Fluorescence Assay Of Avian Influenza Virus In Magnetism-integrated Chips

Continuously epidemics of infectious diseases (such as severe acute respiratory syndrome (SARS), avian influenza, foot-and-mouth disease, dengue disease, and influenza H1N1, influenza H7N9) have caused devastating effects on social economic development, threatened human health and life, and become a global health concern. Therefore, the development of a accurate, simple, rapid method for detecting pathogens is of great importance for preventing and controlling the outbreaks of emerging infectious diseases. So far, a variety of methods have been used to analyze avian influenza virus (AIV), such as viral isolation, immunofluorescence (IF) assay, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (RT-PCR) assay. These techniques have played a significant role in clinical diagnosis. Isolation and identification of virus has high accuracy, and well sensitivity, specificity, and is still the "gold standard" of avian influenza virus identification. But, it is tedious and time-consuming (1-2weeks), and can’t achieve the requirements of rapid diagnosis. ELISA is based on the detection of color change or fluorescence labeled on a secondary antibody. Conventional ELISA is usually performed in a96-well plate and involves a series of tedious processes such as incubation and washing steps. This makes it time-consuming (over4h) and labor-intensive. And the well-trained personnel was required to perform the protocol precisely. The molecular diagnostic assays such as PCR and RT-PCR for the detection of influenza infection provide highly sensitive and selective diagnosis approaches. However, these protocols may not be stable with degrading nucleases, nonspecific stains, and cellular debris existing in clinical samples and usually require sophisticated and expensive instrumentation, as well as complicated and multistep sample preparation, which prohibits potential on-site and real-time practical applications as disease outbreaks.Compared to traditional detection methods, microfluidic chip has some unique advantages such as rapid analysis, low sample and reagent consumption, portability, disposability, high integration and versatility in design. So microfluidic chip can satisfy the demand of simple and rapid detecting pathogens. Superparamagnetic bead is a new type of nano-material developed recently and has attracted a great attention from scientists in a broad field because of its unique advantages such as high surface-to-volume ratio which provids more conjugating sites, functionalization easily with biochemical agents, and manipulation flexible by external magnetic field. Simultaneously, using quantum dots (QDs) as fluorescent marker can improve the detection sensitivity. QDs is a new kind of nano-material as well, and possesses superior optical performances such as high quantum yield, wide excitation spectrum and narrow emission spectrum, highly resistant to light bleaching, and so on. Take advantage of the combination of magnetic beads, fluorescence QDs and microfluidic chip, we developed a kind of simple, rapid, low sample and reagent consumption, high sensitivity microfluidic magnetic fluorescence assay for pathogen detection, which is expected to achieve simple and rapid pathogen detection on-site and clinical.Based on the above background, the major works in this dissertation are as follows:1. In this work, we established a simple rapid and point-of-care magnetic immunofluorescence assay for avian influenza virus (AIV) based on the integration of immunomagnetic target capture, enrichment, and fluorescence detection in the microfluidic chip. By optimizing flow rate and incubation time, we could obtain a limit of detection low down to3.7×104copy/μL with sample consumption of2μL and a total assay time of less than55min. This approach had proved to possess high portable, fast analysis, high specificity, low sample and reagent consumption, high stability and reproducibility with the intra-assay variability and inter-assay variability of2.87%and4.36%, respectively. Meanwhile its well stability also demonstrated by the virus detection in synthetic complicated biological samples with feces and tissues. Take together, this microfluidic system may provide a powerful platform for the simple rapid detection of AIV and may be extended for detection of other viral pathogens, and enable the development of point-of-care diagnostic systems.2. We designed and fabricated a integrated micro-magnetic field microfluidic chip with multiple branch channels to capture different antibodies modified immunomagnetic beads in each branch channel for multiple pathogens(H9N2、H1N1、H3N2) synchronous detection. Our results showed that this method possessed low sample and reagent consumption (only2μL), fast analysis (a total assay time of less than50min), low detection limit (H1N110.98ng/mL, H3N213.76ng/mL, H9N29.35ng/mL), high stability and reproducibility with an intra-assay variability of H1N13.56%, H3N24.44%, H9N23.92%and an inter-assay variability of H1N15.78%, H3N26.84%, H9N25.51%. Furthermore, its well stability also demonstrated by the virus detection in synthetic complicated biological samples with feces and tissues. This method may provide a useful technique platform for the simple rapid on-site multiple synchronous detection and subtyping analysis of AIV.3. We carried out multiple synchronous DNA hybridization analysis to the nucleic acids of the three subtype AIV (H9N2%H1N1、H3N2) in the integrated micro-magnetic field microfluidic chip with multiple branch channels to capture different capture probe DNA modified magnetic beads. Our results exhibited that this method possessed low sample and reagent consumption (only3μL), fast analysis (the whole assay time of less than80min), low detection limit (H1N10.21nM, H3N20.16nM, H9N20.12nM), high stability and reproducibility with an intra-assay variability of H1N15.05%, H3N26.89%, H9N24.98%and an inter-assay variability of H1N16.53%, H3N27.18%, H9N28.88%. This method may provide a powerful technique platform for the simple rapid multiple synchronous detection and subtyping analysis to the internal nucleic acids of AIV.