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 sequence-specific 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.