Rapid Detection Methods For Avian Influenza Virus Based On Immunomagnetic Separation And Enzymatic Amplification

Viral epidemics, such as severe acute respiratory syndrome (SARS), avian influenza (AT) and acquired immune deficiency syndrome (AIDS), seriously threaten the health of human beings. Fast and sensitive detection techniques for viruses are of great importance to provide immediate and appropriate clinical strategies to control the infection and spread of viruses. However, traditional virus-detection methods, such as virus culture, serological tests, and enzyme-linked immunoassays (ELISA) usually require long analysis times with low sensitivity. The polymerase chain reaction (PCR), which is widely accepted as a sensitive method, requires expensive reagents, facilities operators, and sophisticated instruments, is not well suitable for on-site diagnosis.Electrochemical immunoassay is a new kind of immunoassay method which integrates the highly specific immunoreaction with the highly sensitive electrochemical detection. It has attracted considerable interest for its intrinsic advantages such as high sensitivity, low cost, ability to be easily miniaturized and low energy consumption. Nanomaterials exhibit unique electronic, photonic, and magnetic properties, which can greatly enhance the performance of biosensors. Among them, magnetic beads (MBs) are extensively used in immunoassays owing to their excellent properties:such as the increased surface area, the faste assay kinetics as well as the capability of separation and enrichment. The use of MBs as the reaction carrier can not only simplify the pretreatment procedures and reduce the immunoreaction time, but also greatly amplify the detection signal. Therefore, immunomagnetic separation was widly used in cell separation and pathogens detection and so on. A series of virus detection method which suitable for on-site diagnosis were proposed based on the integration of the immunomagnetic separation and the enzymatic amplification. The detail contents are as follows:(1) A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2avian influenza virus particles (H9N2AIV) was designed based on the combination of high-efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin-streptavidin system. The reusable, homemade magneto Au electrode (M-AuE) was designed and used for the direct sensing. Immunocomplex-coated magnetic beads (IMBs) were easily accumulated on the surface of the M-AuE to obtain the electrochemical signal of HRP after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic-bead-based electrochemical immunosensor showed better analytical performance than the planar-electrode-based immunosensor with the same sandwich construction. Amounts as low as10pg mL"1H9N2AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.(2) A novel electrochemical immunosensor based on the integration of immunomagnetic separation and bienzymatic amplification for sensitive detection of virus particles was fabricated in this work. The bienzymatic strategy was realized by using the first enzyme as tracer tagged on immunomagnetic beads which could be accumulated on the magneto controlled home-made Au electrode (m-AuE) and the second enzyme immobilized on the m-AuE by layer-by-layer (LBL) assembly technique. The proposed immunosensor not only provides a rapid, simple and on-site platform with high sensitivity, selectivity, and reproducibility for early diagnosis but also presents a new approach for sensitive magneto immunoassay.(3) A highly sensitive electrochemical immunosensor for avian influenza virus particles detection was porposed by using magnetic bead-based electrochemical immunoassay coupled with enzyme-induced metallization. Alkaline phosphatase (ALP) was used as the signal tag which could form immunocomplex-coated magnetic beads (IMBs) through immunoreaction. The home-made AuNPs modified magneto electrode was used to capture the IMBs and obtain the electrochemical signals. The presence of ALP catalyzed the hydrolysis of p-aminophenyl phosphate monosodium salt (p-APP), which produced a p-aminophenol (p-AP) intermediate to reduce Ag+and then deposited on the magneto electrode. The electrochemical signal was obtained by anodic stripping analysis of the deposited silver in KC1solution. This strategy combines the advantages of the specificity of the enzyme-induced metallization reaction and the high sensitivity of metal stripping voltammetric signal, which could accumulate the enzyme-generated product on the surface of the magneto electorde by means of silver deposition, dramatically enhanced the sensitivity of immunoassay. Furthermore, a bi-electrode signal transduction system was constructed, which could aviod the possible influence of silver ions and silver deposition on the enzyme activity. This strategy could also aviod the deposition of silver on the surface of magnetic bead, thus the quantitation of the target virus was easier to be realized.(4) A novel colorimetric assay method based on enzyme-induced metallization has been proposed for highly sensitive detection of alkaline phosphatase (ALP), and it was further applied to highly sensitive detection of avian influenza virus particles with naked eye coupled with immunomagnetic separation. The enzyme-induced metallization-based color change strategy combined the amplification of the enzymatic reaction with the unique optical properties of metal nanoparticles (NPs), which could lead to a great enhancement in optical signal. The detection limit for ALP detection was0.6amol/50?L which was4-6orders of magnitude more sensitive than other metal NP-based colorimetric methods. Moreover, this technique was successfully employed to a colorimetric viral immunosensor, which could be applied to complex samples without complicated sample pretreatment and sophisticated instruments, and a detection limit as low as17.5pg mL-1was achieved. This work not only provides a simple and sensitive sensing approach for virus detection but also offers an effective signal enhancement strategy for development of a highly sensitive metal NP-based colorimetric assay method.