Nanoassembled SERS Sensing for Complex Biological Systems: From Hotspot Engineering to Interface Regulation

ConspectusSurface-enhanced Raman scattering (SERS) has evolved over the past five decades into a powerful vibrational spectroscopic technique capable of single-molecule sensitivity. Despite significant advances in the rational design and fabrication of SERS substrates with abundant hotspots, SERS still faces formidable challenges in transitioning from laboratory demonstrations to reliable analysis in complex biological environments. In practical sensing scenarios, the achievable enhancement factor is rarely the decisive parameter. Instead, the dominant bottleneck lies at the interface, where analyte transport and capture, as well as signal stability, are governed by dynamic mass transfer and matrix interference. A predictive understanding and rational regulation of analyte-hotspot interactions are therefore urgently needed.In this Account, we present a conceptual shift in SERS substrate design: from passive hotspot engineering to active interface regulation. Rather than focusing solely on maximizing local electromagnetic fields, we integrate hotspot construction with dynamic control over interfacial interactions, establishing a unified framework for SERS sensing in gas, liquid, and solid-state biological systems. We first summarize our efforts in constructing highly ordered and reproducible plasmonic assemblies as a structural foundation for reliable sensing. Traditional hotspot engineering strategies often rely on sophisticated top-down nanofabrication techniques or poorly controlled chemical synthesis methods, limiting their scalability and practical deployment. In contrast, by combining precision nanoscale self-assembly with scalable patterned printing methods (a strategy termed "printing assembly"), the resulting large-area superlattice substrates exhibit uniform hotspot distribution, controlled domain regulation, and mechanical robustness. This printing assembly technology establishes a viable pathway for constructing highly sensitive SERS substrates and achieving high-fidelity spectral acquisition.Building upon this foundation, we introduce interfacial regulation strategies capable of actively tailoring analyte-substrate interactions for SERS sensing in complex systems. For gas-phase systems, aerodynamic modulation, including pore confinement and cavity enrichment, is employed to alter molecular collision dynamics and enhance retention efficiency at plasmonic interfaces. For liquid-phase environments, hydrodynamic manipulation through convective channels, size filtration, and wettability regulation is applied to accelerate convective diffusion, suppress biofouling, and direct analyte enrichment. For volumetrically large or noncontact targets, dielectric-mediated field extension is introduced to mitigate intrinsic near-field decay, enabling detection beyond conventional electromagnetic confinement limits.In summary, these advances demonstrate that interface regulation is equally as important as hotspot engineering in determining SERS sensing performance. Looking forward, the convergence of artificial intelligence (AI) with existing SERS platforms is expected to enable the data-driven inverse design of nanostructures and interfaces as well as AI-assisted spectral interpretation for precise diagnostics, thereby accelerating the evolution of SERS into a robust analytical technology for complex biological systems.