Label-free triplex DNA-based biosensing of transcription factor using fluorescence resonance energy transfer between...

Herein, a new turn-on fluorescent assay was established as a platform for the sensing of transcription factor NF-kB p50 based on triplex DNA labeled with N-doped carbon dots (NCDs) and gold nanoparticles (AuNPs) as donors and acceptors, respectively in the fluorescence resonance energy transfer (FRET) system. The synthetized nanoparticles were studied by different characterization techniques. A labeled DNA molecule was designed to form a triplex when no target protein existence and reported its formation by the change in FRET efficiency. While the triplex DNA was formed, the fluorescence of carbon dots at 503 nm (excitation at 460 nm) was quenched by FRET between NCD and AuNP. However, presence of NF-kB p50 followed by the considerable enhancement in the fluorescence intensity caused by the release of AuNPs labeled single stranded DNA from the triplex DNA structure, used for sensitive determination of the transcription factor. This technique showed a linearity (R2 = 0.9943) in the range of 20-150 pM with a limit of detection of 9 pM for the determination of NF-kB p50. Moreover, the sequence-specific triplex-based biosensor could discriminate NF-kB p50 from the other proteins with high selectively. Our results suggest that the biosensor provides a generalizable platform for rapid detection of NF-kB p50 in synthetic medium, promising in prevention and early diagnosis of cancer.