Study On Magnetic Nanobead Separator For Rapid Detection Of Avian Influenza Virus

In recent years, the development of nanotechnology has provided new means and opportunities for the biological research. The immunomagnetic nanobead separation(IMNBS) technology shows great potential applications for biological materials concentration and separation. The technology can be used for effective elimination and analysis of the interference background so as to achieve the purification and concentration of a wide variety of samples. The IMNBS technology introduces functional groups to the surface of the magnetic nanobead(MB) and combines it with the specific antibody. The new formed MB reacts with the target sample analyte and forms the MB-antibody-antigen compound called immunomagnetic nanobead(IMNB). In the electromagnetic field, only those magnetized IMNBs move accordingly in a specific direction and accumulate. As so,the target analyte on antigen epitope is then separated out from other interference materials and to be extracted, purified and concentrated. The combination of IMNBS technology and impedance immune sensor embedded interdigital micro-electrode can significantly improve the sensitivity of the microorganism rapid detection system. For the study of avian influenza virus(AIV) rapid detection on microelectrode, this study focuses on the design and tests of the MNS for collecting and pretreating the AIV samples on AIV rapid detection platform. Previous arts of published research have shown us the fact that smaller IMNBs are difficult to be separated in systems of small MB with low magnetic susceptibility and high fluid viscosity. There is also some theoretical analysis available for reference that explained the difficulty of such separation efforts. Thus, a high gradient and high intensity magnetic separator(HGHIMS) is required. Integrated and small size MNS is essential to daily practices for its convenience and easy-to-carry also.In terms of MNS design, based on the analysis and simulation of common permanent magnet MNS, an annular MNS with six separation holes was proposed. The MNS was made of neodymium iron boron(Nd Fe B) blocks with special profile and dimension to enhance its magnetic field. Six HGHIMS separation holes on MNS were formed by six outer Nd Fe B tiles and inner Nd Fe B tiles with the like magnetic poles closed together.Permalloy material is used as magnetizer in the middle of closed magnet poles to gatherand conduct the magnetic field. The MNS is shelled by cylindrical aluminum alloy with weak paramagnetic to form a compact structure. In the research, four sets of MNS were designed and fabricated with different structure design elements including MNS volume,material and profile of magnetizer. The intensity and gradient of the magnetic fields in each of separation hole were measured for analysis. A highest MNS magnetic field intensity of1.44 T and a highest MNS magnetic field gradient of 93.4 T/m(865.5 cm3 volume, 4.8 kg weight) were achieved. The measurement average relative error among each hole of the four sets of MNS was between 2.0% to 3.3%. That means it has the same separation effect for six separation holes in the MNS. The research results also showed that MNS volume,material and profile of magnetizer have different effects on the intensity and gradient of magnetic field. The larger MNS or the MNS with permalloy magnetizer or with sidleing-magnetizer can obtained higher intensity and gradient of the magnetic field compared to the smaller MNS or MNS with soft iron magnetizer or MNS with straight-shape magnetizer. Therefore, according to different IMNBS application requirements, it can be obtained the ideal separator easily with different structure elements.In a research to analyze MNS separation efficiency, a samll MNS V2 was used for qualitative and quantitative evaluation. The MNS V2, of which the volume was 401.3 cm3 and the weight was 2.2kg, has a highest magnetic field strength of 1.26 T and a highest magnetic field gradient of 84.4 T/m. Firstly, the qualitative evaluation was conducted using visual inspection experiments, transmission electron microscopy(TEM) observation experiments and Dot-ELISA experiments with 150 nm MB separating inactivated H5N1 AIV from the solution. Secondly, the quantitative evaluation was conducted using the combination of E.coli O157:H7 separation and plate counter method with MBs in size of30 nm, 100 nm and 180 nm. Separation duration, separation sensitivity and separation stability were studied on MNS V2. Lastly, the quantitative evaluation was conducted using fluorescence quantitative RT-PCR technique with 180 nm MB separating activated H5N1 AIV from the solution. The experiment was conducted on AIV rapid detection platform with the system integration. The results of qualitative evaluation experiments with 150 nm MB separating the inactivating H5N1 AIV showed that the MB compound can completely be separated from the solution in 1 minute. The results of the quantitative evaluation experiments with MB in size of 180 nm, 100 nm and 30 nm separating E.coli O157:H7solution, showed capture efficiency was higher than 96.5% if the separation duration was no less than 40 seconds, 60 seconds or 60 minutes. No bacteria residue was found in the solution after MB separation. For the high and low concentration E.coli O157:H7, captureefficiencies of the 180 nm, 100 nm and 30 nm MB were 96.5% ~ 106.9%, 99.7% ~ 114.6%and 98.5% ~ 112.6% respectively. For concentration of 103CFU/m L E.coli O157:H7solution, the relative standard deviation of 20 parallel samples with different sizes of MB separation was between 0.84% ~ 1.4%. All these results showed that the MNS V2 worked well in sensitivity and stability with MB in different sizes. On the AIV rapid detection platform with the system integration, the results of quantitative evaluation by fluorescence quantitative RT-PCR technique with 180 nm MB separating activated H5N1 AIV showed that optimal separation duration was 2 minutes for the AIV impedance threshold determination on impedance biochip. The average capture efficiency was 47.8% ± 3.2%under experimental condition for 102-104EID50/m L H5N1 AIV. The result showed that matching and specificity of the influenza virus antibody and antigen directly affected separation efficiency of IMNBS technology. In conclusion, the research of MNS separation efficiency showed that the MNS V2 had a good potential in the field of rapid detection for both bacteria in micron dimension and virus in nanometer dimension. Thus, the MNS design is a solid foundation for the AIV rapid detection platform.