A Nanotechnology-Enhanced SERS Platform Integrated with Isothermal Amplification for Ultrasensitive Detection of Urinary miR-21 in Early Acute Kidney Injury Diagnosis

Acute kidney injury (AKI) remains a critical clinical condition with high morbidity and mortality, underscoring the urgent need for early and accurate diagnostic tools. Traditional biomarkers such as serum creatinine (Cer) and blood urea nitrogen (BUN) suffer from delayed response and lack of specificity, limiting their utility in early-stage AKI detection. This study presents an innovative nanotechnology-enabled platform combining isothermal nucleic acid amplification with surface-enhanced Raman scattering (SERS) for the direct and ultrasensitive detection of urinary microRNA-21 (miR-21), a promising early biomarker for AKI. The platform leverages a cascade signal amplification strategy: (1) a polymerase/endonuclease-mediated isothermal amplification system for target recognition and primer generation, (2) a hybridization chain reaction (HCR) to self-assemble DNA nanostructures conjugated with gold nanostars core with silver shell and 4-aminothiophenol (AuNS/Ag@4-ATP) SERS nanoprobes, and (3) a polyadenine-functionalized SERS substrate for robust signal capture. This integrated approach achieves a remarkable detection limit of 8.4 femtomolar (fM), surpassing conventional methods like quantitative reverse transcription polymerase chain reaction (qRT-PCR) in sensitivity while eliminating the need for RNA extraction or thermocycling. Clinical validation using patient urine samples demonstrated the platform’s ability to detect miR-21 elevations 24–48 h earlier than traditional biomarkers, with exceptional specificity and reproducibility (relative standard deviation (RSD) < 5.7%). The modular design and direct urine compatibility highlight its potential for point-of-care applications, offering a rapid (<2 h) and equipment-minimized solution for early AKI diagnosis. This work not only advances AKI biomarker detection but also provides an adaptable framework for other disease-related nucleic acid analyses, bridging nanotechnology with clinical diagnostics. • A multiplex isothermal amplification-SERS platform achieves 8.4 fM sensitivity, replacing qRT-PCR. • Enables direct miR-21 detection in unprocessed urine within 2 hours, bypassing RNA extraction. • Detects miR-21 elevations 24–48 hours earlier than SCr/BUN, with high specificity (RSD <5.7%). • Adaptable design for other miRNAs, expanding potential in disease diagnostics.