Enzyme-free dual-hotspot SERS immunosensor via cascaded boronate assembly.

The precise detection of CA19-9 is paramount for early cancer diagnosis and clinical monitoring. However, traditional immunoassays are often hampered by their heavy reliance on matched antibody pairs and the insufficient sensitivity of enzymatic amplification systems. To address these limitations, we report a novel, enzyme-free surface-enhanced Raman scattering (SERS) platform based on a "dual-hotspot amplification" strategy. The core innovation lies in exploiting the dual-connectivity of phenylboronic acid (PBA) to achieve precise hotspot modulation via a multi-cycle assembly. By utilizing D-glucose as a reversible molecular bridge, this strategy successfully breaks the traditional "one-to-one" linear binding limit of PBA. It facilitates a synergistic transition from heterogeneous binding, which anchors nanoprobes to a BSA@CPBA macromolecular scaffold, to homogeneous binding that drives continuous inter-particle cross-linking. This cycle-dependent assembly transforms individual immune recognition events into a highly coupled 3D dendritic plasmonic network, resulting in progressive accumulation of plasmonic hotspots and amplified macroscopic SERS signals. Under optimized conditions, the proposed sensor achieves an exceptionally low limit of detection of 6.2 × 10-5 U/mL and a wide linear dynamic range spanning five orders of magnitude (10-4 to 10 U/mL). Furthermore, the platform demonstrates outstanding specificity and excellent reproducibility (RSD = 8.34%). Practical utility was validated in human serum samples with near-ideal recovery rates (98.67%-103.87%) and excellent agreement with clinical chemiluminescence immunoassays. This modular and cost-effective approach not only enhances detection sensitivity but also provides a useful design concept for the ultrasensitive analysis of carbohydrate-derived biomarkers.