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The overall goal of this project is to develop a nanobead-based portable microfluidic biosensor system for rapid screening and specific detection of avian influenza virus H5N1 for field use. The supporting objectives are to: <ol>
<li> Develop anti-H5 and anti-N1 monoclonal antibodies and monospecific polyclonal antibodies targeting specifically H5N1 AI virus. To make the biosensor minimally prone to false positives we will utilize anti-H5 for capture and anti-N1 for detection, yielding the highest specificity.
<li> Develop magnetic bio-nanobeads, on which the developed anti-H5 monoclonal antibody will be immobilized, to efficiently capture, separate and concentrate H5 subtype of AI virus from a poultry swab sample.
<li> Design and fabricate a microfluidic biochip that consists of microchannel, microsorter, and measurement microchamber with interdigitated array microelectrodes modified with anti-N1 monoclonal antibody to rapidly deliver, capture and detect H5N1 AI virus. <li> Evaluate the biosensor system for rapid screening and specific detection of H5N1 AI virus in poultry swab samples. The performance of the biosensor system will be characterized in terms of specificity, sensitivity, detection limit, detection time, reproducibility and cost. </ol>
NON-TECHNICAL SUMMARY: Highly pathogenic avian influenza virus H5N1 represents a potential danger not only to poultry but also to human health. Therefore, in addition to containment procedures, sensitive detection for early diagnosis is vital to lower the chances of spread and reduce the risk of development into an epidemic. We propose in this research to develop a biosensor for rapid, sensitive, specific and inexpensive screening of H5N1 virus in poultry swab samples. We expect this biosensor technology to be able to detect the target virus at a very low concentration in less than 1 hour, and be convenient for field use.
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APPROACH: The novelty of the proposed technology lies in the integration of <ol>
<li>a novel magnetic bio-nanobead based separation/concentration procedure;
<li> a novel microfluidic biochip with imbedded interdigitated array microelectrodes,
<li> use of red blood cells for biolabeling and signal amplification <li> use of monoclonal antibodies against H5 and N1 to be developed specific to H5N1 virus. </ol> We expect that this biosensor would be able to rapidly, sensitively and specifically detect the presence of any H5N1 virus in less than 1 hour, and to be convenient for field use. <P>
It is estimated that the biosensor testing cost would be less than $10 per sample which would be cheaper than all current detection methods. The bio-nanobead technique may allow for separation and concentration of a target from any types of samples from poultry, including tracheal and cloacal swabs, droppings and blood, which is a very critical pretreatment step in detection of AI virus using a biosensor. In this study, magnetic nanobeads will be coated with anti-H5 monoclonal antibody that will specifically bind only to H5 subtype of AI virus. After mixing a sample with the bio-nanobeads, a magnetic field will be applied to retain the nanobead-H5 AI virus complex and the rest of the sample will then be washed away. This will result in the separation and concentration of target AI virus into a small volume. The resulting nanobead-H5 AI virus complex solution will then be injected into the biosensor, in which anti-N1 monoclonal antibody will have been immobilized for detection of the target H5N1 virus. A novel microfluidic biochip will be designed and fabricated as a flow-through device to deliver H5N1 virus to an interdigitated array microelectrode for impedance measurement. The nanobead-H5 virus complex will be captured by the immobilized N1 monoclonal antibodies on the microelectrode surface, resulting in an impedance change. Red blood cells will then be injected and used as biolabels for amplification of the binding reaction of the antibody-antigen (virus), since they have relatively large diameters (12 micrometer) compared to H5N1 virus (80 nm), and they have specific and strong binding receptor for influenza viruses through the sialic acid-binding sites on the AI virus surface. Finally, the impedance data will be collected and analyzed using a laptop PC. Changes in the impedance of a sample in continuous flow mode will be correlated to the presence of H5N1 AI virus in a poultry sample. We will make interdisciplinary efforts (involving poultry, virology, immunology, chemistry, engineering) and a systematic approach (including sampling and detection) in this research to reach our goal.