Hepatitis B virus (HBV) infection is associated with the progression of liver disease. Occult HBV infection (OBI) is defined as the existence of detectable HBV DNA in HBV surface antigen negative individuals. However, HBV DNA is negative in serum while HBV surface antigen remains positive in incomplete cured chronic HBV infection. Hence, combined detection of HBV surface antigen and HBV DNA is essential for the accurate detection and rehabilitation of HBV infection. Therefore, a multiplex detection strategy based on branched DNA nanostructures was developed. The single-stranded segment of branched DNA nanostructure (segment of S4 and S2) assembled by four single-stranded DNA was hybridized with aptamer of HBV surface antigen and DNA hairpin to construct the DNA nanosensor, which can achieve high specificity identification and highly sensitive fluorescence responding to the targets. The detection limits of the developed nanosensor for HBV and HBV DNA are 50 pM and 5 nM, respectively. The combined detection of HBV surface antigen and HBV DNA provides a new insight for more thorough diagnostic evaluation of HBV infection and rehabilitation.

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