Step toward Superior Nanoscale Biosensing: Investigation of Trace Cytochrome c Using TiO₂ SERS Substrates and Phosphonium-Based Fluorine-Free Ionic Liquid “Linkers”

Titanium dioxide (TiO2) nanostructures exhibit exceptional flexibility for integration into surface-enhanced Raman spectroscopy (SERS) sensing platforms enabling nanoscale trace detection of biomolecules with high sensitivity. However, fabricating TiO2 nanomaterials for large-scale SERS applications remains challenging due to the high cost and complexity of synthesis methods. In this study, we demonstrate a cost-effective and scalable approach using commercial TiO2 P25 nanoparticles as the SERS substrates, functionalized with a phosphonium-based fluorine-free ionic liquid (IL) comprising the trihexyl(tetradecyl)phosphonium cation ([P6,6,6,14]+) with four long alkyl chains and the 2–2-(2-methoxyethoxy)ethoxy anion ([MEEA]−). This IL serves as a nanoscale “linker”, effectively bridging Cytochrome c (Cyt c) molecules with TiO2 nanoparticles, significantly enhancing the Cyt c–TiO2 interactions and SERS signal intensity. The optimized system achieves remarkable sensitivity, enabling the detection of Cyt c concentrations as low as 5 × 10–4 M, with an enhancement factor increased by 1 order of magnitude compared to the control system. This work stresses the importance of nanoscale interactions and offers an alternative straightforward strategy for trace protein detection using commercially available TiO2 P25 nanoparticles, thus circumventing the need for complex nanomaterial fabrication methods.