A new microrheology set-up is described, which allows us to quantitatively measure the transient rheological properties and microstructure of a variety of solvent-responsive complex fluids. The device was constructed by integrating particle tracking microrheology and microfluidics and offers unique experimental capabilities for performing solvent-response measurements on soft fragile materials without applying external shear forces. Transient analysis methods to quantitatively obtain rheological properties were also constructed, and guidelines for the trade-off between statistical validity and temporal resolution were developed to accurately capture physical transitions. With the new device and methodology, we successfully quantified the transient rheological and microstructural responses during gel formation and break-up, and viscosity changes of solvent-responsive complex fluids. The analysis method was expanded for heterogeneous samples, incorporating methods to quantify the microrheology of samples with broad distributions of individual particle dynamics. Transient microrheology measurements of fragile, heterogeneous, self-assembled block copolypeptide hydrogels revealed that solvent exchange via convective mixing and dialysis can lead to significantly different gel properties and that commonly applied sample preparation protocols for the characterization of soft biomaterials could lead to erroneous conclusions about microstructural dynamics. Systematic investigations by varying key parameters, like molecular structure, gel concentration, salt concentration, and tracer particle size for microrheology, revealed that subtle variations in molecular architecture can cause major structural and microrheological changes in response dynamics. Moreover, the results showed that the method can be applied for studying gel formation and breakup kinetics. The research in this thesis facilitates the design of solvent-responsive soft materials with appropriate microstructural dynamics for in vivo applications like tissue engineering and drug delivery, and can also be applied to study the effect of solvents on self-assembly mechanisms in other responsive soft materials, such as polymer solutions and colloidal dispersions.

Due to the large extent to which important biological mechanisms are conserved evolutionarily, the study of a simple soil nematode, C. elegans, has provided the template for significant advances in biology. Use of this model organism has accelerated in recent years as developments of advanced reagents such as synapse localized fluorescent markers have provided powerful tools to study the complex process of synapse formation and remodeling. Even as much routine biology work, such as sequencing, has become faster and easier, imaging protocols have remained essentially unchanged over the past forty years of research. This, coupled with the ability to visualize small, complex features as a result of new fluorescent reagents, has resulted in genetic screens in C. elegans becoming increasingly labor intensive and slow because microscopy mainly relies on manual mounting of animals and phenotyping is usually visually done by experts. Genetic screens have become the rate limiting factor for much of modern C. elegans research. Furthermore, phenotyping of fluorescent expression has remained a primarily qualitative process which has prevented statistical analysis of subtle features. To address these issues, a comprehensive system to allow autonomous screening for novel mutants was created. This was done by developing novel microfluidic devices to enable high-throughput screening, systems-level components to allow automated operation, and a computer vision framework for identification and quantitative phenotyping of synaptic patterns. The microfluidic platform allows for imaging and sorting of thousands of animals at high-magnification within hours. The computer vision framework employs a two-stage feature extraction to incorporate local and regional features and allows for synapse identification in near real-time with an extremely low error rate. Using this system thousands of mutagenized animals were screened to indentify numerous novel mutants expressing altered synaptic placement and development. Fully automated screening and analysis of subtle fluorescent phenotypes will allow large scale RNAi and drug screens. Combining microfluidics and computer vision approaches will have a significant impact on the biological community by removing a significant bottleneck and allowing large-scale screens that would have previously been too labor intensive to attempt.

In the United States and other developed countries, despite great efforts in time and funding for the prevention of foodborne and airborne diseases, there is still an unacceptable level of common pathogens spread via food, water, and air. To this end, lab-on-a-chip (LOC) technologies were developed for field-deployable assays and point of care diagnostics. These devices have potential applications in hospitals, agricultural farms, processing plants, and even on fields of battle. Two successful types of assays in the recent years towards point of care diagnostics are immunoassays and nucleic acid detection assays. In the Appendix A, we demonstrated a complete, field-deployable particle immunoassay encased within a microfluidic chip that detects small quantities of Salmonella Typhimurium in poultry fluid samples. Because the necessary reagents are pre-loaded and the test and negative control channels are fed by a single sample inlet, single pipetting of sample is possible. This assay demonstrated a 10 CFU/mL limit of detection, which is considerably lower than PCR and enzyme-linked immunosorbent assay (ELISA). Total assay time, including sample reading in an integrated handheld device, was 10 minutes, which was much lower than conventional methods. Because of the simplified protocol and assay time, this biosensor has potential in clinical and field diagnostic applications. In Appendix B, we fit the particle immunoassay to test for Influenza A H1N1/2009 virus and included aerosol sampling from a scaled-down mock classroom. To make the assay field deployable, we used an iPhone for signal detection. The detection limit of the assay was 1 pg/mL (10 pg/mL using the iPhone), which is well below the limit of detection for RT-PCR. This protocol demonstrated that immunoassays can be effective in the presence of interfering dust particles and that viruses can be collected from aerosol with minimal sample preparation. In Appendix C, we demonstrated that paper microfluidics, a newer vision of microfluidics, is a cheap and easy method to extract nucleic acid from S. Typhimurium in a variety of samples, including poultry packaging liquid, whole blood, and feces. Fluorescent detection with an iPhone allows for field and clinical testing. This protocol interfaces with rapid PCR and is a true diagnostic tool.

Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.

Digital microfluidics (DMF) has emerged in the past decade as a novel microfluidic paradigm. As a liquid handling technology, DMF facilitates the electrostatic manipulation of discrete nano- and micro- litre droplets across open electrode arrays providing the advantages of single sample addressability, automation, and parallelization. This thesis presents DMF advances toward improved functionality and compatibility for automated miniaturized cell culture in two and three dimensions. Through the development and integration of surface patterning techniques we demonstrate a virtual microwell method for high precision on-device reagent dispensing in one and two plate DMF geometries. These methods are shown to be compatible with two-dimensional culture of immortalized cell lines on ITO, primary cells on coated surfaces, and for co-culture assays. We further extrapolate this method for the formation of microgels on-demand where form micro scale hydrogel structures through passive dispensing in a wide array of geometries. With this system we interrogate three-dimensional cell culture models, specifically for the recapitulation of kidney epthelialization and the analysis of functional cardiac microgels.

Biological cell manipulation and analysis is one of the most investigated applications of microfluidics. In the last decade, researchers have developed means to handle and sort cells, isolate and study single cells, assay whole and lysed cells, and transfect and electroporate in microchannels. Much of this work was motivated by the observation that many external forces and fields scale favorably in the micro-regime; this is especially the case for the electrical field. This dissertation investigates further integration of electrical forces with microfluidic devices, both channel- and droplet-based, in order to generate new, flexible and more efficient tools for studying cell biology. The first part of the dissertation (Chapter 3) explores a new dielectrophoretic particle separation method in microchannels. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a direct current (DC) or low-frequency alternating current (AC) field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. Very effective separation at the low field strength suggests that this method can also be applied to a separation of biological cells that are not sensitive to low electric potential. The second, larger part of the dissertation (Chapters 4 and 5) is focused on digital microfluidics (DMF), which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. It was demonstrated for the first time that DMF can be used as a method for cell culture and analysis. Several cell-based applications were implemented in DMF format including long-term culture, cell passaging, assaying and transfection. The data presented here suggest advanced performance of DMF techniques relative to standard macro-scale techniques. Cell analysis using DMF was found to be advantageous because of greatly reduced reagent and cell use, increased sensitivity, and the potential for multiplexing. Also, DMF technique for cell passaging demonstrated faster and more straightforward manipulation of cells than the standard techniques. In addition, no adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. The new DMF platform for long-term mammalian cell culture represents the first microfluidic implementation of any kind of all of the steps required for mammalian cell culture – cell seeding, growth, detachment, and re-seeding on a fresh surface. In addition, it is the first demonstration of long-term cell culture in nanoliter droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. We anticipate that the DMF cell culture and analysis techniques presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).

Biological cell manipulation and analysis is one of the most investigated applications of microfluidics. In the last decade, researchers have developed means to handle and sort cells, isolate and study single cells, assay whole and lysed cells, and transfect and electroporate in microchannels. Much of this work was motivated by the observation that many external forces and fields scale favorably in the micro-regime; this is especially the case for the electrical field. This dissertation investigates further integration of electrical forces with microfluidic devices, both channel- and droplet-based, in order to generate new, flexible and more efficient tools for studying cell biology. The first part of the dissertation (Chapter 3) explores a new dielectrophoretic particle separation method in microchannels. Current electrodeless dielectrophoretic (DEP) separation techniques utilize insulating solid obstacles in a direct current (DC) or low-frequency alternating current (AC) field, while this novel method employs an oil droplet acting as an insulating hurdle between two electrodes. Since the size of the droplet can be dynamically changed, the electric field gradient, and hence DEP force, becomes easily controllable and adjustable to various separation parameters. Very effective separation at the low field strength suggests that this method can also be applied to a separation of biological cells that are not sensitive to low electric potential. The second, larger part of the dissertation (Chapters 4 and 5) is focused on digital microfluidics (DMF), which is used to actuate nanoliter droplets of reagents and cells on a planar array of electrodes. It was demonstrated for the first time that DMF can be used as a method for cell culture and analysis. Several cell-based applications were implemented in DMF format including long-term culture, cell passaging, assaying and transfection. The data presented here suggest advanced performance of DMF techniques relative to standard macro-scale techniques. Cell analysis using DMF was found to be advantageous because of greatly reduced reagent and cell use, increased sensitivity, and the potential for multiplexing. Also, DMF technique for cell passaging demonstrated faster and more straightforward manipulation of cells than the standard techniques. In addition, no adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. The new DMF platform for long-term mammalian cell culture represents the first microfluidic implementation of any kind of all of the steps required for mammalian cell culture – cell seeding, growth, detachment, and re-seeding on a fresh surface. In addition, it is the first demonstration of long-term cell culture in nanoliter droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. We anticipate that the DMF cell culture and analysis techniques presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).

The work in the dissertation expands the applications of DIEF and describes the development of incorporating DIEF in a microfluidic chip to create a comprehensive proteomics tool. Proof-of-concept DIEF experiments have been done previously, so the focus of this work is to explore the capabilities of DIEF. Dynamic isoelectric focusing (DIEF) is a separation technique invented by Dr. Luke Tolley. It is similar to capillary isoelectric focusing except it uses four high voltage electrodes to form a pH gradient instead of only two. The additional two electrodes are able to manipulate the pH gradient resulting in selection of the region and of the range of pH within a pre-defined sampling or extraction point. One of the first applications described for DIEF was to isolate a single protein from a complex mixture. The protein isolated was a cellulase enzyme capable of degrading multiple cellulose materials over a wide range of environmental conditions. DIEF did isolate the protein in a pH span of 0.005 which is equivalent to 0.075% of the total pH range. Fractions were collected for sequencing analysis, but the fractions were contaminated with keratin both times. DIEF was also successfully performed in an open air channel. Though other electromigration techniques have been successfully done in open air channels, these techniques were severely time and pH limited. In contrast, DIEF in an open air channel is capable of using the entire 3-10 pH range and can perform isolations until the proteins are completely separated. The device developed was also an improvement on increasing sample capacity. The channel was significantly bigger than the traditional glass capillaries used. Since the channel was open, fraction collection was made simpler by collecting using a pipette. This work also demonstrated that DIEF can be made through the use of silicone molding compounds and polyurethane. The amount of milling needed is reduced, the pieces are produced quickly, and a single mold can produce several pieces. Machining pieces with fragile bits is not needed to be done as much since only one acrylic piece is required produce a mold. The mold can produce several polyurethane pieces. This fabrication method has proven useful for making DIEF holders. The next step was to make DIEF a truly comprehensive proteomic tool by incorporating it into a microfluidic chip. Multiple sample fractions are rapidly generated on chip through the use of multiple bubbles simultaneously injected into the separation channel. This stops the separation and, since each droplet is isolated from others by a bubble on each side, the protein peaks are not able to broaden. This novel use of digital microfluidics is still a work in progress, but the fundamentals have been demonstrated. The fabrication protocol for making molds and PDMS casts was developed using materials and procedures that can be done in a common laboratory environment. DIEF is a separation technique still in its infancy, with a wide variety of available applications. DIEF will continue to be tested in other areas and developed into a comprehensive proteomic tool.