A FRET-based biosensor for NO detection

In this paper we explore the use of fluorescently labeled cytochrome c peroxidase (CcP) from baker's yeast for monitoring nitric oxide (NO) down to the sub-micromolar level, by means of a FRET (Forster Resonance Energy Transfer) mechanism. The binding affinity constant (K-d) for the NO binding to CcP was determined to be 10 +/- 1.5 mu M. The rate of NO dissociation from the CcP (k(off)) and the second order rate constant for the NO association (k(on)) were found to be 0.22 +/- 0.08 min(-1) and 0.024 +/- 0.002 mu M-1 min(-1) respectively. The immobilization of fluorescently labeled CcP into a polymeric matrix for use in a solid state NO sensing device was also explored. The results provide proof-of-principle that labeled CcP can be successfully implemented in a fast, simple, quantitative and sensitive NO sensing device. (C) 2010 Elsevier Inc. All rights reserved.