Target-triggered G-quadruplex DNAzyme on electrospun nanofibrous films for multicolor visual detection of uracil-DNA glycosylase.

BACKGROUND

Aberrant expression of UDG in vivo could disturb uracil excision repair process to cause various diseases such as bloom syndrome, human immune deficiency, neurodegeneration, and even cancer. Sensitive, reliable, DNAzyme catalytic, and point-of-care testing technique of uracil DNA glycosylase (UDG) activity is significant for clinical diagnosis and prognostic evaluation monitoring. DNA-functionalized electrospun nanofibrous films (ENFs)-based biosensor could provide possibilities for the portable and reliable detection of UDG. Meanwhile, gold nanobipyramids (Au NBPs) as visually plasmonic probes could provide a vividly visual effect comparing with the traditional ELISA.

RESULTS

In this work, a hairpin DNA-functionalized ENF is constructed for the analysis of UDG activity by coupling catalyzed hairpin assembly (CHA)-DNAzyme systems with Au NBPs as visually plasmonic probes. Firstly, after removing the uracil base by target UDG, the detection probe (H0) was cleaved by the apurinic/apyrimidinic endonuclease (APE1) into two DNA fragments (P1 and P2). The P1 induced the CHA-cycling reaction to form the G-quadruplex DNAzyme with horseradish peroxidase-mimicking activity on ENFs under hemin, catalyzing H2O2 to oxidize3,3',5,5'-tetramethylbenzidine (TMB) into TMB2+ under the acidic environment. Then, the TMB2+ etched the Au NBPs, resulting in the blue shift of their longitudinal absorption wavelength. The etched Au NBPs colloidal solution respectively presented the vivid multicolor variations by naked eyes with the increasing UDG activities. This proposed biosensor enables sensitive, specific, and DNAzyme catalytic analysis of UDG down to 0.077 U/L.

SIGNIFICANCE: This proposed target-triggered CHA-G4-DNAzyme biosensing system on ENFs revealed its applicability for the detection of UDG activity in complex biological samples including real cell lysates and human serums, thereby providing a powerful tool for biomedical research and clinical diagnosis. Multicolor visual effect and target-induced DNAzyme catalytic detection strategy are superior to traditional ELISA methods.