Ligation-Rolling Circle Amplification on Quantum Dot-Encoded Microbeads for Detection of Multiplex G-Quadruplex-Forming Sequences

The combination of microbead array with assay chemistry of isothermal amplification enables the continuous development of nucleic acid detection techniques. Herein we report the implementation of ligation-rolling circle amplification (RCA) reaction on quantum dots-encoded microbead (Qbead) for the detection of multiplex G-quadruplex (G4) forming sequences. The reaction time of RCA on the Qbead was optimized to be 60 min. Zinc phthalocyanine (ZnPc), a molecular "light switch", was selected as the G4-specific label. In the presence of target, the target-triggered ligation-RCA produced long DNA concatemer consisting of tandem repeats of G4-forming sequence, and the labeling helped generate G4/ZnPc nanowires on the Qbead. With the G4/ZnPc nanowires as fluorescent labels, the array of three encoded Qbeads was capable of detecting three G4-forming sequences by flow cytometry in a high-throughput and specific manner. Alternatively, with the G4/ZnPc nanowires as catalytic labels, chemiluminescence of H₂O₂-mediated oxidation of luminol could be used for detecting the target G4-forming sequences with high sensitivity. The catalytic chemiluminescence achieved a limit of detection of 0.5 ng of genomic DNA with 5 logs of linear dynamic range for the detection of the blood sample of a myeloproliferative neoplasms patient. Together the proposed isothermal amplification-on-Qbead assay featured robust detection platform, significant signal amplification, and flexible detection strategy, holding high potential in application in large-scale or "focused" nucleic acid testing.