A FRET-based biosensor for NO detection.

Journal of inorganic biochemistry (2010-03-23)
Maria Strianese, Franco De Martino, Vincenzo Pavone, Angela Lombardi, Gerard W Canters, Claudio Pellecchia
ABSTRACT

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 microM. 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 microM(-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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 620 maleimide, suitable for fluorescence
Sigma-Aldrich
Atto 620, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 620 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)

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