RESUMEN
A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4(Q34S), with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3 HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4(Q34S) to labeled peptide. The detection limit of scFv1F4(Q34S) was 15 nM. In a more complex medium (100 microg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.