title: Polymerase chain reaction-free detection of hepatitis B virus DNA using a nanostructured impedance biosensor.
abstract: A polymerase chain reaction (PCR)-free technique for the effective detection of genomic length hepatitis B virus (HBV) DNA is described in this study. 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 30-nm gold film was sputtered onto the AAO barrier layer surface as the electrode, followed by electrochemical 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 was immobilized on the nanostructured electrode as the capture probe. Target HBV DNA obtained from clinical samples was hybridized to the sensing probes. Detection results illustrate two dynamic linear ranges, 10(2)-10(3) and 10(3)-10(5.1) copies/mL, having R(2) values of 0.801 and 0.996 could be obtained, respectively. The detection limit of the proposed sending scheme was measured to be 111 copies/mL. The total of 45 target samples, including 20 samples with HBV concentration being lower than 10(2) copies/mL and 25 samples with HBV concentration being in the range of 10(3)-10(5.1) copies/mL, were used for real test. The concentration of these 45 HBV DNA samples was measured by the COBAS Ampliprep system. Comparing the measured results of the COBAS Ampliprep and our system, it was illustrated that the HBV DNA concentrations measured by the proposed method in this study had a high linear correlation with the COBAS Ampliprep, having R(2) values of 0.983. The proposed sensing scheme is highly feasible for future clinical applications.
authors.affiliation: Department of Mechanical Engineering, National Chung-Hsing University, Taichung 40227, Taiwan.
authors.forename: Chun-Cheng
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Division of Gastroenterology, Taichung Veterans General Hospital, Taichung 40705, Taiwan; Department of Education and Research, Taichung Veterans General Hospital, Taichung 40705, Taiwan.
authors.forename: Zi-Lun
authors.initials: ZL
authors.lastname: Lai
authors.affiliation: Department of Mechanical Engineering, National Chung-Hsing University, Taichung 40227, Taiwan; Graduate Institute of Biomedical Engineering, National Chung-Hsing University, Taichung 40227, Taiwan; Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung-Hsing University, Taichung 40227, Taiwan.
authors.forename: Gou-Jen
authors.initials: GJ
authors.lastname: Wang
authors.affiliation: Division of Gastroenterology, Taichung Veterans General Hospital, Taichung 40705, Taiwan; Department of Life Sciences, National Chung-Hsing University, Taichung 40227, Taiwan; Department of Public Health and Graduate Institute of Clinical Medicine, China Medical University, Taichung 40402, Taiwan; Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
authors.forename: Chun-Ying
authors.initials: CY
authors.lastname: Wu
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation; D003217:Conductometry / Q000295:instrumentation*; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000295:instrumentation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
chemical_list: D004279:DNA, Viral
keywords: HBV DNA; Nanostructured impedance biosensor; PCR-free detection