Programmable DNA probe-mediated nanopore biosensor for multiplex nucleic acid detection and its application in milk authenticity identification.

Simultaneous and sensitive detection of multiple nucleic acids in complex matrices has always been a significant challenge. Herein, we reported a novel nanopore biosensing strategy that achieved label-free, high-discrimination detection of multiplex nucleic acid targets by ingeniously integrating programmable DNA probe with click chemistry amplified strategy. In this platform, probe DNAs with alkynyl modification at different positions were clicked with azidoadamantane, which were then reacted with cucurbit[6]uril to produce host-guest complexes as signal tags. These reporters showed highly distinguishable electrical signals when analyzed by α-hemolysin (α-HL) nanopore. Coupled with target-specific asymmetric PCR-triggered strand displacement reaction, probe DNAs were released in the presence of target DNA and generated signal tags, which were analyzed with α-HL nanopore to achieve multiplex signal output. Through this strategy, multiple target DNAs could be simultaneously detected with a satisfactory limit of detection (LOD) as low as 18.78 pM. We further applied this method to complex dairy matrices for goat milk authenticity detection, successfully achieving simultaneous detection of adulterated DNA from cow milk and soy milk in goat milk samples. This demonstrated its good applicability in complicated food matrices, and it was expected to be a universal platform for detecting various nucleic acid markers in other matrices.