Graphene oxide and molecular beacons-based multiplexed DNA detection by synchronous fluorescence analysis

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We have developed a multiplexed DNA detection method based on graphene oxide (GO) and molecular beacons (MBs) by synchronous fluorescence analysis, demonstrated it by an oligonucleotide sequence of wild-type HBV (T1) and a reverse-transcription oligonucleotide sequence of the RNA fragment of HIV (T2) as a model system. In the absence of targets DNA, FAM-tagged free MB probes (PHBV) and ROX-tagged free MB probes (PHIV) are adsorbed on GO via π-π interactions between DNA nucleobases and nucleosides, and the π-rich GO brings the fluorophores of MB and GO into close proximity. And then, the fluorescence of fluorophores is quenched by GO. But in the presence of targets DNA, PHBV and PHIV hybridize with their targets DNA resulting in the formation of double-stranded DNA (dsDNA), causing the separation of PHBV and PHIV from the surface of GO and the recovery of the fluorescence of fluorophores (FAM and ROX) simultaneously. The simultaneous detection of T1 and T2 can be realized by measuring fluorescence signals of FAM and ROX, respectively. Under the optimum conditions, the fluorescence intensities of two dyes all exhibit good linear dependence on their target DNA concentration in the range of 5×10−11−5×10−9 M. The detection limit of T1 is 3×10−11 M (3 σ), and that of T2 is 2×10−11 M. Compared with other methods for DNA detection based on GO, the proposed method has some advantages including higher selectivity and shorter analytical time.