Enhancing RBP4 protein detection in clinical urine samples with solid-state nanopores through optimized sandwich immunoassay techniques.

Nanopore technology is a promising single-molecule sensing platform that can identify substances through the precise monitoring of changes in ion currents. However, protein detection in clinical samples using solid-state nanopores remains challenging due to their heterogeneously charged spherical structure, which results in signals with extremely low signal-to-noise ratios (SNR) and low capture rates that are difficult to analyze. In this study, we employed a double-antibody sandwich technique to specifically capture and amplify the target antigen, which significantly improves the SNR and effectively distinguishes the target signal from background interference. Key factors including buffer composition, voltage, antibody concentration, and pore dimensions were systematically optimized to further improve capture efficiency. The optimized approach enabled precise and reliable detection of retinol-binding protein 4 (RBP4) with an excellent linear response within the range of 55 fM to 5.5 pM. Moreover, our method facilitates quantitative detection of RBP4 in clinical urine samples within 40 min, and achieves 100% accuracy in distinguishing between 11 urine samples from chronic kidney disease (CKD) patients and healthy donors, highlighting its robustness and specificity. Our research not only paves a new pathway for efficient RBP4 detection, but also provides valuable insights into the application of nanopore technology for the clinical diagnosis of protein biomarkers.