Synthesis of Fluorescent SiO2 Nanoparticles

Fluorescent SiO₂ NPs were synthesized as previously reported [34 (link)]. Briefly, N-1-(3-trimethoxysilylpropyl)-N′-fluoresceyl thiourea (FITC-APTMS) conjugate solution was prepared by dissolving 2 mg fluorescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, MO, USA) in 1 mL anhydrous ethanol (Carlo Erba Reagents S.r.l., Milano, Italy) and immediately mixed with 10 µL of (3-Aminopropyl)trimethoxysilane (APTMS) (Sigma-Aldrich, St. Louis, MO, USA) under shaking at room temperature for 4 h in the dark. Under nitrogen atmosphere, 25 mL of ethanol was added to 1 mL of aqueous ammonia (28%) (Sigma-Aldrich, St. Louis, MO, USA) and stirred. Then, 950 µL Tetraethyl orthosilicate (TEOS) (Sigma-Aldrich, St. Louis, MO, USA) was mixed with the conjugate solution, and the reaction was stirred at 600 rpm at room temperature for a further 20 h in darkness. The resulting particles were washed three times by centrifugation and resuspension in EtOH using bath sonication and ultimately resuspended in EtOH to reach a final particle concentration of 10 mg/mL.