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 (Förster Resonance Energy Transfer) mechanism. The binding affinity constant (K_d) for the NO binding to CcP was determined to be 10 ± 1.5 µM. 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 µM^− 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.