Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches

Abstract

Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequencespecific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.

Nanopore sensing specific enhancement technique for single molecule detection

“…DNA纳米开关在基于凝胶电泳的核酸传感中 被广泛应用 [96,97] , Zhu等 [98] 随着DNA结构自组装技术的发展, 越来越多 的二维、三维DNA结构被设计出来 [99] , 研究者们 期望通过多样化的构象来获得更好区分的电流信 号 [100,101] . 最近, Wang等 [93] .…”

Silica Nanowires-Filled Glass Microporous Sensor for the Ultrasensitive Detection of Deoxyribonucleic Acid

“…Nanopore detection technology, as a novel single molecule detection method, can achieve molecular recognition by analyzing the ion current signals generated when molecules pass through nanopores with high sensitivity and specificity. − Solid-state nanopores have attracted much attention due to their controllable pore shape and size, excellent mechanical and chemical stability, and unique ion transport properties. − Due to the relatively excellent detection performance of solid-state nanopores, they have been widely used as a highly efficient detection device for ions, DNA, proteins, and so on. − Dekker et al first inserted α-hemolysin, which bound to DNA strands, into solid-state pores based on electrophoretic effects to form hybridized nanopores for the detection of single-stranded DNA . Seker et al used DNA probes labeled with nanoporous gold and methylene blue with different thicknesses and pore shapes for DNA target detection .…”

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore

“…In addition, designing an additional polymer chain, to which the target attached, has proven to be an effective way to improve the analysis using nanopores. For instance, DNA carriers were used to detect the size and shape of various DNA nanostructures, including protrusions, hairpins, dumbbells, − rings, cubes, , tetrahedrons, , helix bundles, , and multiway junctions, facilitating the advancement of DNA data storage, , artificial nanoscale rotary motors, and respiratory infection diagnosis . The design of the DNA carrier may offer a smart strategy for improving molecular ping-pong by increasing the recapture probability.…”