Design and Characterization of a Microfluidic Flow Cytometer

The development and characterization of a microfluidic flow cytometer is described in this dissertation for rapid, inexpensive fluorescence and scatter based cellular enumeration for biomedical diagnostic and monitoring applications. Microfluidic cytometry enables a portable, economical platform for performing point-of-care cellular assays that are impractical with large conventional flow cytometry systems. Microchips were fabricated from glass using standard photolithography and wet chemical etching techniques. These chips were used in conjunction with static optical and electronic components to construct a robust instrument capable of accepting disposable microchips. Flow cytometry measurements on-chip have demonstrated cellular analysis at rates greater than 2 kHz using vacuum driven flow, and positive pressure driven flow cytometry has been performed to measure cells at 12 kHz. Measurement precision has been investigated using commercially available calibration beads, and results demonstrate that the precision of chip based cytometry measurements is close to that seen using conventional instrumentation. Cell discrimination based on immunophenotyping has been demonstrated in mixtures of cultured cells, peripheral blood mononuclear cells, and whole blood to demonstrate the applicability of this instrument for clinical cellular diagnostic and monitoring applications. Comparison studies have been performed using conventional cytometry instrumentation to assess the accuracy of our microfluidic system for immunophenotyping applications. Efforts have also been made to incorporate erythrocyte lysis on-chip in order to rapidly analyze whole blood in an automated fashion. Chemical erythrocyte lysis followed by leukocyte cytometry has been integrated on a single monolithic device to demonstrate the ability to accurately measure cell subtype ratios from a whole blood sample while reducing off-chip sample processing steps. Further, applications in blood dosimetry are described to reveal potential advantages of performing rapid blood cell counts for therapeutic monitoring and bioterrorism applications using only a fingerstick of whole blood. The technology described in this dissertation may be applied to numerous applications requiring the measurement of relative cell populations from biological fluids. Further development and optimization of this instrument has the potential to increase the accessibility of clinical cellular analyses in resource poor settings by providing rapid, low-volume, inexpensive clinical testing at the point-of-care.Doctor of Philosoph