Self-calibrating SERS sensor for multi-component urinalysis based on plasmonic nanocubes with embedded internal standards

Accurate and multiplexed detection of metabolic small molecules is essential for the management of metabolic and renal diseases. However, due to the diverse chemical structures of metabolic molecules and the wide range of their concentrations, the accurate analysis becomes extremely challenging. Herein, a reliable ratiometric surface-enhanced Raman scattering (SERS) sensing platform based on Au@4-MBA@Ag core-shell nanocubes is reported, which effectively resolves the SERS signal fluctuations and severe background interference during the measurements, enabling the quantitative analysis of uric acid (UA), creatinine (CRE), and pH in human urine. By confining 4-mercaptobenzoic acid (4-MBA) reporters within a sub-nanogap between the Au core and Ag shell, a shielded internal standard is established, effectively mitigating external environmental and matrix interference. Furthermore, a morphology-selective purification process elevates nanocube purity to over 90%, which effectively mitigates systematic errors induced by morphological heterogeneity, providing the substrate with uniform electromagnetic hotspots and reliable signal reproducibility. Upon functionalizing the outer Ag shell with pH-sensitive 4-mercaptopyridine (4-MPY) probes, this spatially decoupled nanoplatform enables self-calibrating pH monitoring within the 4.0–7.0 range. Concurrently, it achieves the highly sensitive quantification of UA and CRE, with limits of detection reaching 2.16 × 10⁻⁹ M and 2.29 × 10⁻⁸ M, respectively. Coupled with a facile pretreatment, the sensor demonstrates robust matrix tolerance in real human urine. This proof-of-concept validation yields recoveries of 93%–102%, confirming the method's accuracy. The blind-test results show high concordance with clinical gold standards, which hold great potential for the non-invasive detection of metabolic and renal diseases.