Dumbbell DNA-mediated rolling circle amplification for visual biosensing of intracellular glutathione

Herein, a visual nanoplatform has been constructed for ultrasensitive and accurate analysis of glutathione (GSH) based on the dumbbell DNA-medicated rolling circle amplification (RCA). This system consists of one nicked dumbbell probe (H1/H2) and one single-stranded probe (L0). Upon addition of GSH, the L0 probe containing the disulfide bond is cleaved into L1 and L2, which further hybridize to the loop of H1, respectively. Thus, after circularization of the dumbbell probe as template, the RCA reaction is initiated, in which the HRP can be encapsulated into the three-dimensional (3D) DNA flowers (DFs) by a one-pot method. Notably, the biocatalytic activity of HRP in DFs is significantly improved due to the negatively charged phosphates of DNA backbone. To reduce the high background induced by excess HRP, the complementary sequence of streptavidin (SA) aptamer is encoded in the dumbbell probe, and the SA-coated magnetic beads (SA-MBs) are employed to capture the as-assembled HRP-DFs with high affinity and specificity. Through converting the detection of unstable GSH into versatile HRP-DFs with excellent stability and enhanced enzyme activity, the proposed biosensor has achieved ultra-high sensitivity and accuracy, which achieves the successful determination of GSH in different cancer cells. • Enzymes-encapsulated three-dimensional DNA flowers are assembly by rolling circle amplification. • The catalytic activity of HRP in DNA flowers is enhanced. • Magnetic beads are employed to reduce the background. • This method is applied for accurate determination of GSH in different cancer cells.