Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin

• A versatile dumbbell molecular probe was designed and employed for bioassays.
• The proposed biosensor is of high sensitivity and selectivity.
• The proposed strategy was applied in complex matrix and real sample analysis.
• The strategy was integrated with droplet-based microfluidics for visual detection.

In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5′-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3′-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design.