Optimisation of dielectrophoretic based molecular targeting for blood chronobiology.

Dielectrophoresis (DEP) is an electrostatic technique which can be used to examine cellular electrophysiology. This method offers many advantages in characterising a cell population over conventional methods; however, it has yet to see mainstream pharmacological application. This work demonstrates a DEP assay, 3DEP, as a viable method in the electrophysiological measurement of cells, its application for measuring drug effects, and more specifically, its use as a molecular targeting tool in circadian research. The reliability of the assay was investigated measuring DEP response of several cell types with high resolution spectra and the operational limits of automatic fitting algorithms were determined for single and multiple populations. It was found that reliable readouts from 3DEP could be achieved in as little as 6 s. The ability of 3DEP to test drug cytotoxicity on Jurkat cells was tested with doxorubicin. DEP measured IC50 values compared well with previoiusly measured colorimetric assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) experimental results, suggesting 3DEP could provide an alternative way to measure molecular events due to drug intervention. With 3DEP having been appropriately optimised, it was then applied to the study of RBC circadian rhythm through both human and vole in vitro and in vivo studies. Disruption of these rhythms can lead to negative health outcomes related to areas such as metabolism, cardiovascular health, and mental health. Since RBCs are anucleated, the mechanism cannot rely on the typically accepted transcription-translation feedback loop found for other cells. 3DEP detected robust rhythmicity in the electrophysiological properties of RBCs in isolated and whole blood samples. These rhythms were altered when introduced to pharmacological intervention. Taken together, these findings suggest a model for the non-transcriptional clock in RBCs. Many applications of the 3DEP system were investigated with respect to molecular targeting highlighting 3DEP’s utility and limitations as a biological tool in research