Aptamer-Functionalized Silica Particles for FRET-Based Fluorescence Switching

Aptamers conjugated to particles are known to suffer from limited stability due to nuclease cleavage, chemical degradation (hydrolysis or oxidation), and fluorescence loss through quenching under physiological conditions. To address these challenges, we developed a functionalized particle by covalently anchoring azide- and FITC (Fluorescein-isothiocyanate) labeled aptamers onto PEI (Polyethylenimine) coated silica particles (120 ± 5 nm), which is expected to improve construct integrity compared to noncovalent adsorption. This strategy enhances structural integrity and fluorescence preservation under physiological conditions. Alkyne groups were covalently introduced onto the surface of silica particles via carbodiimide-assisted coupling of 10-undecynoic acid to the PEI molecules, while to maintain aptamer conformation, azide-functionalized, FITC-labeled aptamers were conjugated through triazole ring formation with CuAAC click chemistry under mild reaction conditions. To switch fluorescent emission, a black hole quencher labeled complementary strand (BHQ) was hybridized with a FITC-labeled aptamer, quenching FITC emission (∼3-fold) via Förster resonance energy transfer (FRET) and establishing a nonemissive (quenched) state. Upon lysozyme binding, aptamer-protein interactions disrupted FRET, resulting in a 2.2-fold increase in fluorescence, and ∼74% fluorescence recovery compared to the quenched state. The fluorescent particles demonstrate potential for target-responsive detection of various protein biomarkers by replacing the aptamer sequence. To the best of our knowledge, it is the first modular FRET-based fluorescent switch built on aptamer-conjugated silica functionalized via click chemistry.