A FRET-based biosensor for NO detection

This paper reports 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 (Kd) for the NO binding to CcP was determined to be 10±1.5 μM. The rate of NO dissociation from the CcP (koff) and the second order rate constant for the NO association (kon) 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