Microfluidics for the detection of Cryptosporidium

This thesis details the development of microfluidics for the label-free sorting and/ or identification of waterborne pathogens which are commonly detected in contaminated drinking-water supplies using the United States Environmental Protection Agency method 1623.1 (USEPA 1623.1). This method recovers and detects pathogens of the Cryptosporidium and Giardia species, which can cause human gastroenteritis upon ingestion. USEPA 1623.1 is employed universally in developed regions (e.g., Europe, North America, Australia, New Zealand). Specifically, this thesis describes microfluidic systems that were developed with the objective of rapidly discriminating viable (i.e., intact and apparently infectious), humanpathogenic Cryptosporidium oocysts from non-viable, human-pathogenic oocysts and/ or species which are considered non-hazardous to human health. Such a system would reduce the overall detection time and allow a more accurate assessment of the risk posed to human health. A microfluidic setup incorporating dielectrophoresis was designed and employed for the viability-based sorting and enumeration of a human-pathogenic species of Cryptosporidium. This device enabled the sorting of untreated (live) and heat-inactivated (non-viable) sub-populations of the human pathogenic Cryptosporidium parvum with over 80% efficiency. Existing Microfluidic Impedance Cytometry (MIC) and Microfluidic-enabled Force Spectroscopy (MeFS) technologies were adapted for the enumeration, detection and viability determination of human-pathogenic Cryptosporidium oocysts, plus the discrimination of Cryptosporidium species which pose a major risk to human health from those which pose little to no risk. Using MIC, it was possible to discriminate untreated and heat-inactivated C. parvum with over 90% certainty. Furthermore, populations of C. parvum, Cryptosporidium muris (low-risk, human pathogen) and Giardia lamblia (also recovered using USEPA 1623.1) were discriminated from one another with over 90% certainty. Using MeFS, it was possible to differentiate temperature-inactivated (either by freeze- or heat-treatment) C. parvum from live C. parvum with a minimum of 78% efficiency. Finally, the high-risk, human pathogenic C. parvum was discriminated from C. muris with over 85% efficiency. Upon further validation, i.e., the analysis of other Cryptosporidium species and of oocysts which have been inactivated by other means (e.g., ozonation, ultraviolet radiaton), it is hoped that water utilities will employ such method(s) to more accurately characterise the human risk associated with contaminated supplies

USE OF HIGH SURFACE NANOFIBROUS MATERIALS IN MEDICINE

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Theoretical and experimental investigations in acoustofluidic manipulation of bioparticles

With the maturation of micro and nano processing technologies and the development of microchip laboratories, the acoustofluidic manipulation of particles has been developed rapidly. Microfluidic chips as miniature experimental platforms are revolutionizing research tools in several disciplines. Acoustically driven microfluidic chips is a current research hotspot with the advantages of non-invasiveness, high strength, and good biocompatibility. This thesis demonstrates acoustofluidic platforms based on static surface acoustic waves for particle manipulation, including the design of interdigital transducers (IDTs), cleaning room fabrication and integration with microfluidic technologies, electronics, and mechanical systems. I present a numerical model to predict the motion of particles under microfluidic manipulation. I employ a perturbation approach where the flow variables are divided into first and second-order fields. Impedance boundary conditions are used to model microchannel walls and displacement boundary conditions are used to model acoustic actuation. This model is verified to be accurate by comparison with published numerical studies. Furthermore, to extend the generalizability of the model, I present two microfluidic systems, conventional PDMS-SAW system, and the GaN system. By comparing numerical predictions with experimental results, I validate the GaN system's ability to manipulate particles with high throughput and verify the accuracy of the model for different piezoelectric materials and working frequencies. Based on this simulation model, I developed two new acoustic fluid configurations: a microchannel sandwiched between two identical surfaces acoustic wave (SAW) transducers (SAW-SAW) or between a piezoelectric transducer (PZT) and a SAW transducer (PZT-SAW). I have numerically simulated the distribution of acoustic pressure, time-averaged flow velocity, and particle trajectories in these devices and compared the simulation results for different acoustic fluid configurations. The results show that the SAW-SAW and PZT-SAW configurations produce significantly higher acoustic pressures and particle velocities in the microchannel. In addition, I have developed a novel Filled Tilt Angle (FTA) acoustic fluidic device to be applied to the mechanophenotyping of live cells. The FTA 3 device consists of an interdigital transducer placed at an angle along the microfluidic channel in the direction of fluid flow. Pressure nodes formed within the acoustic fluid field of the microchannel cause biological cells to deviate from their original flow pattern based on their mechanical properties, including volume, compressibility, and density. The threshold power that allows the cells to converge to the pressure node fully is used to calculate the acoustic contrast factor. To demonstrate the role of FTA in the mechanophenotyping of cells and to distinguish between different cell types, further experiments were performed by using A549 (lung cancer cells), MDB-MA-231 (breast cancer cells), and leukocytes. The obtained acoustic contrast factors for lung and breast cancer cells differed from those of leukocytes by 55.1% and 17.8%, respectively. These results show that this method can successfully distinguish between different cell types based on the acoustic contrast factors, which has tremendous clinical implications for identifying, for example, epithelial cells in the circulation. Finally, this thesis extends the understanding of acoustofluidic units through the study of innovative microfluidic systems. It will further advance the knowledge of SSAW based cell manipulation techniques and enable further development for high precision cell manipulation and biosensor applications

Development of Microwave/Droplet-Microfluidics Integrated Heating and Sensing Platforms for Biomedical and Pharmaceutical Lab-on-a-Chip Applications

Interest in Lab-on-a-chip and droplet-based microfluidics has grown recently because of their promise to facilitate a broad range of scientific research and biological/chemical processes such as cell analysis, DNA hybridization, drug screening and diagnostics. Major advantages of droplet-based microfluidics versus traditional bioassays include its capability to provide highly monodispersed, well-isolated environment for reactions with magnitude higher throughput (i.e. kHz) than traditional high throughput systems, as well as its low reagent consumption and elimination of cross contamination. Major functions required for deploying droplet microfluidics include droplet generation, merging, sorting, splitting, trapping, sensing, heating and storing, among which sensing and heating of individual droplets remain great challenges and demand for new technology. This thesis focuses on developing novel microwave technology that can be integrated with droplet-based microfluidic platforms to address these challenges. This thesis is structured to consider both fundamentals and applications of microwave sensing and heating of individual droplets very broadly. It starts with developing a label-free, sensitive, inexpensive and portable microwave system that can be integrated with microfluidic platforms for detection and content sensing of individual droplets for high-throughput applications. This is, indeed, important since most droplet-based microfluidic studies rely on optical imaging, which usually requires expensive and bulky systems, the use of fluorescent dyes and exhaustive post-imaging analysis. Although electrical detection systems can be made inexpensive, label-free and portable, most of them usually work at low frequencies, which limits their applications to fast moving droplets. The developed microwave circuitry is inexpensive due to the use of off-the-shelf components, and is compact and capable of detecting droplet presence at kHz rates and droplet content sensing of biological materials such as penicillin antibiotic, fetal bovine serum solutions and variations in a drug compound concentration (e.g., for Alzheimer’s Disease). Subsequently, a numerical model is developed based on which parametrical analysis is performed in order to understand better the sensing and heating performance of the integrated platform. Specifically, the microwave resonator structure, which operates at GHz frequency affecting sensing performance significantly, and the dielectric properties of the microfluidic chip components that highly influence the internal electromagnetic field and energy dissipation, are studied systematically for their effects on sensing and heating efficiency. The results provide important findings and understanding on the integrated device operation and optimization strategies. Next, driven by the need for on-demand, rapid mixing inside droplets in many applications such as biochemical assays and material synthesis, a microwave-based microfluidic mixer is developed. Rapid mixing in droplets can be achieved within each half of the droplet, but not the entire droplet. Cross-center mixing is still dominated by diffusion. In this project, the microwave mixer, which works essentially as a resonator, accumulates an intensive, nonuniform electromagnetic field into a spiral capacitive gap (around 200 μm) over which a microchannel is aligned. As droplets pass by the gap region, they receive spatially non-uniform energy and thus have non-uniform temperature distribution, which induces non-uniform Marangoni stresses on the interface and thus three-dimensional (3D) chaotic motion inside the droplet. The 3D chaotic motion inside the droplet enables fast mixing within the entire droplet. The mixing efficiency is evaluated by varying the applied power, droplet length and fluid viscosity. In spite of various existing thermometry methods for microfluidic applications, it remains challenging to measure the temperature of individual fast moving droplets because they do not allow sufficient exposure time demanded by both fluorescence based techniques and resistance temperature detectors. A microwave thermometry method is thus developed here, which relies on correlating fluid temperature with the resonance frequency and the reflection coefficient of the microwave sensor, based on the fact that liquid permittivity is a function of temperature. It is demonstrated that the sensor can detect the temperature of individual droplets with ±1.2 °C accuracy. At the final part of the thesis, I extend my platform technology further to applications such as disease diagnosis and drug delivery. First, I develop a microfluidic chip for controlled synthesis of poly (acrylamide-co-sodium acrylate) copolymer hydrogel microparticles whose structure varies with temperature, chemical composition and pH values. This project investigates the effects of monomer compositions and cross-linker concentrations on the swelling ratio. The results are validated through the Fourier transform infrared spectra (FTIR), SEM and swelling test. Second, a preliminary study on DNA hybridization detection through microwave sensors for disease diagnosis is conducted. Gold sensors and biological protocols of DNA hybridization event are explored. The event of DNA hybridization with the immobilized thiol-modified ss-DNA oligos and complimentary DNA (c-DNA) are monitored. The results are promising, and suggests that microwave integrated Lab-on-a-chip platforms can perform disease diagnosis studies

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals

Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.Portuguese Science Foundation - (SFRH/BD/44258/2008), “SMART-EC” projec

Device for pressure-driven plug transport comprising microchannel with traps

The present invention provides microfabricated substrates and methods of conducting reactions within these substrates. The reactions occur in plugs transported in the flow of a carrier-fluid

Method for conducting an autocatalytic reaction in plugs in a microfluidic system

The present invention provides microfabricated substrates and methods of conducting reactions within these substrates. The reactions occur in plugs transported in the flow of a carrier-fluid

Method of performing PCR reaction in continuously flowing microfluidic plugs

The present invention provides microfabricated substrates and methods of conducting reactions within these substrates. The reactions occur in plugs transported in the flow of a carrier-fluid