent dyes, fluorescein and C-SNARF 1, have been used to generate pH maps of microfluidic devices with a time-gated camera and a time-and-space-correlated single photon counting (TSCSPC) detector, respectively. Using time-gated detection and fluorescein, the fluorescence lifetime images allow for direct reading of the pH. The relative contribution to fluorescence of the acid and basic forms of C-SNARF 1 was spatially resolved on the basis of pre-exponential factors, giving quantitative mapping of the pH in the microfluidic device. Three dimensional maps of solvent composition have been generated using 2-photon excitation FLIM (2PE-FLIM) in order to observe the importance of gravitational effects in microfluidic devices. Two fluidic systems have been studied: glycerol concentration in the microfluidic device was measured using Kiton red; water concentration in a methanolic solution was measured using ANS. The density mismatch between two solutions of different composition induced a rotation of the interface between two streams travelling side by side in a microchannel. The experiment has provided evidence of non-negligible gravitational effects in microflows. 2PE-FLIM has superior capability than methods used previously to assess similar phenomena. FLIM and micro-particle imaging velocimetry (μ-PIV) have been implemented on a custom-built open frame microscope and used simultaneously for multimodal mapping of fluid properties and flow characteristics. It has been shown that viscosity mismatch between two streams induces a non-constant advective transport across the channel and results in a flow profile that deviates from the usual Poiseuille profile, characteristic of pressure driven flow in microfluidic devices.

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;

Drop Towers provide brief terrestrial access to microgravity environments. When used for capillary fluidics research, a drop tower allows for unique control over an experiment's initial conditions, which enables, enhances, or otherwise improves the study of capillary phenomena at significantly larger length scales than can normally be achieved on the ground. This thesis provides a historical context for the introduction of a new, highly accessible, 2.1s tower design used for capillary research and presents a variety of demonstrative experimental results for purely capillarity-driven flows leading to bubble ingestion, sinking flows, multiphase flows, and droplet ejections. The focus of this thesis is paid to capillarity-driven droplet ejection including historical significance, mathematical models, criteria for ejection and experimental validation. A scale analysis provides a single parameter Su+ which is used to predict the flow velocity at the base of the nozzle. By simplifying the flow in the nozzle we identify two criteria for auto-ejection, the nozzle must be `short' and the velocity of the flow must be sufficient to invert the liquid meniscus and overpower surface tension at the nozzle tip such that We⁺ > 12. Drop tower experiments are conducted and compared to analytical predictions using a regimemap. This thesis also includes results from experiments experiments conducted in a stationary ground-based laboratory and aboard the International Space Station which clearly demonstrate droplet ejection in regimes from transient liquid jets to large isolated drops. Droplets generated in a microgravity environment are 106 times larger than 1g₀ counter-parts.

In this paper a novel stimuli responsive hydrogel material, methacrylated sodium alginate beta-cyclodextrin (Alg-MA-β-CD), was used in combination with a microfluidic device to create microspheres. Currently there is no reliable method for fabricating homogeneous stimuli-responsive microspheres, in-house microfluidic devices are not reliable in manufacture quality or long-term use. Alginate hydrogels have many attractive characteristics for bioengineering applications and are commonly used to mimic the features and properties of the extracellular matrix (ECM). Human mesenchymal stem cells (hMSCs) are of top interest to tissue engineers. hMSCs are widely available and can be harvested and cultured directly out of human bone marrow. hMSCs have the ability to differentiate into osteoblasts, adipocytes, chondrocytes, muscle cells, and stromal fibroblasts depending on mechanical signals transmitted through surrounding ECM. The biomechanical properties of alginate based stimuli-responsive hydrogels can be tuned to match those of different types of tissues. When trying to transport and control the differentiation of hMSCs into generating new tissues or regenerating damaged tissues, it is highly beneficial to encapsulate the cells inside a microsphere made from these hydrogels. The proposed research objectives are: 1) To optimize fabrication techniques and create functional microfluidic devices; 2) Analyze the effects of flow parameters on microsphere production; and 3) Encapsulate viable hMSCs inside multi-stimuli responsive alginate microspheres using the fabricated microfluidic devices (MFDs). In this study, photolithography microfabrication methods were used to create flow-focusing style MFDs. The hydrogel materials were characterized via rheological methods. Syringe pumps controlled flow rates of fluids through the devices. Active droplets formation was monitored through a camera attached to an inverted microscope, where images were analyzed. Microsphere production was analyzed optically and characterized. Alg-MA-β-CD polymer solutions containing hMSCs were encapsulated, and a live/dead florescence assay was preformed to verify cell viability. Using a modified fabrication process it was possible to manufacture Alg-MA-β-CD microspheres and encapsulate and maintain viable hMSCs inside.

No description available.

Cavity enhanced absorption spectroscopies have earned themselves a place as one of the methods of choice for sensitive absorption measurements on gas-phase samples, but their application to liquid samples has so far been more limited. Sensitive short pathlength analysis of liquid samples is required for online analysis of microfluidic samples, which are processed in channels with dimensions of tens to hundreds of micrometres. Microfluidics is important for a range of applications including drug discovery and environmental sensing. This thesis explores the application of cavity enhanced absorption spectroscopies to short pathlength (0.010 mm to 2 mm) analysis of sub-microlitre volumes of liquids. Three experimental set-ups have been been examined. Firstly, a single-wavelength cavity ringdown (CRD) spectrometer operating at 532 nm was assembled using two 99.8% reflectivity mirrors. High optical quality flow cells with short pathlengths ranging from 0.1 mm to 2 mm were inserted into this cavity at Brewster’s angle. The detection limit of the set-up with each inserted flow cell was established using a concentration series of aqueous potassium permanganate (KMnO₄) solutions. For the 1 mm flow cell, a detection limit of 29 nM KMnO₄ or 1.4 x 10⁻⁴ cm⁻¹ was established. Several different types of microfluidic devices were also inserted into the cavity, and it was found that the losses arising from the inserted chip were highly dependent on the method of chip manufacture. The CRD set-up with inserted 1 mm flow cell was applied to the detection of two important species, nitrite and iron(II), via analyte-specific colourimetric reactions. Detection limits of 1.9 nM nitrite and 3.8 nM iron(II) were established. The second experimental set-up utilised broadband, supercontinuum light generated in a 20 m length of nonlinear photonic crystal fibre. Broadband mirrors with around 99% reflectivity over the wavelength range from 400 to 800 nm were used to form the cavity, and a miniature spectrometer was used to wavelength-resolve the time-integrated cavity output. Flow cells and microfluidic chips were inserted into the cavity either at normal incidence or at Brewster’s angle. This set-up was employed for reaction analysis of an iron complexation reaction with bathophenanthroline, and for a model organic reaction, the Diels-Alder reaction between anthracene and 4-phenyl-1,2,4-triazoline-3,5-dione. The same broadband set-up was also used for pH measurements using bromocresol green indicator solution. Using dual-wavelength CRD spectroscopy, the pH sensitivity was established to be around a few milli pH units. Finally, an alternative type of cavity, formed from a loop of optical fibre has been investigated. A novel light-coupler was designed and fabricated in 365 μm core diameter multimode optical fibre. Sample designs employing both direct and evanescent wave absorption were investigated in small-core and large-core optical fibres, and the lowest detection limit of 0.11 cm⁻¹ was determined in direct absorption measurements, with a pathlength of 180 μm, using our novel light coupler in 365 μm core diameter optical fibre.