In this study, a PCR free technique for effective detection of hepatitis B virus (HBV) DNA obtained directly from clinical samples was presented. The honeycomb-like barrier layer of an anodic aluminum oxide (AAO) film having a uniform nanohemisphere array was used as the substrate of the sensing electrode. A gold thin film about 30 nm thick was radio-frequency (RF) magnetron sputtered onto the AAO barrier-layer surface as the electrode for the successive deposition of gold nanoparticles (GNPs) on the hemisphere surface. A specially designed single-strand 96-mer gene fragment of the target genomic DNA of HBV based on the genome sequences of HBV from the National Center for Biotechnology Information (NCBI) was immobilized on the nanostructured electrode as the capture probe. Complementary target HBV DNA (3020–3320 mer) obtained from clinical samples were further hybridized to the sensing probes. Detection results through electrochemical impedance spectroscopy (EIS) illustrate that two dynamic linear ranges, 0–103 and 103–105 copies/mL, having R2 values of 0.973 and 0.998, respectively, could be obtained. A detection limit of 186 copies/mL could be achieved. The proposed simple and high performance HBV DNA detection technique in this study is highly feasible for future clinical applications.

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