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.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Recent approaches in analytical separations are being advanced towards the “lab-on-a-chip” concept in which multiple lab functions are integrated into micro/nano fluidic platforms. Among the variety of separation techniques that can be implemented on microfluidic devices, capillary electrophoresis is the most popular as it provides high efficiency, simple, fast and low cost separations. In addition, integrating miniaturized fluid manipulation tools into microfluidic devices with separations is essential for a variety of biological applications. Chapter 1 discusses the fundamentals of capillary electrophoresis and miniaturized fluid manipulation tools and provides an over view of single cell analysis in microfluidics. In chapter 2, the integration of miniaturized peristaltic pumps into multilayer microfluidic platforms is discussed. In addition, device characterization, precise fluid control and high throughput single cell analysis are discussed. As a proof of principle, T-lymphocytes were loaded with two fluorescent probes Carboxyfluorescein diacetate (CFDA) and Oregon green (OG). Thousands of single cells were automatically transported, lysed on these devices and analytes from the lysate were electrophoretically separated. 1120 cells were analyzed over the course of 80 min (14 cells/min) and separation characteristics of analytes released from individual cells were investigated. In the third chapter, the development of microfluidic platforms for the electrochemical detection of nitric oxide (NO) and other reactive nitrogen species (RNS) at the single cell level is discussed. A microfluidic system was developed to perform rapid cell lysis followed by electrochemical detection. Miniaturized microband electrodes were designed and integrated with a microfluidic separation channel. Three alignment techniques (in-channel, end-channel and off-channel configurations) were used to detect the electrochemical response of the analyte of interest. Furthermore, a model analyte (CFDA) was used to demonstrate the potential of performing the simultaneous dual detection with electrochemical and laser induced fluorescence detection. In addition, the same microfluidic platform was adapted to detect intracellular superoxide using laser induced fluorescence. In the fourth chapter, the off-chip integration of optical fiber bridges with multilayer microfluidic chips is discussed. A multimode optical fiber (~10cm long) was integrated between the single cell lysing spot and a spot downstream of the separation channel in order to detect both intact cells and the analyte in the lysate. This technique was used to create two detection spots on the microfluidic platform with the use of a single excitation source and single detector. Fluorescently labeled T-lymphocytes were automatically transported and lysed in a manner similar to that described in chapter 2. Hundreds of single cells were analyzed and the absolute migration time was determined for the analytes in the lysate. In addition, the separation characteristics of fluorescently labeled protein kinase B peptide substrates were investigated. Furthermore, this technique was used to measure cell size and the velocity of intact cells (discussed in 5th chapter) by making use of a light tunneling concept available in multimode optical fibers. All the experiments presented in this dissertation exploit the use of multilayer microfluidic platforms to investigate intracellular components in single cells in a high throughput manner that has several advantages over current conventional techniques.

The goal of this study is to characterize the mixing that occurs in a microfluidic chip. To characterize the mixing, the minimum length to complete mixing and evolution of mixing will be investigated. There are two types of mixing that occur within a microfluidic channel, diffusion and advection. In the beginning of the microfluidic chip, diffusion is the dominant form of mixing, and in the later portion of the microfluidic chip advection is the dominant form of mixing. The type of design used for this experiment was a zig zag geometry microfluidic chip with channel dimensions of 60 µm X 500 µm X 522 mm. The minimum length for complete mixing was 361 ± 3.475 µm at a flow rate of 25 mL/hr. The mixing was measured using optical light microscopy. For all flow rates less than 20 mL/hr the flow rate was too low to mix the two fluids. The pressure produced by the 30 mL/hr flow rate caused the microfluidic chip to fail.