| Cell analysis plays a key role in many biomedical applications both in research laboratories and in clinical settings. Because assays at the cellular level can provide more accurate information about the cellular behavior as compared to molecular assays, they have become increasingly important. Although much progress has been made, there is still room for novel and more accurate cell characterization techniques.;In this thesis, we demonstrate a highly-sensitive and label-free method for characterizing cells based on cell-surface receptors. The method involves measuring a current pulse generated when an individual cell moves under the effect of a pressure-driven flow through an artificial pore having a diameter comparable to the size of the cell. When the pore is functionalized with proteins, specific interactions between a cell-surface marker and the functionalized proteins retard the cell, thus leading to an increased residence time in the pore. This effect results in an increased pulse duration that indicates the presence of that specific biomarker. During its flow across the pore, the cell doesn't bind completely to the pore wall due to the hydrodynamic flow, but experiences only transient binding events. We employed soft-lithography and traditional photolithography to fabricate the devices that we used in our experiments, while we coupled the proteins to the pore walls using a UV-activable cross-linker that can bind to any protein through a nitrene group.;After analyzing the flow of cell populations in an unfunctionalized pore, we successfully screened murine erythroleukemia cells based on their CD34 surface marker in both a single and mixed population of cells. We also used our method to sense CD71 receptor on human stem cells differentiating into erythrocytes and sea-1 receptor on murine myoblasts derived from satellite stem cells (precursors of muscle cells). As a further example, we performed an apoptosis assay. Particularly, we employed annexin V-functionalized pores to detect phosphatidylserine on the surface of apoptotic Jurkat cells and we showed that we are able to distinguish between early and late apoptotic cells without requiring any cell labeling. |
