Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor.

Although the analyses of HBV genomic DNA have traditionally been performed with commercial techniques, the high cost and long time consumed have hindered their applications in routinely diagnosis and prognosis of infection. We construct peptide nucleic acid (PNA) piezoelectric biosensor for real-time monitoring of hybridization of hepatitis B virus (HBV) genomic DNA. The PNA probe can combine to target DNA sequences more effectively and specifically than a DNA probe. The PNA probe was designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The hybridization assay was completed in 50 min. The detection limit was 8.6 pg/L and the clinical specificity was 94.44% compared with real time-PCR (RT-PCR). The PNA probe was able to distinguish sequences that differ only in one base. Detection sensitivity can be improved and detection time can be decreased by adding RecA protein-coated complementary ssDNA which complement to HBV gene regions. The QCM system we designed has the advantages of being rapid, label-free and highly sensitive and can be a useful supplement to commercial assay methods in clinical chemistry.

[1]  P. Laugier,et al.  Monitoring cell adhesion processes on bioactive polymers with the quartz crystal resonator technique. , 2005, Biomaterials.

[2]  Y. Okahata,et al.  Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance. , 1998, Biochemistry.

[3]  J. Gordon,et al.  Frequency of a quartz microbalance in contact with liquid , 1985 .

[4]  J. Goedert,et al.  Comprehensive Analysis of Class I and Class II HLA Antigens and Chronic Hepatitis B Virus Infection , 2003, Journal of Virology.

[5]  S Tombelli,et al.  Coupling of a DNA piezoelectric biosensor and polymerase chain reaction to detect apolipoprotein E polymorphisms. , 2000, Biosensors & bioelectronics.

[6]  Manna Zhang,et al.  Rapid detection of hepatitis B virus mutations using real‐time PCR and melting curve analysis , 2002, Hepatology.

[7]  B. P. Belotserkovskii,et al.  Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes. , 2002, Biochemistry.

[8]  Y. Okahata,et al.  Design and characterization of asparagine- and lysine-containing alanine-based helical peptides that bind selectively to A.T base pairs of oligonucleotides immobilized on a 27 mhz quartz crystal microbalance. , 2001, Biochemistry.

[9]  F. Caruso,et al.  Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic Acid sensor development. , 1997, Analytical chemistry.

[10]  S. Kowalczykowski.  Initiation of genetic recombination and recombination-dependent replication. , 2000, Trends in biochemical sciences.

[11]  M. Cox,et al.  The bacterial RecA protein and the recombinational DNA repair of stalled replication forks. , 2002, Annual review of biochemistry.

[12]  Myron F. Goodman,et al.  The importance of repairing stalled replication forks , 2000, Nature.

[13]  D. Vergani,et al.  The role of HBV DNA quantitative PCR in monitoring the response to interferon treatment in chronic hepatitis B virus infection. , 1999, Journal of hepatology.

[14]  Tae Song Kim,et al.  Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever. , 2005, Biosensors & bioelectronics.

[15]  Hans Wolf,et al.  An aptamer-based quartz crystal protein biosensor. , 2002, Analytical chemistry.

[16]  T. Horii,et al.  Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA. , 2003, Journal of molecular biology.

[17]  M. Cox,et al.  A RecA filament capping mechanism for RecX protein. , 2004, Molecular cell.

[18]  Fan Yu,et al.  A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin. , 2004, Biosensors & bioelectronics.

[19]  Sara Tombelli,et al.  Piezoelectric biosensors: strategies for coupling nucleic acids to piezoelectric devices. , 2005, Methods.

[20]  J Wang,et al.  Mismatch-sensitive hybridization detection by peptide nucleic acids immobilized on a quartz crystal microbalance. , 1997, Analytical chemistry.

[21]  R. Yu,et al.  Immunophenotyping of acute leukemia using an integrated piezoelectric immunosensor array. , 2004, Analytical chemistry.

[22]  M. Wulfsohn,et al.  Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. , 2003, The New England journal of medicine.

[23]  Sara Tombelli,et al.  Detection of β-thalassemia by a DNA piezoelectric biosensor coupled with polymerase chain reaction , 2003 .

[24]  S. Hadziyannis,et al.  Oral ganciclovir treatment in chronic hepatitis B virus infection: a pilot study. , 1999, Journal of hepatology.

[25]  Gustavo Rivas,et al.  Detection of point mutation in the p53 gene using a peptide nucleic acid biosensor , 1997 .

[26]  Fredrik Höök,et al.  Characterization of PNA and DNA Immobilization and Subsequent Hybridization with DNA Using Acoustic-Shear-Wave Attenuation Measurements , 2001 .