Development of electrochemical methods to enzymatically detect traumatic brain injury biomarkers.

As awareness of traumatic brain injury (TBI) increases, the need to detect mild forms and distinguish between the different severities of TBI becomes more apparent. The goal of this work is to develop a point-of-care sensor to detect whole blood biomarkers for rapid and sensitive diagnosis of TBI severity. Presented herein is the enzymatic detection of norepinephrine through the use of immobilization chemistry and impedance techniques. Sustained elevation of norepinephrine concentrations in the blood has been correlated to negative long-term outcomes in TBI cases, often resulting in permanent cognitive or physical deficits. Novel analysis techniques have been used to identify an optimal binding frequency (371.1 Hz) of norepinephrine to the immobilized enzyme on a gold disk electrode. This form of analysis yielded a logarithmic fit characterized by exceptional responsivity (20.89 Ω/pg mL(-1)), reproducibility (R(2)=0.96), and lower limit of detection (98 pg/mL) first in purified samples, then in rabbit whole blood solutions. Once the optimal binding frequency was determined, the preliminary use of an impedance-time technique was attempted in this work. This technique more closely resembles the amperometric detection method used in commercial self-monitoring blood glucose meters, allowing for continuous or instantaneous measurement of blood borne biomarkers without compromising sensitivity. Future directions include exploration of simultaneous multi-marker detection with the impedance-time technique and experimentation with novel mesoporous materials to filter large blood components.

Amperometric sensing of norepinephrine at picomolar concentrations using screen printed, high surface area mesoporous carbon.

Norepinephrine (NE) is detected amperometrically using the enzyme Phenylethanolamine N-methyl transferase and cofactor S-(5'-Adenosyl)-l-methionine chloride dihydrochloride with disposable screen printed mesoporous carbon electrodes. The role of internal surface area and pore size of the mesoporous carbon is systematically examined using soft-templated, mesoporous silica-carbon powders with highly microporous walls obtained from etching of the silica to produce powders with surface areas ranging from 671-2339 m(2)g(-1). As the surface area increases, the sensitivity of the biosensor at very low NE concentrations (0-500 pg mL(-1)) in phosphate buffered saline (PBS) increases just as the current signal increases with respect to the NE concentration of 81-1581 µA mL ng(-1) cm(-2) for the mesoporous carbons. The best performing electrode provides similar sensitivity in whole rabbit blood in comparison to PBS despite no membrane layer to filter the non-desired reactants; the small (<5 nm) pore size and large internal surface area acts to minimize non-specific events that decrease sensitivity.