Femtosecond Laser Two‐Photon Polymerization and Reduction of Line‐Contact SERS Structures via Capillary Self‐Assembly for Microfluidic 3D Biosensing

Surface‐enhanced Raman scattering (SERS) technology has emerged as a crucial analytical tool due to its high sensitivity in detecting trace substances. However, traditional SERS substrates, constrained by two‐dimensional fabrication approaches, cannot meet the demands for in situ analysis in complex microenvironments. To address this challenge, a three‐dimensional SERS structure fabrication strategy based on the synergistic interaction between hydrogel micropillars and silver nanoparticles (Ag NPs) was proposed. Low‐stiffness hydrogel micropillars were first fabricated via femtosecond laser two‐photon polymerization (TPP). By integrating two‐photon reduction and capillary force‐driven self‐assembly, the “line‐contact” assembly structure featuring high‐density nanogaps was manufactured. The hydrogel's strong adsorption captures more Ag NPs to serve as SERS hotspots, boosting the electromagnetic field enhancement. Additionally, its low Young's modulus improves assembly morphology and increases hotspot density. Mechanical and electromagnetic simulations confirm that the line‐contact structure has a broader enhancement region than traditional point‐contact self‐assembled SERS structures. Experimental results demonstrate that substrate achieves a limit of detection (LOD) of 10 − 8 mol/L for Rhodamine 6G (R6G), with a enhancement factor (EF) of 10 7 and excellent reproducibility (relative standard deviation, RSD = 8.99%). Furthermore, the fabricated 3D SERS structure successfully achieves multi‐component sensing of mixed antibiotic solutions, demonstrating its potential for complex biochemical analysis.