A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1.

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.

[1]  M. Abe,et al.  Expediting magnetic separation using Ni Wires,Applicable for robot-manipulated bio-screening systems , 2005, IEEE Transactions on Magnetics.

[2]  Ralph Weissleder,et al.  Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes. , 2002, Bioconjugate chemistry.

[3]  H. Tsai,et al.  Detection of C-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles. , 2007, Analytical chemistry.

[4]  M. Peiris,et al.  Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus , 1998, The Lancet.

[5]  R. Webster,et al.  Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus , 1998, The Lancet.

[6]  Franz L Dickert,et al.  Sensing picornaviruses using molecular imprinting techniques on a quartz crystal microbalance. , 2009, Analytical chemistry.

[7]  R. Huebner,et al.  INACTIVATION OF THE INFECTIVITY OF VIRAL HEMAGGLUTINATING ANTIGENS WITH THE USE OF BETAPRONE. , 1964, The Journal of laboratory and clinical medicine.

[8]  Tzong-Zeng Wu,et al.  Piezoelectric immunochip for the detection of dengue fever in viremia phase. , 2005, Biosensors & bioelectronics.

[9]  A. Ribbe,et al.  Self-assembly of cobalt nanoparticle rings. , 2002, Journal of the American Chemical Society.

[10]  Yong Poovorawan,et al.  Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. , 2006, Journal of virological methods.

[11]  Tzong-Zeng Wu,et al.  Recognition of dengue virus protein using epitope-mediated molecularly imprinted film. , 2005, Analytical chemistry.

[12]  Bo Zhang,et al.  Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor. , 2008, Biosensors & bioelectronics.

[13]  I. Donatelli,et al.  A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control , 2006, BMC infectious diseases.

[14]  Sun,et al.  Monodisperse FePt nanoparticles and ferromagnetic FePt nanocrystal superlattices , 2000, Science.

[15]  Xiaoli Zhang,et al.  Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification , 2009 .

[16]  G. Tannock,et al.  The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance , 2009, Journal of Virological Methods.

[17]  M. L. Havens,et al.  Influenza Virus Purification with the Zonal Ultracentrifuge , 1966, Science.

[18]  Pedro Estrela,et al.  Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences. , 2008, Analytical biochemistry.

[19]  Kevin W Plaxco,et al.  Microfluidic device architecture for electrochemical patterning and detection of multiple DNA sequences. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[20]  Ting-Feng Wu,et al.  Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[21]  F. K. Ng,et al.  Detection of H5 Avian Influenza Viruses by Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody , 2007, Clinical and Vaccine Immunology.

[22]  G. Whitesides,et al.  Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold , 1989 .

[23]  M. Goldstein,et al.  Effect of formalin, beta-propiolactone, merthiolate, and ultraviolet light upon influenza virus infectivity chicken cell agglutination, hemagglutination, and antigenicity. , 1970, Applied microbiology.

[24]  Long Jiang,et al.  Construction of effective receptor for recognition of avian influenza H5N1 protein HA1 by assembly of monohead glycolipids on polydiacetylene vesicle surface. , 2009, Bioconjugate chemistry.

[25]  G. Storch.  Essentials of Diagnostic Virology , 1999 .

[26]  M. Muhammed,et al.  Superparamagnetism of magnetite nanoparticles: dependence on surface modification. , 2004, Langmuir : the ACS journal of surfaces and colloids.

[27]  J. T. Mayo,et al.  Low-Field Magnetic Separation of Monodisperse Fe3O4 Nanocrystals , 2006, Science.

[28]  C. Cardona,et al.  Commercial Immunoassay Kits for the Detection of Influenza Virus Type A: Evaluation of Their Use with Poultry , 2005, Avian diseases.

[29]  X. Mao,et al.  A nanoparticle amplification based quartz crystal microbalance DNA sensor for detection of Escherichia coli O157:H7. , 2006, Biosensors & bioelectronics.

[30]  Gustaaf Borghs,et al.  Magnetic particles as labels in bioassays : Interactions between a biotinylated gold substrate and streptavidin magnetic particles , 2007 .

[31]  Bing Xu,et al.  Using biofunctional magnetic nanoparticles to capture vancomycin-resistant enterococci and other gram-positive bacteria at ultralow concentration. , 2003, Journal of the American Chemical Society.

[32]  Robin H. Liu,et al.  Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. , 2006, Analytical chemistry.

[33]  Ku-Shang Chang,et al.  A Novel Method for Determination of Cattle Bovine Ephemeral Fever Virus in Liquid with Quartz Crystal Microbalance Immunosensor , 2005 .

[34]  Ronghui Wang,et al.  Interdigitated array microelectrode based impedance immunosensor for detection of avian influenza virus H5N1. , 2009, Talanta.

[35]  F. Pariente,et al.  A quartz crystal microbalance assay for detection of antibodies against the recombinant African swine fever virus attachment protein p12 in swine serum , 1998 .

[36]  Yibin Ying,et al.  QCM immunosensor with nanoparticle amplification for detection of Escherichia coli O157:H7 , 2007 .

[37]  Ku-Shang Chang,et al.  Application of a flow type quartz crystal microbalance immunosensor for real time determination of cattle bovine ephemeral fever virus in liquid. , 2005, Talanta.

[38]  A. Limaye,et al.  Surface plasmon resonance detection using antibody-linked magnetic nanoparticles for analyte capture, purification, concentration, and signal amplification. , 2009, Analytical chemistry.

[39]  James A. Smagala,et al.  Experimental Evaluation of the FluChip Diagnostic Microarray for Influenza Virus Surveillance , 2006, Journal of Clinical Microbiology.

[40]  T. Pellegrino,et al.  Magnetic-fluorescent colloidal nanobeads: preparation and exploitation in cell separation experiments. , 2009, Macromolecular bioscience.

[41]  Christopher J. Bardeen,et al.  Microgravimetric immunosensor for direct detection of aerosolized influenza A virus particles , 2007, Sensors and Actuators B: Chemical.

[42]  Tzong-Zeng Wu,et al.  Artificial receptors in serologic tests for the early diagnosis of dengue virus infection. , 2006, Clinical chemistry.

[43]  N. Cox,et al.  Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. , 1998, Science.

[44]  N. Hieu,et al.  Impact parameters on hybridization process in detecting influenza virus (type A) using conductimetric-based DNA sensor , 2009 .

[45]  Xiaoli Zhang,et al.  Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification , 2010 .

[46]  J. Kwang,et al.  Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies , 2009, Clinical and Vaccine Immunology.

[47]  Quan Cheng,et al.  Detection of influenza virus: traditional approaches and development of biosensors , 2005, Analytical and bioanalytical chemistry.

[48]  D. Akin,et al.  Real-time detection of airborne viruses on a mass-sensitive device. , 2008, Applied physics letters.

[49]  Zhiqiang Gao,et al.  A DNA biosensor based on the detection of doxorubicin-conjugated Ag nanoparticle labels using solid-state voltammetry. , 2009, Biosensors & bioelectronics.

[50]  Fook Tim Chew,et al.  Detection of two orchid viruses using quartz crystal microbalance (QCM) immunosensors. , 2002, Journal of virological methods.

[51]  V. Martin,et al.  Origin and evolution of highly pathogenic H5N1 avian influenza in Asia , 2005, Veterinary Record.