Electrochemical measurement of nitric oxide from biological systems

Nitric oxide (NO) is known to be involved in a number of physiological processes, including the immune response. As such, its role in severe infection and sepsis has been investigated, but previous measurement techniques have relied on complicated instrumentation or the quantification of NO byproducts (e.g., nitrate and nitrite). Herein, the fabrication of a microfluidic amperometric sensor for the direct detection of NO in whole blood is described. These sensors were used to evaluate the potential of NO and nitrosothiols (a stable transporter) as prognostic and/or diagnostic biomarkers for infection and sepsis. The microfluidic devices facilitated the selective electrochemical measurement of NO in small volumes of blood at the point-of-care, with adequate sensitivity and limits of detection achieved in buffer, wound fluid, and whole blood. A green (530 nm) light-emitting diode was coupled to the device to enable photolysis of S-nitrosothiol species with subsequent NO detection. While inefficient photolysis prevented the measurement of nitrosothiols in whole blood, detection in serum was achieved. A porcine model of sepsis permitted monitoring of temporal changes in NO and nitrosothiols throughout disease progression. While increases in NO were observed concurrently with other indicators (e.g., increased blood lactate and base deficit), the accumulation of nitrosothiols was observed hours prior to the onset of other symptoms, despite a dramatic drop in the circulating white blood cells that produce NO. A murine model of sepsis was utilized to understand the effects of bacterial virulence and immune suppression on NO during an infection. A non-lethal pneumonia with Pseudomonas aeruginosa resulted in elevated NO levels at 72 h that returned to baseline concentrations after 1 wk. A more virulent bacterium, Klebsiella pneumoniae, resulted in much greater increases in NO, reflecting its pathogenicity. Conversely, in a murine model of post-burn immune suppression and infection, blood NO concentrations remained unchanged relative to uninfected animals despite increased infection severity. Nitric oxide-selective microelectrodes were also used to study NO release at the single cell level, from both immune cells and neurons. Upregulation of carbon monoxide production by the macrophages was demonstrated to inhibit their ability to release NO following immune stimulation. Additionally, the concentration and kinetics of NO release from neurons were determined.Doctor of Philosoph