abstract: Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and &beta-lactoglobulin; protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis. / Dissertation/Thesis / Ph.D. Chemistry 2011

The number of new cancer cases is projected to rise to 23.6 million by 2030 according to the National Cancer Institute. Obesity & cardiovascular diseases are among the leading causes of death worldwide according to recent reports. Biomarkers— any molecules found within a human body that can be used to monitor an individual's health — have been shown to play a significant role in the detection of cancer, obesity, and cardiovascular diseases. Recent studies have shown that in the diagnosis and screening of various human diseases, including cancer, obesity and cardiovascular diseases, circulating microRNAs (miRNAs) are important biomarkers. A crucial roadblock to using microRNA in screening applications is the lack of effective and low-cost microRNA detection. To address this issue, in this study, we have developed a viable method that combines the dielectrophoresis and electrical impedance. Results show this approach can measure very small concentrations of label-free microRNAs (1pM).

Firstly, this work demonstrates that contactless dielectrophoresis (cDEP) was useful to detect a reversal in the electrical phenotype of late-stage ovarian cancer cells to a profile similar to that of slow-growing early-stage ovarian epithelial cells after treatment with a non-toxic bioactive metabolite, sphingosine. Current chemotherapeutics are highly toxic to patients and can cause severe adverse side effects, so non-toxic treatments that could slow or reverse cancer growth would be advantageous. This is the first instance of cDEP for detecting induced changes in cell structure, showing its potential as a rapid, non-biomarker-based drug screening platform. Specifically, low frequency contactless dielectrophoresis devices previously designed by Sano et al were used to extract the crossover frequency and specific membrane capacitance of early and late stage mouse ovarian surface epithelial (MOSE-E and MOSE-L) cells when untreated, treated with the anti-cancer sphingosine (So) metabolite and with a generally cancer-supporting sphingosine-1-phosphate (S1P) metabolite. The specific membrane capacitance of MOSE-L cells treated with So decreased and the normalized crossover frequency increased to levels matching MOSE-E cells. Secondly, a new multilayer cDEP device featuring curved interdigitated electrode channels overlaying a straight sample channel for the purpose of cell sorting was designed, computationally modeled, fabricated, and tested. The goal of this design was to achieve continuous multi-stream sorting of cells, and preliminary testing demonstrated that prostate cancer PC3 cells were continuously deflected toward the top of the channel under an electric field, as predicted by the numerical model. / Master of Science

No description available.

Placement of single walled carbon nanotubes is demonstrated through massively parallel indirect dielectrophoresis (MPID). MPID is shown to be able to control the placement of carbon tubes as well as the number of tubes placed. Lumped element analysis for AC circuits is used to model MPID. This model allows for predictions of the number of tubes that will be captured in a trap. This model has been consistent with experimental data of numbers of nanotube placed in a junction. Carbon nanotubes placed with MPID are shown to be electrically active.

Dielectrophoresis has been used as a technique for the parallel localization and alignment of both semiconducting and metallic carbon nanotubes (CNTs) at junctions between electrodes. A variation of this technique known as Floating Potential Dielectrophoresis (FPD) allows for a self-limiting number of CNTs to be localized at each junction, on a massively parallel scale. However, the smallest FPD geometries to date are restricted to conductive substrates and have a lower limit on floating electrode size. We present a geometry which eliminates this lower limit and enables FPD to be performed on non-conducting substrates. We also discuss experiments clarifying the self-limiting mechanism of CNT localization and how it can be used advantageously as devices are scaled downward to smaller sizes.

No description available.

Doctor of Philosophy / Over 1.9 million new cases of cancer will pop up just this year alone. The prevalence of cancer, however, has not been met with the same magnitude of effective treatments, resulting in over 600,000 deaths in the United States. Before current treatments can be improved and new treatments can be developed, it is critical that we increase our understanding of what drives cancer to be so aggressive and maintain a fighting chance within the body despite our complex immune systems. The severity of cancer is not just a product of the cancer cell itself, but rather the components that make up the cell that define and drive metastatic behaviors and drug resistance. In order to improve diagnoses, prognoses, and treatment planning, the intracellular drivers of the disease must be better understood. Cells, electrical circuits in nature, reflect unique electrical properties dictated by their biophysical composition. These electrical properties can be revealed and exploited to characterize and treat contributors to disease progression. Using electric fields applied in several modalities, this work explores the electrical entities of malignant cell types towards improving in vitro treatment planning and developing a treatment modality cognizant of subcellular drivers. This dissertation details the use of dielectrophoresis and electroporation to detect and treat intracellular changes associated with poor prognosis.

No description available.

Electrokinetics is a family of effects that induces motion of a liquid or a particle within a liquid in response to an external electric field. Using the intrinsic electrical properties of bacteria and of breast cancer cells, electrokinetics can be used to manipulate these particles for two different types of applications: tissue engineering and breast cancer detection. The first application studied the effects of electric fields on bacteria cells as well as calcium ions to potentially create a meniscus scaffold with hydroxyapatite ends for anchoring. In response to the electric field, calcium ions were able to deposit locally and simultaneously with cellulose growth. Bacteria cells were also studied to determine their response under an AC field. At low frequencies, bacteria demonstrated controlled movement caused by electroosmosis and dielectrophoresis with a net motion caused by a dielectrophoretic force. In the second application, the separation capabilities of different stages of breast cancer cells from the same cell line were tested using contactless dielectrophoretic (cDEP) devices. The electric field gradients in cDEP devices were altered to optimize selectivity and to determine an estimated membrane capacitance for each. From the results, the membrane capacitance of the early to intermediate stages proved to be very similar; however, late stage breast cancer cells have potential in being separated from early and intermediate stages. / Master of Science