In recent years, BioMEMS/NEMS have been primary elements associated with the research and development efforts in the bioengineering area. International and federal funding has effected an enormous increase in the development of state-of-the-art bioengineering and biomedical technologies. Most of the BioMEMS/NEMS related applications are associated with diagnostics, sensing and detection. Procedures for separation and manipulation of biological components play a paramount role in the function of these bioengineering mechanisms. This research was concerned with the development of a novel BioMEMS device for cell manipulation. The functioning of the device is based on the use of thermally responsive polymer networks, which differs dramatically from existing approaches. This approach is cost effective, requires low power and uses a minimal amount of on-device area, which makes it suitable for personal medical diagnostics and battle field scenarios. The device integrates the technologies associated with reversibly binding surfaces and dielectrophoresis, (DEP). The DEP field drives a sample into contact with a binding surface. This surface can be controlled to provide different levels of target selectivity. This system provides a separation strategy that does not suffer from fouling issues. The binding surfaces are fabricated from LCST polymers. The LCST polymers experience hydration-dehydration changes in response to temperature fluctuations. Therefore, separation efficiency can be "dialed in" as a function of temperature to prompt the selection of targets. Furthermore, size-exclusion "trenches" were patterned into the binding surfaces. The trenches permit the passage of the small objects in order to provide size-exclusion separations. In order to expand the discrimination size range from the micron to the submicron scale, two techniques for submicron patterning of cross-linked reversibly binding surfaces were investigated. The patterning techniques associated with electron-beam lithography and the combination of softlithography and a focused ion beam patterning were found to generate well-defined patterns that retained their thermo-responsiveness. The combination of DEP and reversibly binding surfaces for bio-particle manipulation is a significant contribution to microfluidic based separations in BioMEMS/NEMS. The developments associated with this research provide a novel technology platform that facilitates separations, which would be difficult to achieve by any other existing methods.

Exocytosis is a fundamental cellular process where membrane-bound secretory granules from within the cell fuse with the plasma membrane to form fusion pore openings through which they expel their contents. This mechanism occurs constitutively in all eukaryotic cells and is responsible for the regulation of numerous bodily functions. Despite intensive study on exocytosis the fusion pore is poorly understood. In this research micro-fabrication techniques were integrated with biology to facilitate the study of fusion pores from cells in the anterior pituitary using the atomic force microscope (AFM). In one method cells were chemically fixed to reveal a diverse range of pore morphologies, which were characterised according to generic descriptions and compared to those in literature. The various pore topographies potentially illustrates different fusion mechanisms or artifacts caused from the impact of chemicals and solvents in distorting dynamic cellular events. Studies were performed to investigate changes in fusion pores in response to stimuli along with techniques designed to image membrane topography with nanometre resolution. To circumvent some deficiencies in traditional chemical fixation methodologies, a Bioimprint replication process was designed to create molecular imprints of cells using imprinting and soft moulding techniques with photo and thermal activated elastomers. Motivation for the transfer of cellular ultrastructure was to enable the non-destructive analysis of cells using the AFM while avoiding the need for chemical fixation. Cell replicas produced accurate images of membrane topology and contained certain fusion pore types similar to those in chemically fixed cells. However, replicas were often dehydrated and overall experiments testing stimuli responses were inconclusive. In a preliminary investigation, a soft replication moulding technique using a PDMS-elastomer was tested on human endometrial cancer cells with the aim of highlighting malignant mutations. Finally, a Biochip comprised of a series of interdigitated microelectrodes was used to position single-cells within an array of cavities using positive and negative dielectrophoresis (DEP). Selective sites either between or on the electrode were exposed as cavities designed to trap and incubate pituitary and cancer cells for analysis by atomic force microscopy (AFMy). Results achieved trapping of pituitary and cancer cells within cavities and demonstrated that positive DEP could be used as a force to effectively position living cells. AFM images of replicas created from cells trapped within cavities illustrated the advantage of integrating the Biochip with Bioimprint for cellular analysis.

abstract: A new challenge on the horizon is to utilize the large amounts of protein found in the atmosphere to identify different organisms from which the protein originated. Included here is work investigating the presence of identifiable patterns of different proteins collected from the air and biological samples for the purposes of remote identification. Protein patterns were generated using high performance liquid chromatography (HPLC). Patterns created could identify high-traffic and low-traffic indoor spaces. Samples were collected from the air using air pumps to draw air through a filter paper trapping particulates, including large amounts of shed protein matter. In complimentary research aerosolized biological samples were collected from various ecosystems throughout Ecuador to explore the relationship between environmental setting and aerosolized protein concentrations. In order to further enhance protein separation and produce more detailed patterns for the identification of individual organisms of interest; a novel separation device was constructed and characterized. The separation device incorporates a longitudinal gradient as well as insulating dielectrophoretic features within a single channel. This design allows for the production of stronger local field gradients along a global gradient allowing particles to enter, initially transported through the channel by electrophoresis and electroosmosis, and to be isolated according to their characteristic physical properties, including charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. Thus, different types of particles are simultaneously separated at different points along the channel distance given small variations of properties. The device has shown the ability to separate analytes over a large dynamic range of size, from 20 nm to 1 μm, roughly the size of proteins to the size of cells. In the study of different sized sulfate capped polystyrene particles were shown to be selectively captured as well as concentrating particles from 103 to 106 times. Qualitative capture and manipulation of β-amyloid fibrils were also shown. The results demonstrate the selective focusing ability of the technique; and it may form the foundation for a versatile tool for separating complex mixtures. Combined this work shows promise for future identification of individual organisms from aerosolized protein as well as for applications in biomedical research. / Dissertation/Thesis / Ph.D. Chemistry 2011

abstract: Due to heterogeneity at the cellular level, single cell analysis (SCA) has become a necessity to study cellomics for the early detection of diseases like cancer. Development of single cell manipulation systems is very critical for performing SCA. In this thesis, electrorotation (ROT) chips to trap and rotate single cells using electrokinetic forces have been developed. The ROT chip mainly consists of a set of closely spaced metal electrodes (60µm interspacing between opposite electrodes) that forms a closed electric field cage (electrocage) when driven with high frequency AC voltages. Cells were flowed through a microchannel to the electrocage where they could be precisely trapped, levitated and rotated in 3-D along the axis of interest. The dielectrophoresis based ROT chip design and relevant electrokinetic effects have been simulated using COMSOL 3.4 to optimize the design parameters. Also, various semiconductor technology fabrication process steps have been developed and optimized for better yield and repeatability in the manufacture of the ROT chip. The ROT chip thus fabricated was used to characterize rotation of single cells with respect to the control parameters namely excitation voltage, frequency and cell line. The longevity of cell rotation under electric fields has been probed. Also, the Joule heating inside the ROT chip due to applied voltage has been characterized to know the thermal stress on the cells. The major advantages of the ROT chip developed are precise electrorotation of cells, simple design and straight forward fabrication process. / Dissertation/Thesis / M.S. Electrical Engineering 2012

abstract: DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system. / Dissertation/Thesis / Presentation for Lin Gan's thesis defense (orginally in pptx exported in PDF) / Doctoral Dissertation Chemistry 2015

abstract: X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm – ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm – ~20 μm crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion. Additionally, a passive mixer was created to generate unique solution concentrations within isolated nanowells to crystallize phycocyanin and lysozyme. Crystal imaging with brightfield microscopy, UV fluorescence, and SONICC coupled with numerical modeling allowed quantification of crystal growth conditions for efficient phase diagram development. The developed microfluidic tools demonstrated the capability of improving samples for protein crystallography, offering a foundation for continued development of platforms to aid protein structure determination. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

abstract: This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS. / Dissertation/Thesis / Ph.D. Chemistry 2013

abstract: Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation. / Dissertation/Thesis / Ph.D. Chemistry 2014

No description available.

As the world population approaches seven billion, a greater strain is put on the resources necessary to sustain life. One of the most basic and essential resources is water and while two thirds of the earth is covered by water, the majority is either salt water (oceans and seas) or it is too contaminated to drink. The purpose of this project is to develop a portable device capable of testing whether a specific source of water (i.e. lake, river, well…) is potable. There are numerous filtration techniques that can remove contaminants and make even the dirtiest water clean enough for consumption but they are for the most part, very time consuming and immobile processes. The device is not a means of water purification but rather focuses on determining the content of the water and whether it is safe. Particles within the water are separated and trapped using a combination of a Taylor Couette fluid flow system and Dielectrophoretic electrodes. This paper explores Taylor Couette flow in a large gap and low aspect ratio system through theory and experimentation with early stage prototypes. Different inner cylinder radii, 2.12cm, 1.665cm and 1.075cm, were tested at different speeds approaching, at and passing the critical Taylor number, 3825, 4713 and 6923 respectively for each cylinder. Dielectrophoretic (DEP) electrodes were designed, fabricated, coated and tested using latex beads to determine the method of integrating them within the fluid flow system. Taylor Couette theory, in terms of the formation of vortices within the large gap, small aspect ratio system, was not validated during testing. The flow pattern generated was more akin to a chaotic circular Couette flow but still served to move the particles toward the outer wall. Fully integrated tests were run with limited success. Recommendations were made to pursue both circular Couette flow as the basis for iv particle separation and dimensional changes in the setup to allow for the formation of Taylor vortices by increasing the radius ratio but still allowing for a larger volume of fluid.