Detection of HIV/HCV virus DNA with homogeneous DNA machine-triggered in situ formation of silver nanoclusters

Currently, the early disease screening is still the most effective way to increase the survival rate of the HIV/HCV infected patients. However, the trace analysis remains a great challenge for early disease screening. Herein, a novel silver nanocluster (AgNCs) is in situ generated and served as fluorescence probes for the ultrasensitive HIV/HCV DNA detection by combing Exo III-assisted target recycling amplification (ERA) with rolling circle amplification (RCA). As an important key element of this sensor, the padlock probes (PLP) of RCA are exquisitely designed to compose of a guanine-rich (G-rich) region. Target DNA firstly hybridizes with the stem segment of the hairpin DNA (hp-DNA) and triggered the Exo III digestion, which releases and recycles the target and generate numerous ssDNA fragments. The generated ssDNA fragments act as ligation probes, which hybridize with PLP to trigger the RCA process, generating numerous periodically repeated cytosine-rich (C-rich) units. In the presence of NaBH4 and AgNO3, numerous AgNCs are in situ generated, thereby producing a strong fluorescent signal. Under optimal conditions, an ultrasensitive DNA machine is constructed for the detection of HIV DNA with a detection limit of 1.4 fM and a linear range of 10 fM to 100 pM. More fascinatingly, this DNA machine could intelligently distinguish multiple virus DNAs by the construction of analog AND and OR logic circuits.