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

Titanium dioxide (TiO 2 ) 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 TiO 2 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 TiO 2 P25 nanoparticles as the SERS substrates, functionalized with a phosphonium-based fluorine-free ionic liquid (IL) comprising the trihexyl(tetradecyl)phosphonium cation ([P 6,6,6,14 ] + ) 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 TiO 2 nanoparticles, significantly enhancing the Cyt c–TiO 2 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 TiO 2 P25 nanoparticles, thus circumventing the need for complex nanomaterial fabrication methods.