Encodable DNA Hairpin Probes for Nanopore Multiplexed Target Detection

Owing to the co-occurrence of hazardous compounds, it is crucial to build multiple highly discriminative probe libraries for simultaneous determination. Drawing inspiration from nucleic acid barcodes, we developed a probe system that is exclusively based on the nucleic acid secondary structure's hairpin structure, which can be directly read by nanopores. The highly distinguishable hairpin probes were constructed, and a detailed explanation of the possible patterns in their design was provided. These probe-representative events measured through the α-hemolysin (α-HL) nanopores were both distinguished, either through visual observation or comparison of the nanopore parameters. Besides, the potential design pattern for probes with unique telegraphic switching between the two levels was also unveiled. Finally, these probes were utilized to realize simultaneous, ultrasensitive mycotoxin multiple-detection, and their prospective applications for the detection of proteins and microRNAs were presented, indicating their suitability for a wide range of sensing applications.