Integrated SERS-magnetic capture platform for multiplex detection of early tubular injury biomarkers in diabetic kidney disease progression

• Addressing a Critical Clinical Gap in DKD Early Diagnosis • Novel Biosensor Design: Integrating Magnetic Enrichment and SERS Enhancement • Efficient Multiplex Detection with Clinical Translational Potential • Laying a Foundation for DKD Diagnostic Innovation Early detection of tubular injury biomarkers in diabetic kidney disease (DKD) is critical for timely intervention. This study presents an innovative magnetic separation-assisted surface-enhanced Raman spectroscopy (SERS) immunoassay for ultrasensitive, multiplex detection of three tubular injury biomarkers: retinol-binding protein 4 (RBP4), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM1). The platform combines three key technological innovations: (1) Raman internal standard (4-mercaptobenzoic acid)-embedded Fe 3 O 4 -AuAg core-satellite nanoparticles with optimized plasmonic-magnetic properties; (2) Spectrally distinct Raman probes using Au nanorods functionalized with three reporter molecules (2-naphthalenethiol, 2-mercaptopyridine, 4-nitrobenzenethiol); and (3) An automated magnetic separation workflow greatly reduces sample enrichment time. The assay demonstrated unprecedented sensitivity with limits of detection of 10 fg/mL (RBP4), 10 fg/mL (NGAL), and 1 pg/mL (KIM1) in PBS solution, surpassing traditional common methods by 3-4 orders of magnitude. Clinical validation with 24 samples showed excellent correlation with the conventional Luminex assay method (Pearson's r=0.96-0.99), and the coefficient of variation was less than 9.28%, with Bland-Altman analysis confirming 95% agreement. This integrated SERS-magnetic approach enables simultaneous, high-sensitivity detection of multiplex tubular injury biomarkers, offering significant potential for early DKD diagnosis and progression monitoring. The demonstrated analytical performance and clinical concordance position this technology as a promising tool for precision nephrology.