DEVELOPMENT OF HIGH-THROUGHPUT IMPEDANCE SPECTROSCOPY-BASED MICROFLUIDIC PLATFORM FOR DETECTING AND ANALYZING CELLS AND PARTICLES

Impedance spectroscopy based microfluidics have the capability to characterize the dielectric properties of mediums, particles, cellular and sub-cellular contents in response to stimulating voltage signals over a frequency range. This label-free technology has broad ranges of applications in life sciences where there is a need for high-throughput, label-free, non-contact, and low-cost microsystems. To address these limitations, three innovative impedance spectroscopy microfluidic platforms have been developed and presented in this dissertation. The first platform was developed for detecting and characterizing the transverse position of a single cell flowing within a microfluidic channel using a single impedance spectroscopy electrode pair. Regardless of the cell separation methods used, identifying and quantifying the position of cells and particles within a microchannel are important, as these information indicate both the degree of separation as well as how many cells are separated into each position. Using a single pair of non-parallel surface microelectrodes, five different transverse positions of single cells flowing through a microfluidic channel were successfully identified at a throughput of more than 400 particles/s using the detected impedance peak height and width. The second platform utilizes the above technology to count and quantify cells flowing through multiple outlets of microfluidic cell separation systems. A single pair of step-shaped electrodes was developed by integrating five different electrode-to-electrode gaps within a single pair of electrodes. Using this platform, an overall misclassification error rate of only 1.85% was achieved. The result shows the technology’s capability in achieving efficient on-chip cell counting and quantification, regardless of the cell separation methods used, making it a promising on-chip, low-cost and label-free quantification method for cell and particle sorting and separation applications. The third platform was developed for counting cells and particles encapsulated in water-in-oil emulsion droplets using microfluidic based impedance spectroscopy systems. Impedance signal peak height and width were utilized to successfully quantify the number of cells encapsulated within a droplet, and was successfully applied for various cell types and growth media. In addition, the developed platform has been also successfully tested for identifying and discriminating filamentous fungal cell growth, where single fungal spores and filamentous fungi of different lengths could be discriminated inside droplets. Overall in this research, several impedance spectroscopy based microfluidic systems have been successfully developed to solve current limitations in technologies that need high-throughput, low-cost and label-free detection and characterization method for a broad range of cell/particle screening applications

The Role of Micro fluidic Systems in Biological and Medical Sciences

Micro fluidics is a young discipline. During its beginning, it was mainly an academic field in where researchers study the behavior of fluids at micro scale and how it can be modified with operative and experimental variables. Then, the focus was placed on studying device fabrication process and how to optimize them to lower costs and time, and to enhance system features. After a period of maturation, micro fluidic researchers began to evaluate system usefulness and the possibility of use them in different areas. Micro fluidics became a multidisciplinary field combining concepts of biological and medical sciences and engineering. Diagnostic test, micro particles fabrications, contaminant detection, and medical analyses were first goals. Then, its uses expanded exponentially to other areas opening a world of possibilities. With the advances in miniaturization and material sciences as well as the boom in micro and nanotechnology, manufacturing process became highly precise. New applications in biochemistry, biotechnology, biology and medical sciences were appearing attracting the interest of the industrial sector. Since then, projects are aimed to develop micro fluidic systems with industrial applications. The present contribution describes the characteristics of the three major type of micro fluidic systems, chip-based, capillary-based and paper-based systems. Advantages and limitations of each one are mentioned. In addition, their most important applications in biological and medical sciences are presented.Fil: Helbling, Ignacio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico Para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico Para la Industria Química; ArgentinaFil: Luna, Julio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico Para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico Para la Industria Química; Argentin

Analysis of Bacterial Communities Using Droplets Based Millifluidics

Microbes typically form highly complex and diverse communities that account for a significant portion of life's genetic diversity. Analysis of living systems, e.g. bacterial or cell population, plays a significant role in detecting and identifying pathogens, testing antibiotic susceptibility, and the fundamental research of population diversity and evolution. This work focuses on the analysis of bacterial communities using droplets based millifluidics. To monitor the bacteria growth, we designed an optofluidic system, combining the encapsulation of bacteria in numerous emulsion droplets to monitor their long-term behavior and relationship in a co-culture environment using fluorescent signals. In the first part of this work, we co-encapsulated and cultured two isogenic strains of Escherichia coli (E. coli) in numerous emulsion droplets to reveal their competition and cooperation relationship. Since two strains of E. coli express blue and yellow fluorescent proteins (BFP and YFP, respectively), we quantified their growth by integrating a fluorescence detection system. We analyzed the following parameters: doubling time, population yield, final biomass ratio, correlation map of doubling time and competition coefficient to characterize and compare the bacterial growth kinetics and behavior in mono and co-cultures. In addition, the experimental observations were compared with the predictions from a single growth model. Finally, we employed the millifluidic device to verify the appearance of cross-protection between antibiotic-sensitive bacteria and antibiotic-resistant bacteria. It is one of the mechanisms by which different bacteria, sharing the same environment, protect each other to survive in the presence of antibiotics. For this purpose, the E.coli YFP strain was chosen as an antibiotic-sensitive group. Simultaneously, the E.coli BFP strain with β-lactam and its mutations were selected as resistant strains. Combining the millifluidic droplet reactor method with other detection strategies, e.g. fluorescence microscopy, fluorescence flow cytometry, and plate reader, we proved the appearance of cross-protection by detecting the filamentary cells, the fluorescence of cell-free media, viable cell rates, cell shape and size, as well as β-lactamase activity. All these results obtained by millifluidic devices proved that this strategy could be used in a high-throughput bacterial coexistence study. In addition, the research of these general fields, such as bacterial community and antibiotic impact, can help us to reveal the interaction between microbial species and determine the right dose of antibiotics to inhibit bacterial growth in a co-existent environment efficiently

Online oxygen monitoring using integrated inkjet-printed sensors in a liver-on-a-chip system

The demand for real-time monitoring of cell functions and cell conditions has dramatically increased with the emergence of organ-on-a-chip (OOC) systems. However, the incorporation of co-cultures and microfluidic channels in OOC systems increases their biological complexity and therefore makes the analysis and monitoring of analytical parameters inside the device more difficult. In this work, we present an approach to integrate multiple sensors in an extremely thin, porous and delicate membrane inside a liver-on-a-chip device. Specifically, three electrochemical dissolved oxygen (DO) sensors were inkjet-printed along the microfluidic channel allowing local online monitoring of oxygen concentrations. This approach demonstrates the existence of an oxygen gradient up to 17.5% for rat hepatocytes and 32.5% for human hepatocytes along the bottom channel. Such gradients are considered crucial for the appearance of zonation of the liver. Inkjet printing (IJP) was the selected technology as it allows drop on demand material deposition compatible with delicate substrates, as used in this study, which cannot withstand temperatures higher than 130 °C. For the deposition of uniform gold and silver conductive inks on the porous membrane, a primer layer using SU-8 dielectric material was used to seal the porosity of the membrane at defined areas, with the aim of building a uniform sensor device. As a proof-of-concept, experiments with cell cultures of primary human and rat hepatocytes were performed, and oxygen consumption rate was stimulated with carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), accelerating the basal respiration of 0.23 ± 0.07 nmol s-1/106 cells up to 5.95 ± 0.67 nmol s-1/106 cells s for rat cells and the basal respiration of 0.17 ± 0.10 nmol s-1/106 cells by up to 10.62 ± 1.15 nmol s-1/106 cells for human cells, with higher oxygen consumption of the cells seeded at the outflow zone. These results demonstrate that the approach of printing sensors inside an OOC has tremendous potential because IJP is a feasible technique for the integration of different sensors for evaluating metabolic activity of cells, and overcomes one of the major challenges still remaining on how to tap the full potential of OOC systems.Peer ReviewedPostprint (author's final draft

Microfluidic devices interfaced to matrix-assisted laser desorption/ionization mass spectrometry for proteomics

Microfluidic interfaces were developed for off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI). Microfluidic interfaces allow samples to be manipulated on-chip and deposited onto a MALDI target plate for analysis. For this research, microfluidic culturing devices and automated digestion and deposition microfluidic chip platforms were developed for the identification of proteins. The microfluidic chip components were fabricated on a poly(methyl methacrylate), PMMA, wafer using the hot embossing method and a molding tool with structures prepared via micromilling. One of the most important components of the chip system was a trypsin microreactor. An open channel microreactor was constructed in a 100 µm wide and 100 µm deep channel with a 4 cm effective channel length. This device integrated frequently repeated steps for MALDI-based proteomics such as digestion, mixing with a matrix solution, and depositing onto a MALDI target. The microreactor provided efficient digestion of proteins at a flow rate of 1 µL/min with a residence time of approximately 24 s in the reaction channel. An electrokinetically driven microreactor was also developed using a micropost structured chip for digestion. The micropost chip had a higher digestion efficiency due to the higher surface area-to-volume ratio in the channel. Also, the electrokinetic flow eliminated the need for an external pumping system and gave a flat flow profile in the microchannel. The post microreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with trypsin immobilized on an array of 50 µm in diameter micropost support structures with a 50 µm edge-to-edge inter-post spacing. This micropost reactor was also used for fingerprint analysis of whole bacterial cells. The entire tryptic digestion and deposition procedure for intact bacteria took about 1 min. A contact deposition solid-phase bioreactor coupled with MALDI-TOF MS allowed for low-volume fraction deposition with a smaller spot size and a higher local concentration of the analyte. A bacterial cell-culturing chip was constructed for growing cells on-chip followed by off-line MALDI analysis. Coupling MALDI-TOF MS whole cell analysis with microfluidic culturing resulted in more consistent spectra as well as reduction of the total processing time. The microfluidic cell culturing was performed in a PMMA chip with a polydimethylsiloxane (PDMS) cover to allow gas permeation into the culture channel, which contained a 2.1 μL volume active culture chamber. After incubation of E. coli in a microfluidic culture device at 37 ℃ for 24 h, the cultured cells were analyzed with MALDI MS. Also, a microfluidic cell culture device containing continuous perfusion of culture medium was developed using a polycarbonate membrane. This microfluidic culturing format was improved with a fluidic manifold and thermostatted microheaters. Fingerprint mass spectra distinguishing E. coli strains tested were obtained after a 6 h incubation time, which was shorter compared to the 24 h incubation time using conventional culturing techniques. In addition, an enhanced identification procedure for bacteria was achieved by integrating on-chip digestion of cultured bacteria

DEVELOPMENT OF HIGH-THROUGHPUT IMPEDANCE SPECTROSCOPY-BASED MICROFLUIDIC PLATFORM FOR DETECTING AND ANALYZING CELLS AND PARTICLES

Impedance spectroscopy based microfluidics have the capability to characterize the dielectric properties of mediums, particles, cellular and sub-cellular contents in response to stimulating voltage signals over a frequency range. This label-free technology has broad ranges of applications in life sciences where there is a need for high-throughput, label-free, non-contact, and low-cost microsystems. To address these limitations, three innovative impedance spectroscopy microfluidic platforms have been developed and presented in this dissertation. The first platform was developed for detecting and characterizing the transverse position of a single cell flowing within a microfluidic channel using a single impedance spectroscopy electrode pair. Regardless of the cell separation methods used, identifying and quantifying the position of cells and particles within a microchannel are important, as these information indicate both the degree of separation as well as how many cells are separated into each position. Using a single pair of non-parallel surface microelectrodes, five different transverse positions of single cells flowing through a microfluidic channel were successfully identified at a throughput of more than 400 particles/s using the detected impedance peak height and width. The second platform utilizes the above technology to count and quantify cells flowing through multiple outlets of microfluidic cell separation systems. A single pair of step-shaped electrodes was developed by integrating five different electrode-to-electrode gaps within a single pair of electrodes. Using this platform, an overall misclassification error rate of only 1.85% was achieved. The result shows the technology’s capability in achieving efficient on-chip cell counting and quantification, regardless of the cell separation methods used, making it a promising on-chip, low-cost and label-free quantification method for cell and particle sorting and separation applications. The third platform was developed for counting cells and particles encapsulated in water-in-oil emulsion droplets using microfluidic based impedance spectroscopy systems. Impedance signal peak height and width were utilized to successfully quantify the number of cells encapsulated within a droplet, and was successfully applied for various cell types and growth media. In addition, the developed platform has been also successfully tested for identifying and discriminating filamentous fungal cell growth, where single fungal spores and filamentous fungi of different lengths could be discriminated inside droplets. Overall in this research, several impedance spectroscopy based microfluidic systems have been successfully developed to solve current limitations in technologies that need high-throughput, low-cost and label-free detection and characterization method for a broad range of cell/particle screening applications

Developing Droplet Based 3D Cell Culture Methods to Enable Investigations of the Chemical Tumor Microenvironment

Adaptation of cancer cells to changes in the biochemical microenvironment in an expanding tumor mass is a crucial aspect of malignant progression, tumor metabolism, and drug efficacy. In vitro, it is challenging to mimic the evolution of biochemical gradients and the cellular heterogeneity that characterizes cancer tissues found in vivo. It is well accepted that more realistic and controllable in vitro 3D model systems are required to improve the overall cancer research paradigm and thus improve on the translation of results, but multidisciplinary approaches are needed for these advances. This work develops such approaches and demonstrates that new droplet-based cell-encapsulation techniques have the ability to encapsulate cancer cells in droplets for standardized and more realistic 3D cell culture and cancer biology applications. Three individual droplet generating platforms have been designed and optimized for droplet-based cell encapsulation. Each has its own advancements and challenges. Together, however, these technologies accomplish medium to high-throughput generation (10 droplets/second to 25,000 droplets/second) of biomaterial droplets for encapsulation of a range of cell occupancies (5 cells/droplet to 400 cells/droplet). The data presented also demonstrates the controlled generation of cell-sized small droplets for biomolecule compartmentalization, droplets with diameters ranging between 100-400 μm depending on device parameters, and the generation of instant spheroids. Standardized assays for analyzing cells grown within these new 3D environments include proliferation assays of cells grown in mono- and co-cultures, the generation of large and uniform populations of scaffold supported multicellular spheroids, and a new system for culturing encapsulated cells in altered environmental conditions

Development of a PDMS Based Micro Total Analysis System for Rapid Biomolecule Detection

The emerging field of micro total analysis system powered by microfluidics is expected to revolutionize miniaturization and automation for point-of-care-testing systems which require quick, efficient and reproducible results. In the present study, a PDMS based micro total analysis system has been developed for rapid, multi-purpose, impedance based detection of biomolecules. The major components of the micro total analysis system include a micropump, micromixer, magnetic separator and interdigitated electrodes for impedance detection. Three designs of pneumatically actuated PDMS based micropumps were fabricated and tested. Based on the performance test results, one of the micropumps was selected for integration. The experimental results of the micropump performance were confirmed by a 2D COMSOL simulation combined with an equivalent circuit analysis of the micropump. Three designs of pneumatically actuated PDMS based active micromixers were fabricated and tested. The micromixer testing involved determination of mixing efficiency based on the streptavidin-biotin conjugation reaction between biotin comjugated fluorescent microbeads and streptavidin conjugated paramagnetic microbeads, followed by fluorescence measurements. Based on the performance test results, one of the micromixers was selected for integration. The selected micropump and micromixer were integrated into a single microfluidic system. The testing of the magnetic separation scheme involved comparison of three permanent magnets and three electromagnets of different sizes and magnetic strengths, for capturing magnetic microbeads at various flow rates. Based on the test results, one of the permanent magnets was selected. The interdigitated electrodes were fabricated on a glass substrate with gold as the electrode material. The selected micropumps, micromixer and interdigitated electrodes were integrated to achieve a fully integrated microfluidic system. The fully integrated microfluidic system was first applied towards biotin conjugated fluorescent microbeads detection based on streptavidin-biotin conjugation reaction which is followed by impedance spectrum measurements. The lower detection limit for biotin conjugated fluorescent microbeads was experimentally determined to be 1.9 x 106 microbeads. The fully integrated microfluidic system was then applied towards immuno microbead based insulin detection. The lower detection limit for insulin was determined to be 10-5M. The total detection time was 20 min. An equivalent circuit analysis was performed to explain the impedance spectrum results

REVERSE INSULATOR DIELECTROPHORESIS: UTILIZING DROPLET MICROENVIRONMENTS FOR DISCERNING MOLECULAR EXPRESSIONS ON CELL SURFACES

Lab-on-a-chip (LOC) technologies enable the development of portable analysis devices that use small sample and reagent volumes, allow for multiple unit operations, and couple with detectors to achieve high resolution and sensitivity, while having small footprints, low cost, short analysis times, and portability. Droplet microfluidics is a subset of LOCs with the unique benefit of enabling parallel analysis since each droplet can be utilized as an isolated microenvironment. This work explored adaptation of droplet microfluidics into a unique, previously unexplored application where the water/oil interface was harnessed to bend electric field lines within individual droplets for insulator dielectrophoretic (iDEP) characterizations. iDEP polarizes particles/cells within non-uniform electric fields shaped by insulating geometries. We termed this unique combination of droplet microfluidics and iDEP reverse insulator dielectrophoresis (riDEP). This riDEP approach has the potential to protect cell samples from unwanted sample-electrode interactions and decrease the number of required experiments for dielectrophoretic characterization by ~80% by harnessing the parallelization power of droplet microfluidics. Future research opportunities are discussed that could improve this reduction further to 93%. A microfluidic device was designed where aqueous-in-oil droplets were generated in a microchannel T-junction and packed into a microchamber. Reproducible droplets were achieved at the T-junction and were stable over long time periods in the microchamber using Krytox FSH 157 surfactant in the continuous oil FC-40 phase and isotonic salts and dextrose solutions as the dispersed aqueous phase. Surfactant, salts, and dextrose interact at the droplet interface influencing interfacial tension and droplet stability. Results provide foundational knowledge for engineering stable bio- and electro-compatible droplet microfluidic platforms. Electrodes were added to the microdevice to apply an electric field across the droplet packed chamber and explore riDEP responses. Operating windows for droplet stability were shown to depend on surfactant concentration in the oil phase and aqueous phase conductivity, where different voltage/frequency combinations resulted in either stable droplets or electrocoalescence. Experimental results provided a stability map for strategical applied electric field selection to avoid adverse droplet morphological changes while inducing riDEP. Within the microdevice, both polystyrene beads and red blood cells demonstrated weak dielectrophoretic responses, as evidenced by pearl-chain formation, confirming the preliminary feasibility of riDEP as a potential characterization technique. Two additional side projects included an alternative approach to isolate electrode surface reactions from the cell suspension via a hafnium oxide film over the electrodes. In addition, a commercially prevalent water-based polymer emulsion was found to adequately duplicate microchannel and microchamber features such that it could be used for microdevice replication