Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O 6 -Methylguanine DNA Methyltransferase in Breast Tissues.
Yun HanDong-Ling LiQian HanFei MaShuangshuang ZhangPublished in: Analytical chemistry (2024)
O 6 -Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O 6 -methylguanine modification (O 6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O 6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10 -8 to 0.1 ng/μL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.
Keyphrases
- single molecule
- living cells
- circulating tumor
- label free
- sensitive detection
- nucleic acid
- gene expression
- cell free
- fluorescent probe
- single cell
- high throughput
- quantum dots
- air pollution
- loop mediated isothermal amplification
- circulating tumor cells
- energy transfer
- risk assessment
- photodynamic therapy
- pet imaging
- human health
- amino acid
- pet ct