Self‐Assembled Hydrophobic Confinement Coupled with Calcium Coordination Enables Selective and Label‐Free SERS Detection of Urinary Biomarkers in Complex Samples

Reliable and label‐free detection of small molecule biomarkers in urine remains a significant challenge due to signal suppression caused by abundant interfering macromolecules such as proteins. Here, a surface‐enhanced Raman spectroscopy (SERS) platform is developed based on self‐assembled hydrophobic molecular layers combined with calcium‐assisted molecular recognition to selectively enrich and enhance representative urinary biomarkers, including creatinine and albumin fragments. Hydrophobic monolayers exclude polar or high molecular weight interferences from plasmonic interfaces, while calcium ions coordinate with functional groups on target molecules to enhance their affinity and orientation at SERS hotspots. The experimental results demonstrate that the platform provides highly reproducible and selective signals in synthetic urine without any chemical labeling or separation. Density functional theory simulations further confirm the dual enhancement mechanism by showing improved adsorption energy and vibrational mode alignment for the calcium‐coordinated creatinine complex. This strategy enables ultrasensitive, interference‐resistant detection of clinically relevant metabolites, providing a general approach to biomarker sensing in real biological fluids.