Antibody-Responsive Ratiometric Fluorescence Biosensing of Biemissive Silver Nanoclusters Wrapped in Switchable DNA Tweezers

Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezerlike DNA architecture is assembled to populate fluorescent greenand red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid fulcrum, in which g-AgNCs are lighted up due to G18 proximity (green-on), and r-AgNCs away from G18 are still dark (red-off). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for green-off and bringing r-AgNCs close to G18 for red-on. As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.

Automated summary

The study successfully demonstrated a ratiometric fluorescence biosensing method using tweezerlike DNA architecture and dual-emitting AgNCs for specific detection of anti-Dig, providing reliable quantization at the picomolar level.