Synthesis and characterization of N‐rich fluorescent bio‐dots as a reporter in the design of dual‐labeled FRET probe for TaqMan PCR: A feasibility study

DNA-based analytical techniques have provided an advantageous sensing assay in the realm of biotechnology. Bio-inspired fluorescent nanodots are a novel type of biological staining agents with excellent optical properties widely used for cellular imaging and diagnostics. In the present research, we successfully synthesized bio-dots with excellent optical properties and high-quantum yield from DNA sodium salt through the hydrothermal method. We conjugated the bio-dots with 3' Eclipse Dark Quencher (Eclipse)-labeled single-strand oligodeoxyribonucleotide according to carbodiimide chemistry, to design a fluorescence resonance energy transfer (FRET) probe. The results confirmed the prosperous synthesis and surface functionalization of the bio-dot. Analysis of size, zeta potential, and FTIR spectroscopy verified successful bioconjugation of the bio-dots with probes. UV-visibility analysis and fluorescence intensity profile of the bio-dot and bio-dot@probes represented a concentration-dependent quenching of fluorescent signal of bio-dot by Eclipse after probe conjugation. The results demonstrated that TaqMan PCR was not feasible using the designed bio-dot@probes. Our results indicated that bio-dot can be used as an efficient fluorescent tag in the design of fluorescently labeled oligonucleotides with high biocompatibility and optical features.