Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase.

Abstract

Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its inability to bind oxygen, and its high affinity for nitric oxide make it well-suited for sensor applications. The heme domain has been labeled with a fluorescent reporter dye and changes in this dye's intensity are observed based on the sGC heme domain's characteristic binding of nitric oxide. The current sensors are prepared with 100-microns optical fiber but could also be prepared using submicrometer fiber tips. These sensors have fast, linear, and reversible responses to nitric oxide and are unaffected by numerous common interferents, such as oxygen, nitrite and nitrate. The sensor limit of detection is 1 microM nitric oxide. Glutathione has been shown to decrease the sensitivity of the sensor; however, the sensor response remains linear and can be calibrated on the basis of the glutathione concentration present in the biological environment of interest. The sensors have been used to measure extracellular nitric oxide production by BALB/c mouse macrophages. Minimal nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 microM in a given cell culture.