Highly Sensitive Detection Of Avian Influenza Virus Based On Quantum Dots And Immunomagnetic Separation

Sensitive and reliable virus detection is becoming greatly important in various areas, such as food and environmental safety, clinical diagnosis, and anti-bioterrism. Although new technologies have markedly improved the ways of pathogen detections, there still exits a general need for robust, sensitive, and fast methods for clinics at the point-of-care. Taking avian influenza viruses (AIV), for example, they are type A influenza viruses which have caused several worldwide outbreaks of influenza-like illness in the recent decade. So far, the acknowledged standard detection method of AIV is virus isolation in cultured cells, eggs, or laboratory animals, subsequently with the serological methods for final identification, but the outcomes may take1-2weeks. Other commercial and widely used methods in lab diagnoses are enzyme-linked immunosorbent assays (ELISAs) and polymerase chain reaction (PCR) methods. However, detections at the point-of-care are met with more severe technical challenges due to low concentration of target organisms in a complex biological sample. What’s more, point-of-care oriented methods, which may be carried out at the patient beside or self-home service, are expected to achieve low-concentration, rapid, highly selective and reliable detection of complex biological samples and meet the requirements of portability, low cost, less-skilled, and easy manipulation without well-training staff. Therefore, the ELISAs and PCR methods are not well suitable for point-of-care test.For clinical samples, recognition and separation of targets are usually considered as essential and key steps for accurate detection. Polymer micro/nanobeads especially consisting of maghemite (γ-Fe2O3) or magnetite (Fe3O4) as superparamagnetic beads have unique advantages for this usage. The approach of combining immunomagnetic micro/nanobeads and fluorescence QDs for target pathogen detection is promising for on-site application at the point-of-care due to its high sensitivity and selectivity, easy manipulation, versatility, no pretreatment of samples, and no need for well-trained staff. However, robustness and reproducibility of nanoparticle-based methods are still needed to be improved for clinical applications. Thus, main works in this dissertation are as follows:1. In this work, a robust approach for highly sensitive point-of-care avian influenza virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2subtype, H9N2viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. High-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immunosandwich mode. This method gets a limit of detection low up to60copies of viruses in200μL. This method is robust for application at the point-of-care due to its very good specificity, precision and reproducibility with an intra-assay variability of1.35%and an inter-assay variability of3.30%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with30throat swab samples with a coincidence of96.7%with the expected results.2. We study the capture kinectics to find out that the capture kinectics could be perfectly fitted with a first-order bimolecular reaction when the concentration of the nanobeads was exceeded. The capturing rate constant kf was calculated to be4.25×109(mol/L)-1s-13. Inspired by the selective binding between hemagglutinins (HA) of avian influenza virus and the acceptor of α2,3-sialic acid-Gal on the surface of cell, we modified the Neu5Ac to the QD-Gla surface via sialic acid α-2,3-Gla linkage with the help of α-2,3-Sialyltransferase to get the α2,3-sialic acid-QDs, and tried to label H9N2avian influenza virus with this α2,3-sialic acid-QDs.4. A new method for detection of H9N2avian influenza virus was established by identifying H9N2virus with α2,3-sialic acid-QDs after they were captured by the immunomaganetic nanobeads.