Orthogonal nanopores cross-validation for multiplex single-molecule profiling.

Multiplex biomolecule profiling provides comprehensive molecular insights essential for precision health diagnosis. Yet, achieving simultaneous analysis of diverse biomolecule types, such as DNAs, RNAs, peptides, oligosaccharides, and metabolites, remains challenging due to their fundamentally diversity including size, charge, structure, and chemical composition. We present a nanopore multiplex sensor (NMS) based on a bioelectronic microchip that enables concurrent profiling of multiple biomolecules in a single experiment, which was previously unattainable. Cross-validation using multiple types of orthogonal nanopores with complementary recognition abilities offered the simultaneous detection and relative quantification of five major types of biomolecules relevant to non-small-cell lung cancer. Our work establishes a robust and generalizable nanopore platform for rapid and parallel biomarker sensing, providing an important step toward addressing the vital challenge of single-molecule multiomics.