Unambiguous Discrimination of Multiple Protein Biomarkers by Nanopore Sensing with Double-Stranded DNA-Based Probes

Biological research and clinical testing frequently require simultaneous quantification of multiple biomarkers for accurate disease diagnosis. In particular, the application of nanopore technology to real-sample analysis is limited in terms of discriminating multiple analytes in a mixture. In this study, we designed a series of highly specific double-stranded DNA-based probes and used them for synchronous detection of and discrimination among three protein biomarkers associated with lung cancer in a single nanopore. The probe could specifically identify and bind to the target, releasing an output DNA hybrid that generated signature current signals upon translocation through the nanopore. DNA hybrids with different hybridization stability and structure produced blockade currents clearly distinguishable in nanopore recordings, which could be unequivocally assigned to each analyte, thereby enabling accurate differentiation among multiple biomarkers. This assay based on the excellent discrimination ability of the probes facilitates unambiguous identification and quantification of multiple analytes on a natural serum background, thus presenting a powerful tool for biomedical research and clinical practice.