In this work, we propose an ultrasensitive fluorescent strategy for DNA detection. This method utilizes a molecular beacon (MB), a hairpin probe (HP), and an enzyme to trigger dual-cycling reactions (cycles I and II). In cycle I, the target is repeatedly used to amplify the fluorescence emission through hybridizations with the MB and cleavage reactions achieved by the enzyme. In cycle II, hybridization reactions between the HP and a segment of the MB continuously regenerate the target to trigger more cycle I reactions, leading to enhanced fluorescent signal. The detection limit of the method is determined as low as 50 fM within 45 min, which is 2 to 3 orders of magnitude lower than the conventional fluorescent strategies. The method also shows a high selectivity over mismatched and random DNA sequences. The signal amplification mechanism of the strategy offers insights in constructing efficient and ultrasensitive biosensors for various applications.

Ultrasensitive fluorescent DNA detection through signal amplification and target regeneration via dual-cycling reactions

2019 Fall National Meeting

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