Orthogonal Nanopores Cross-Validation for Multiplex Single-Molecule Profiling

Abstract

Multiplex biomolecule profiling provides comprehensive molecular insights essential for precision health diagnosis. However, the difficulty arises from the fundamental diversity of biomolecules. Their sizes, charges, structures, and chemical compositions differ dramatically that achieving a simultaneous molecular profiling of multiple biomolecules, such as DNAs, RNAs, peptides, oligosaccharides, and metabolites remains inherently challenging. Herein, we present a Nanopore Multiplex Sensor (NMS) based on bioelectronic microchip that enables concurrent profiling of multiple biomolecules in a single experiment, which was previously unattainable. Further cross-validation using multiple types of orthogonal nanopores with complementary recognition abilities offers the simultaneous detection and relative quantitation 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 deciphering molecular diversity through single-molecule multi-omics.