Detection and Quantification of Rare Analytes in Biological Samples using Dielectrophoretic Spectroscopy

Abstract

Novel techniques for Dielectrophoresis (DEP) crossover frequency calculation, positive dielectrophoretic spectroscopy and negative dielectrophoretic spectroscopy are proposed in this dissertation. A novel automated immunoassay based on negative dielectrophoretic spectroscopy for the detection and quantification of rare-analytes in a biological sample is also presented. All of these techniques are based on a custom made automated software “DEP spectroscopy application” for Microsoft Windows that was designed and developed for this research project. The techniques for DEP crossover frequency calculation and dielectrophoretic spectroscopy were validated through experiments with blue colored polystyrene beads with 1000 nm diameter. The techniques for positive dielectrophoretic spectroscopy and negative dielectrophoretic spectroscopy were validated through experiments with fluorescent polystyrene beads with 500 nm diameter. An increase in negative DEP force was observed in response to the increase in the frequency of the applied electric field. This increased DEP force resulted in higher speed of repulsion of functionalized polystyrene beads from the edge of the electrode. The speed of repulsion was measured for 0%, 0.8%, 50% and 100% conjugation of avidin with biotin functionalized polystyrene beads with the automated software through real-time image processing. A significant difference in the velocity of the beads was observed among different avidin-biotin conjugation concentrations that can be used to quantify rare analytes in a biological sample. Using this technique, as little as 80 molecules of avidin per biotin functionalized bead can be detected in a sample. This technology can be applied to the detection and quantification of rare analytes that can be useful in the diagnosis and treatment of diseases like cancer and myocardial infarction with the use of polystyrene beads functionalized with antibodies for the target biomarkers.