Simultaneous Detection of Multiple Biomarkers by Peptide Nucleic Acids‐Based Triplex Molecular Beacon in a Nanopore

Abstract In this study, we propose an enhanced nanopore sensing strategy that utilizes a peptide nucleic acid (PNA)‐based triplex molecular beacon sensor to achieve the simultaneous detection of multiple biomarkers with a high degree of sensitivity. The sensor is a triplex switch composed of a triplex‐forming DNA strand and an oligo‐arginine‐tagged PNA strand, serving as the target recognition moiety and signal output element, respectively. Upon target binding to the recognition element of the sensor, the PNA signal output strand is released and a hybrid complex of the target‐DNA recognition strand is formed simultaneously. Due to the positive charges carried by the PNA‐Arg strands, they could be driven through the nanopore under positive electric field, effectively eliminating interferences from co‐existing target‐DNA complexes. This approach enables label‐free, one‐step detection of targets without requiring complex treatments and procedures. Leveraging the modular properties of DNA recognition strand, this method can be applied universally, and here, we successfully demonstrate its application using three SARS‐CoV‐2 related biomarkers.