Nanowire Switch and Nanoelectrode/Nonchannel Based Impedance Biosensor for Rapid Screening of Avian Influenza Virus

NON-TECHNICAL SUMMARY: Continuous outbreaks of highly pathogenic H5N1 avian influenza (AI) and cases of human infection have caused increasing international concerns, resulting in an urgent need for new technology to rapidly detect H5N1 AI virus. Current AI detection methods such as ELISA, diagnostic test kits and RT-PCR are either poor in specificity, low in sensitivity, time consuming, too expensive, or require a laboratory and a highly trained technician. The goal of this project is to develop a nanowire and nanoelectrode/nanochannel based nano-biosensor for rapid screening of AI virus. The specific objectives are to: (1) synthesize and characterize gold nanowires for their use in a nano-biosensor; (2) functionalize and surface-modify the gold nanowires; (3) conjugate the gold nanowire with anti-H5 or anti-N1 monoclonal antibodies; (4) design and fabricate interdigitated nanoelectrode and nanochannel, and integrate them into a nano/microfluidic chip; (5) immobilize anti-H5 or anti-N1 monoclonal antibodies in the nanochannel; and (6) evaluate the nano-biosensor for screening of H5N1 and other H5 subtypes of AI virus in comparison with gold standard methods. The novelty of the proposed nano-biosensor is the integration of a novel nanowire switch, innovative interdigitated nanoelectrode/nanochannel biochip, and new anti-H5 and anti-N1 monoclonal antibodies for highly effective capture of target AI virus and ultra sensitive signal generation. Multidisciplinary efforts (chemistry, virology, mechanical and biological engineering) will be made to reach the goal of this proposed research. We expect that this nano-biosensor will be able to more rapidly, specifically and sensitively screen AI virus in a poultry sample in less than 30 min. This research addresses the CSREES goals to enhance protection and safety of the Nation?s agriculture and food supply and to improve the Nation?s nutrition and health.

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ACCESSION NO: 0216030 SUBFILE: CRIS
PROJ NO: ARK02220 AGENCY: CSREES ARK
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2009-35603-05063 PROPOSAL NO: 2008-01425
START: 01 JAN 2009 TERM: 31 DEC 2010 GRANT YR: 2009
GRANT AMT: $455,308

INVESTIGATOR: Li, Y.; Ruan, C. M.; Huang, T. J.; Lu, H.

PERFORMING INSTITUTION:
BIOLOGICAL & AGRICULTURAL ENGINEERING
UNIVERSITY OF ARKANSAS
FAYETTEVILLE, AR 72703

NANOWIRE SWITCH AND NANOELECTRODE/NANOCHANNEL BASED IMPEDANCE BIOSENSOR FOR RAPID SCREENING OF AVIAN INFLUENZA VIRUS

NON-TECHNICAL SUMMARY: Continuous outbreaks of highly pathogenic H5N1 avian influenza (AI) and cases of human infection have caused increasing international concerns, resulting in an urgent need for new technology to rapidly detect H5N1 AI virus. Current AI detection methods such as ELISA, diagnostic test kits and RT-PCR are either poor in specificity, low in sensitivity, time consuming, too expensive, or require a laboratory and a highly trained technician. The goal of this project is to develop a nanowire and nanoelectrode/nanochannel based nano-biosensor for rapid screening of AI virus. The specific objectives are to: (1) synthesize and characterize gold nanowires for their use in a nano-biosensor; (2) functionalize and surface-modify the gold nanowires; (3) conjugate the gold nanowire with anti-H5 or anti-N1 monoclonal antibodies; (4) design and fabricate interdigitated nanoelectrode and nanochannel, and integrate them into a nano/microfluidic chip; (5) immobilize anti-H5 or anti-N1 monoclonal antibodies in the nanochannel; and (6) evaluate the nano-biosensor for screening of H5N1 and other H5 subtypes of AI virus in comparison with gold standard methods. The novelty of the proposed nano-biosensor is the integration of a novel nanowire switch, innovative interdigitated nanoelectrode/nanochannel biochip, and new anti-H5 and anti-N1 monoclonal antibodies for highly effective capture of target AI virus and ultra sensitive signal generation. Multidisciplinary efforts (chemistry, virology, mechanical and biological engineering) will be made to reach the goal of this proposed research. We expect that this nano-biosensor will be able to more rapidly, specifically and sensitively screen AI virus in a poultry sample in less than 30 min. This research addresses the CSREES goals to enhance protection and safety of the Nation?s agriculture and food supply and to improve the Nation?s nutrition and health.

OBJECTIVES: This project is proposed to integrate nanotechnologies with microanalytical device to develop an innovative nano-biosensor for in-field rapid screening of avian influenza virus (AIV) infection in poultry flocks. The specific objectives of this research are: 1. To synthesize and characterize colloidal gold nanowires. Two methods will be employed to fabricate the gold nanowires with a length less than 100 nm and a diameter of 10~20 nm. One is to synthesize gold nanowires using wet chemistry approach in the presence of cetyltrimethylammonium bromide (CTAB). Another method is to use anodic alumina oxide (AAO) template to synthesize gold nanowires electrochemically, which usually results in more uniform nanowires. 2. To functionalize and modify colloidal gold nanowires. Colloidal gold nanowires prepared from both wet chemistry and electrochemistry need to be further functionalized for biological application because there are no any functional groups on the surfaces of CTAB-capped nanowires. This aim will be to modify the gold nanowires with poly(acrylic acid) to introduce functional groups (carboxyl groups) that can be easily used for further modification. 3. To conjugate the gold nanowires with anti-H5 or anti-N1 antibodies. Gold nanowires with carboxyl acid as terminal groups will be further modified with anti-H5 or anti-N1 monoclonal antibodies to more selectively bind to AI virus. 4. To design and fabricate interdigitated nanoelectrode and nanochannel (less than 100 nm), and then integrated them into a microfluidic biochip that will provide a platform for sample flow, target AIV capture, nanowire labels and impedance measurement. 5. To immobilize anti-H5 or anti-N1 antibodies on the bottom of the nanochannels for highly effective capture of target AI virus. 6. To evaluate the nano-biosensor for detection of AIV H5N1 as well as H5 subtypes in comparison with gold standard methods such as viral isolation, RT-PCR and ELISA tests. It is expected that the nano-biosensor will be able to detect AIV H5N1 at a concentration less than 10 EID50/mL in less than 30 min.

APPROACH: <BR> 1. Synthesis and Characterization of Colloidal Gold Nanowires: Monodispersed gold nanowires will be synthesize in aqueous using two methods. One method is based on modified multi-step seeding protocol. The prepared gold nanowires will be positively charged, highly dispersed in aqueous solution with high mobility and good biocompatibility which are required application in bioassays. Another method to synthesize gold nanowires with uniform structure will be based on electrochemical deposition of gold on AAO template. <BR> <BR> 2. Functionalization of Colloidal Gold Nanowires: Aliquots of as-prepared CTAB-capped gold nanowires will be placed in microcentrifuge tubes and centrifuged. A pellet of gold nanowires will be formed at the bottom of the microcentrifuge tubes. The supernatant colorless solution will be slowly removed without disturbing the pellet. For poly(acrylic acid) coating experiments, two pellets will be combined to double the nanowire concentration. Stock solutions of PAA will be prepared. To the microcentrifuge tubes containing the gold nanowire pellets will be added PAA stock solution. After adsorption time, the excess polymer in the supernatant fraction will be removed by centrifuging and rinsing , and finally redispersing the pellet in deionized water. <BR> <BR> 3. Conjugation of the Gold Nanowires with Anti-N1 or Anti-H5 Antibodies: Stable and water-soluble amphiphilic polymer-coated gold nanowires with carboxylic acid groups exposed outside have no selectivity to target AI virus. The high capture efficiency for bacterial cells can be achieved exploiting of streptavidin-biotin system to conjugate polyclonal anti-bacteria antibodies to particle surfaces. <BR> <BR> 4. Design and Fabrication of Interdigitated Nanoelectrodes and Nanochannels: During incubation the species of interest are transported to the surface of the sensing elements through molecule diffusion, and subsequently captured by surface immobilized antibody. This step often accounts for a good percentage of the total assay time and the target capture efficiency through molecule diffusion is low. A combination of AC-electroosmosis and Dielectriphoresis will be exloided using the same IDT electrode for impedance spectroscopy sensing to realize rapid pre-concentration viral particles. <BR> <BR> 5. Immobilization of Anti-H5 or Anti-N1 Antibodies on the Bottom of the Nanochannels: An oriented immobilization method will be used. The nanoelectrode chip surface will be silanized with ATES followed by a treatment with GD, and then protein A or G will be covalently immobilized on the surface via their amine groups. Then the antibody will be oriented on the surface due to the natural affinity of protein A or G towards the Fc region of antibody molecules. <BR> <BR> 6. Evaluate the Detection of AI Virus H5N1 and Other H5 Subtypes: The detection of H5N1 AI virus with the nanowire-based biosensor will be demonstrated with killed H5N1 isolates to be obtained from the USDA. Also the effective detection of H5 subtypes including H5N1, H5N2, H5N3, and H5N9 viruses will be tested using H5 subtype-specific MAbs with the biosensor. The LPAI H5N2 virus will be used initially as a mode for the development of test procedures.