RESUMEN
Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca(2+) ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.
