A FRET-based biosensor for NO detection

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.

Using photochemistry to understand and control the production of reactive oxygen species in biological environments

“…7). [62][63][64][65][66][67][68][69][70] In the overall turn on, the probes are made of a donor molecule linked to a suitable acceptor molecule, which does not exhibit fluorescence (Fig. 7a).…”

“…In one study, a donor molecule, Atto620, a proprietary fluorescent dye, was attached to the N-terminus of cytochrome c peroxidase (CcP) from baker's yeast. [65] The latter protein is acting as the acceptor molecule since CcP exhibits a characteristic high spin marker band at 645 nm due to the presence of a non-covalently bound heme with a five-coordinate high spin iron. [65] Since CcP characteristic absorption band overlaps significantly with the emission spectrum of Atto620, no fluorescence of the dye could be detected due to efficient RET, the probe is said in the "off" state.…”

“…[65] The latter protein is acting as the acceptor molecule since CcP exhibits a characteristic high spin marker band at 645 nm due to the presence of a non-covalently bound heme with a five-coordinate high spin iron. [65] Since CcP characteristic absorption band overlaps significantly with the emission spectrum of Atto620, no fluorescence of the dye could be detected due to efficient RET, the probe is said in the "off" state. In the presence of NO  , the sixth coordination position of the CcP heme is filled, resulting in the disappearance of the 645 nm absorption band.…”

“…In the presence of NO  , the sixth coordination position of the CcP heme is filled, resulting in the disappearance of the 645 nm absorption band. [65] By consequence, the quenching of the donor fluorescence due to (F)RET is stopped, and the probe is turned "on". Another NO  probe was achieved by coating the surface of fluorescent CdSe/ZnS quantum dots (QDs) with the ferric complex Fe(III)(DTC) 3 .…”

Estudios de la regulación de la actividad de cloroperoxidasas inmovilizadas mediante el uso de nanopartículas activadas con luz UV

“…; as well as for the determination of environmental concerning compounds. Indeed, nitric oxide (NO) was detected by a FRET-based NO biosensor using a fluorescent-labeled CcP immobilized on a silica matrix (102).…”