Here, we report a double stranded and dual anchored fluorescent aptamer on reduced graphene oxide (rGO) for sensitive and selective detection of interferon-gamma (IFN-g) in biological samples. The aptasensor is composed of IFN-g specific fluorescent aptamer with BHQ1 as one anchoring moiety, which forms double stranded sequences with complementary oligonucleotide sequence with BHQ1 as the other anchoring moiety, and rGO nanosheets. The double stranded and dual anchored fluorescent aptamer on rGO (AptasensorGO) showed 7.3 and 4.7-fold higher fluorescence intensity compared to single stranded and single anchored fluorescent aptamer on rGO, and double stranded and single anchored fluorescent aptamer on rGO, respectively. The fluorescence intensities of AptasensorGO were influenced by the length of DNA aptamer sequences, showing the highest intensity at 36 bp. AptasensorGO was specifically sensitive to IFN-g in buffer and human serum. AptasensorGO detected IFN-g in buffer and human with linearity ranges over 5 orders of magnitude (from 100 pg/ml to 10 mg/ml), showing the regression coefficient of 0.9982 and 0.9838, respectively. Moreover, AptasensorGO showed fluorescence intensity changes to IFN-g, but not to non-target proteins such as interluekin-2 and tumor necrosis factor-a. Only after 30 min of incubation with AptasensorGO, the levels of IFN-g in human immunodeficiency virus-positive patient plasma samples were quantified to range from 250 to 500 ng/ml. Taken together, the nano-rGO platform of double stranded and dual anchored fluorescence aptasensor provides new opportunities for detection of cytokines such as IFN-g and could be applied to rapidly monitor the cytokine levels in human patient samples.
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