Fluctuation-resilient ratiometric SERS enabled by programmable aptamer disassembly for pump-free microfluidic AKI detection.

Acute kidney injury (AKI) requires rapid and reliable biomarker quantification, yet early assessment is hindered by delayed biomarker kinetics and signal fluctuation under decentralized testing conditions. Here, we developed a pump-free microfluidic aptamer-ratiometric surface-enhanced Raman scattering (SERS) platform for simultaneous quantification of neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (Cys C). The assay uses a Y-shaped aptamer architecture to convert target binding into programmed structural disassembly and controlled release of Au@Ag SERS tags. A substrate-embedded internal standard enables self-calibrated readout through the I1617/I2228 ratio, thereby improving signal robustness against substrate heterogeneity and environmental interference. Integrated into a capillary-driven microfluidic chip, the platform completed dual-biomarker analysis within 15 min. It achieved detection limits of 1 pg/mL for NGAL and 34 pg/mL for Cys C, with excellent linearity (R2 > 0.99). In a cisplatin-induced AKI rat model, both biomarkers increased significantly at 4-6 h after injury, preceding oxidative stress imaging and histopathological changes. Analysis of clinical serum samples showed good agreement with enzyme-linked immunosorbent assay and recovery rates above 99%. These results demonstrate a rapid and reliable strategy for early AKI assessment and quantitative point-of-care biomarker analysis.