Tethered Split-Aptamer Biosensor for Plasmon-Enhanced Fluorescence-Based Continuous Monitoring of Vancomycin.

A split aptamer assay with plasmon- enhanced fluorescence readout is reported for continuous monitoring of low-molecular-weight analytes. The sensor was implemented by using a biointerface with the first split aptamer segment (S1) anchored to the metal sensor surface via a DNA-based flexible polymer linker (FPL) and the second split aptamer segment (S2) directly attached to this surface. The affinity interaction of S1 and S2 with the target analyte modulates the distance between the metallic surface and a fluorophore attached to the FPL outer end. This translates to detected variations in fluorescence signal intensity due to the competing quenching and plasmonic enhancement of emitted fluorescence light. By controlling the surface concentrations of split aptamer segments, the affinity interaction between S1, S2, and the target analyte can be tuned, and key assay characteristics can be modulated over a wide range. For the target antibiotic vancomycin, the respective effective equilibrium dissociation constant was varied by two orders of magnitude from nM to μM concentrations. Despite a fast dissociation rate being necessary for continuous monitoring of the target analyte, the limit of detection could be tuned to low nM concentrations, constituting a concept that can be straightforwardly adapted for other assays that take advantage of reversible affinity interactions with surface-confined split aptamers.