Optimizing the conditions for functionalizing glass with fluorescently labeled oligonucleotides for the creation of reproducible DNA nanosensor and photonic devices

Optimization of glass surface functionalization is a critical step in developing highly sensitive DNA nanosensors and photonic devices. In this study, a systematic comparative analysis was performed to evaluate the efficiency of four glass surface activation protocols - two chemical and two physico-chemical – in combination with different concentrations of (3-aminopropyl)triethoxysilane (APTES: 1%, 2%, and 4% v/v). For the first time, a universal molecular beacon (UMB) labeled with fluorescein (FAM) was used as the immobilized oligonucleotide. The efficiency of each method was assessed by contact angle measurements and fluorescence analysis, including the signal-to-background (S/B) ratio calculation. It was shown that oxygen plasma-based protocols produced the most hydrophilic surfaces (contact angle 4.9$^\circ$ – 5.5$^\circ$) but yielded lower S/B ratios compared to chemical activation. The best results were obtained using Protocol 1 (chemical activation with sulfuric acid and hydrogen peroxide) combined with 2% APTES, providing the highest S/B ratio (5.2 $\pm$ 0.9). Thus, the optimal protocol for UMB immobilization on glass was identified, providing a reliable foundation for the development of advanced DNA nanosensors and photonic devices.