Date of Award
Doctor of Philosophy (PhD)
Eric J. Guilbeau
This study describes the design, fabrication and applications of a novel thermoelectric microfluidic bio-sensor. The bio-sensor is used for real time detection of the L-glutamate (L-glu) dynamics and metabolism for brain tumor cells immobilized in a microfluidic device. The microfluidic device is fabricated using a polymer/glass laminating technique (Xurography). An antimony-bismuth thin-film thermopile (primary sensing element) is integrated to the microfluidic device. The brain tumor cells are immobilized over the thermopile covering measuring and reference junctions of the thermopile using a poly-l-lysine coating layer. L-glutamate oxidase (L-GLOD) is immobilized over the measuring junctions of the thermopile prior to the immobilization of the cells using Layer-by-layer self-assembly. The thermoelectric L-glu sensor measures the heat produced due to the reaction of L-glu released from immobilized brain tumor cells in the presence of L-GLOD. The immobilized brain tumor cells are stimulated using potassium chloride (kcl) and ionomycin to study the dynamics of L-glu release from the cells. The stimulators increase the release of L-glu from the brain cells, which mimics pathological conditions. The thermoelectric sensor is also used to measure the heat produced by normal metabolic activity of the brain tumor cells. The metabolism from the cells is measured by detecting the decrease in the metabolic rate followed by stopping the glucose supply.
The novel design of the microfluidic biosensor has two inlets, in which the fluid from one inlet is hydro dynamically focused by the fluid from the other inlet. The width of the focused fluid depends on the ratio of the inlet's flow rates. This way the hydro dynamically focused fluid flows only over measuring junctions of the thermopile without controlling the temperature of the reference junctions (the thermopile has measuring and reference junctions). Hence, the reaction zone in the microfluidic device is limited to the measuring junctions of the thermopile.
The measured sensitivity of the thermopile is 7 mV (° C)-1 and the heat sensitivity of the biosensor device is 0.165 W (° C)-1 or 23.57 W (V)-1. The thermoelectric L-glu sensor system is calibrated by injecting known concentrations of L-glu samples. The effect of multiple layers of immobilized L-GLOD layers is studied. Two layers of L-GLOD produced a higher response than one layer of L-GLOD. The sensitivity of the thermoelectric L-glu sensor is 0.6 μVs (mg dL -1)-1 in the linear range of 0.1-50 mg dL-1 with an R2 value of 0.9998. The lowest detection limit of the L-glu sensor is 0.1 mg dL-1. The L-glu sensor detected the release of L-glu from the cultured brain tumor cells after stimulating with 50 mM potassium chloride. The cells released L-glu for 30 seconds after the stimulus was applied. The release and uptake of 1.22 mg dL-1 L-glu concentration is detected. The results of L-glu released from the cells were also compared with the fluorescence-based assay tests. Experiments were also performed to detect the heat from brain tumor cell metabolism. The heat production of the brain tumor cells metabolism in anaerobic conditions detected by the thermoelectric metabolism sensor is 54 pW per cell. The developed thermoelectric biosensor sensor that employs simple fabrication, is highly sensitive, and operates at low sample volumes.
Tangutooru, Siva Mahesh, "" (2012). Dissertation. 321.