Optimizing FRET on Aluminum Surfaces via Controlled Attachment of Fluorescent Dyes

Forster resonance energy transfer (FRET) between fluorescent dyes is a frequently applied technique for analyzing concentrations and conformations of biomolecules. Optimizing FRET by controlled dye-surface functionalization is an important requirement to develop sensors based on surface-biomolecule interactions. Here, we investigate the silanization of silica with aminosilanes ((3-aminopropyl)-triethoxysilane, APTES) and their subsequent functionalization with commercial organic fluorophores (ATTO-550 and ATTO-647N) for controlling the fluorescence intensity and FRET interaction between the dyes. Owing to the growing application of aluminum in plasmonics and the possibility to enhance FRET with aluminum nanostructures, we used plasma-enhanced atomic layer deposition (PEALD) to cover aluminum layers with thin silica coatings (similar to 4 nm) as a prototypical system to apply and characterize our controlled APTES-dye functionalization procedure. Detailed spectroscopic and fluorescence imaging analyses were used to optimize the silanization, control the dye functionalization, and rule out aluminum-related fluorescence quenching. The optimized protocol was then used to attach both dyes on the same surface, which enabled efficient FRET. As PEALD is in principle applicable to different substrates, we believe that our controlled FRET-functionalization approach may be adaptable to many other surfaces and nanostructures and may become a useful tool to advance the development of fluorescence biosensors.