Multiplexed Detection of Pancreatic-Specific Nucleic Acids and Protein Biomarkers Using a Logic Nanofluidic Platform.

Early detection of pancreatic cancer is vital for patient survival. However, current diagnostic approaches remain constrained by insufficient precision and specificity inherent to single-biomarker detection strategies. Herein, we develop a nanochannel biosensing platform implementing cooperative dual-signal detection of pancreatic-specific biomarkers CA19-9 and miRNA-196a. Using liquid-liquid interface self-assembly, we constructed anodic aluminum oxide (AAO)-Au hybrid nanochannels integrated with a surface-modified double-key DNA nanolock (DDN). The conformational switch of DDN logic gating triggered by miRNA-196a exposes the CA19-9-aptamer, enabling specific target recognition and consequent ion current signal attenuation. Simultaneously, released miRNA-196a is quantified by catalytic hairpin assembly and hybridization chain reaction-mediated cascade amplification. Experiments show that the present DDN-based logic nanofluidic platform could achieve an ultralow detection limit of 0.000027 U·mL-1 for CA19-9 and 4.74 aM for miRNA-196a, which is 2-3 orders of magnitude higher than traditional ELISA/qPCR methods. Finally, clinical sample analysis confirms the high specificity of this platform in distinguishing pancreatic cancer and acute pancreatitis from healthy individuals. This DDN-functionalized nanofluidic biosensor provides valuable insights into designing precision detection platforms for pancreatic cancer, highlighting its significant potential for clinical diagnostics.