Microfluidic Reduction of Osmotic Stress in Oocyte and Zygote Vitrification.

Microfluidic cryoprotectant exchange enables vitrification of murine zygotes with superior morphology as indicated by a smoother cell surface and higher developmental competence compared to conventional methods. Bovine oocyte vitrification also benefit as evidenced by higher lipid retention. Experimental observations and mathematical analysis demonstrate that the microfluidic advantage arise predominantly from eliminating high shrinkage rates associated with abrupt and uneven exposure to vitrification solutions that readily occur in current manual protocols. The microfluidic cryoprotectant exchange method described has immediate applications for improving animal and human oocyte, zygote, and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sub-lethal damage should be broadly useful for cryobiology.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107056/1/davlai_1.pd

On-line laser desorption/ionization mass spectrometry

Interfaces for on-line laser desorption/ionization mass spectrometry are presented in this dissertation. An on line laser desorption interface allows samples to be introduced directly into the mass spectrometer for high-throughput applications. For this research, a linear time of flight mass spectrometer was constructed with an ionization source designed to accept various laser desorption interfaces. A ball inlet interface was used for continuous analyte deposition at atmospheric pressure and vacuum ionization. A solvent-based cleaning system and a separate capillary for MALDI matrix delivery was utilized for continuous on line sampling with the ball inlet interface. Microfluidic devices were brought into contact with the ball inlet and eluant was electrokinetically driven and deposited onto the ball inlet sampling surface. Polymer based microfluidic chips were engineered with open ended channels for on-line coupling, and peptide mixtures were separated on chip and mass detected on line. A non contact deposition method was studied using the ball inlet interface and a single droplet generator. Single droplets with 100 picoliter volume were ejected by a piezoelectric actuated droplet generator and deposited onto the ball inlet. Analyte droplets were placed onto a pre deposited matrix layer during on line analysis, and protonated molecule signal was obtained from as little as 10 fmol analyte. Two contact methods for fast sampling were studied with the ball inlet interface and a direct desorption/ionization interface. Direct contact with the ball inlet was achieved with an indented ball surface for solid, non crystalline, samples. Analytes were brought into direct contact with the ball inlet allowing deposits to settle into micromachined wells on the ball surface. The wells prevented scraping-off of the analyte by the vacuum gasket due to mechanical forces. The analysis of plant tissue, which is not suited for ball inlet sampling, was performed with the direct desorption/ionization interface

Droplet-based separation tools for multidimensional biological separations

Proteins have been extensively studied over the last decade as comprehensive understanding of the proteome can definitely lead to the discovery of novel biomarkers, early-stage disease diagnoses and the development of diagnostic tools and novel drug therapies. One of the crucial and fundamental processes in protein analysis is protein separation, which is usually performed as multidimensional separations to achieve high resolution and high peak capacity. However, high performance analyses are difficult to achieve due to the challenges involved in efficiently integrating different dimensions. In this work, we present the development of a microfluidic device for the effective transfer of protein droplets into the second separation dimension. Consequently, the device provides a stable, reproducible, easy to operate, portable and flexible system to connect a first dimension separation to the downstream second dimension analysis via droplets. The droplets act to preserve the resolution during transfer between separation techniques. In summary, a fluorescently labeled protein ladder serving as a representative of proteins separated from the first dimension is compartmentalized into droplets using the robotic droplet generator. These protein droplets are then transferred via the interfacing microdevice into the second dimension where the released proteins are further separated using capillary gel electrophoresis. Herein, several designs of interfacing microdevices were evaluated for the successful transfer of droplet contents (droplet injection) into the second dimension. The buffer for capillary gel electrophoresis was developed to achieve high-speed and high-resolution separations of proteins in droplet-based injection format. Several fluorescent dyes were also examined for protein labeling to achieve high fluorescent intensities necessary when using this droplet format. Successful droplet-based separation of proteins necessitates the seamless integration of all the developed components. This has been demonstrated here. This interface automates the oil depletion process, minimizes dead volume, prevents dispersion of analyte bands and reduces sample loss at the interface between separation dimensions. Furthermore, optimization of the entire system used in conjunction with the interfacing microdevice provided for ease of operation and more efficient droplet injections. Moreover, droplet injection into parallel separation channels was achieved, highlighting the interfaces capacity for high-throughput analyses.Open Acces

Doctor of Philosophy

dissertationMicrofluidics is an emerging field that deals with the technology and science of manipulation of fluid in microchannels. Since its birth in the 1990s, it has now gradually matured into an enabling technology, like microelectronics and software engineering. A majority of current applications of microfluidics are in life sciences. Polydimethylsiloxane (PDMS) is a soft elastomer and a popular material for fabricating microfluidic devices. This is due to PDMS's unique set of material properties and low cost. Furthermore, the unique mechanical properties of thin PDMS layers/membranes (< 200 µm) can be used to increase the functionality of PDMS-based microfluidic systems. In this presentation, three unique neuroscience applications of PDMS-based microfluidic devices are presented. The working principle behind each of these devices depends on the unique properties of thin PDMS layers. In the first project a fabrication protocol was developed to stack 30 patterned 10-um thick PDMS layers on top of each other without any trapped air bubbles or wrinkles. Each PDMS layer was patterned by spin-coating uncured PDMS on a photolithographic micromold at very high spin speeds and thermally curing the layer later. The layer stacking procedure was done manually using no specialized tools and did not cause any layer deformation to inhibit functionality. This fabrication protocol was used to develop the first ever microfluidic Magnetic Resonance Imaging Phantom to stimulate brain white matter. In the second project, laser ablation was used to rapidly prototype micromolds and by using these micromolds a unique fabrication protocol was developed and characterized to build microvalve arrays (consisting of 100s of microvalves) without access to any cleanroom facility. This was achieved by manipulating the stiffness of thin PDMS layers that are inherent part of pneumatic microvalves. These microvalve arrays were used to build a microfluidic platform for manipulation of C. elegans (a type of a small round worm), which are used extensively for neuronal behavioral analysis. In the last project using similar fabrication techniques (as described in the second project) microfluidic genotyping devices are developed for zebrafish embryos that are less than 2 days old. The unique advantage of the microfluidic zebrafish genotyping devices is that they enable researchers to collect genetic material (for genotyping) from a zebrafish embryo (1 to 2 days old) without causing any harm to its health. This capability is not possible with any other model multicellular organism to date. The working principle behind one of the presented genotyping devices depends on the controlled actuation of PDMS membranes

Finite element modeling in the design and optimization of portable instrumentation

Finite element modeling method (FEM) is a powerful numerical analysis method that is widely used in various engineering and scientific domains. In this thesis, we have utilized FEM to study structural analysis, heat transfer, and fluid flow in the instrumentation design and optimization. In particular, we have designed and optimized a portable micro-dispenser for bio-medical applications and a portable enclosure device for industrial applications. In the micro-dispenser study, our proposed model is comprised of a permanent mainframe and a disposable main tank, which can hold a bulk volume of sample fluid as an off-chip reservoir. The height of the micro-dispenser and the diameter of the passive valve have been analytically designed upon the physical properties of the fluid sample. A Peltier thermoelectric device supported by a fuzzy logic controller is dedicated to controlling the temperature within the micro-dispenser. As an extension, we have also explored another piezoelectric-based actuator, which is further optimized by genetic algorithm and verified by FEM simulations. Furthermore, in the enclosure study, we have proposed a design and optimization methodology for the self-heating portable enclosures, which can warm up the inner space from -55°C for encasing the low-cost industrial-class electronic devices instead of expensive military-class ones to work reliably within their allowed operating temperature limit. By considering various factors (including hardness, thermal conductivity, cost, and lifetime), we have determined to mainly use polycarbonate as the manufacturing material of the enclosure. The placement of the thermal resistors is studied with the aid of FEM-based thermal modeling. In summary, despite the distinct specialties and diverse applications in this multi-disciplinary research, we have proposed our design methodologies based on FEM. The design efficacy has been not only demonstrated by the FEM simulations, but also validated by our experimental measurements of the corresponding prototypes fabricated with a 3D printer

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Microfabrication Technology for Isolated Silicon Sidewall Electrodes and Heaters

This paper presents a novel microfabricationtechnology for highly doped silicon sidewall electrodesparallel to – and isolated from – the microchannel. Thesidewall electrodes can be utilised for both electricaland thermal actuation of sensor systems. Thetechnology is scalable to a wide range of channelgeometries, simplifies the release etch, and allows forfurther integration with other Surface ChannelTechnology-based systems. Furthermore, thefabrication technology is demonstrated through thefabrication of a relative permittivity sensor. The sensormeasures relative permittivity values ranging from 1 to80, within 3% accuracy of full scale, including waterand water-containing mixtures

Microfluidics for Single Molecule Detection and Material Processing

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week