Confined Internal Standard Core–Gap–Shell Nanoprobes for Ratiometric SERS Sensing of Urine pH

Urine pH is an important biomarker related to metabolic status and urinary system health, but reliable SERS quantification in real urine remains limited by matrix interference, heterogeneous hotspot distribution, and the narrow response range of single pH-responsive molecules. Here, we report a core–gap–shell Au@1,4-BDT@Au@4-MBA/MPY nanoprobe for ratiometric SERS detection of urine pH. 1,4-BDT was confined within the gap between the gold core and shell as an internal standard, while 4-MBA and 4-MPY were co-assembled on the outer gold shell to provide complementary protonation/deprotonation responses. The internal standard-corrected ratio I1004/I1400/I731 reduced signal fluctuation and enabled segmented linear fitting over pH = 1.0–7.0 and pH = 7.0–10.0, with coefficients of determination of 0.98806 and 0.99989, respectively. The sensing platform also maintained stable ratiometric responses under different interference conditions. In real urine samples from five volunteers, SERS-predicted pH values agreed well with commercial pH meter measurements, with relative accuracies of 98.71–101.9% and RSD values below 2.1%. This confined internal standard and dual-molecule ratiometric strategy provides a feasible approach for urine pH sensing in complex biofluid matrices.