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

Abstract

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.

Ultrasound-enhanced fluorescence imaging and chemotherapy of multidrug-resistant tumors using multifunctional dendrimer/carbon dot nanohybrids

“…4 c). 2, 7-Dichlorofluorescin diacetate (DCFH-DA) can be oxidized to generate DCF in the presence of ROS to emit green fluorescence, thus can be used as an ROS probe [ 51 ]. MCF-7/ADR cells treated with the dual-drug (G5-TPGS@y-CDs)-DOX complexes display significantly stronger fluorescence intensity than those treated with the single-drug (G5- m PEG@y-CDs)-DOX and G5-TPGS@y-CDs complexes at the same DOX or equiv.…”

Circular Nucleic Acids: Discovery, Functions and Applications

“…The target DNA was used as a primer to initiate RCA for the synthesis tandem G‐quadruplex, which could form complex with hemin and catalyze the oxidation of ABTS by H 2 O 2 . This RCA‐based peroxidase‐active G‐quadruplex synthesis can also be coupled with other biochemical reaction, chemiluminescence, electrochemical methods, immunoassays, microchips, specific DNA structures, paper devices, and quantum dots for the detection of DNA, protein, small molecules, and cell components. Moreover, in order to further improve the detection sensitivity of the G‐quadruplex producing RCA method, different signal amplification strategies can be adopted.…”

A Fluorescent DNA Hydrogel Aptasensor Based on the Self-Assembly of Rolling Circle Amplification Products for Sensitive Detection of Ochratoxin A

“…However, the sensitivity of DNA hydrogel-based biosensors needs to be improved. As an effective signal amplification element, rolling circle amplification (RCA) has been widely used in the fabrication of biosensors for easy operation and is highly designable. − RCA is an enzymatic amplification reaction, during which long single strand DNA with tens to hundreds of tandem repeats can be produced. DNA hydrogels can be generated by self-assembly of the produced single strand DNA, which has been used widely for the relatively simple sequence design and easy operation …”