| In the tide of information era, the attention given to health problem has reached another level, changing the way to obtain data of human body. As a kind of in vitro diagnostic technologies, lateral flow immunoassay, rooting deeply on its advantages of fast testing, low cost, and simplicity, is on a top of tide of development. However, the traditional lateral flow immunoassays based on gold colloid or latex has met their ceiling, because they are suffering from unfriendly manufactory process, poor repeatability and limited sensitivity. Therefore, finding a suitable material as the label for lateral flow immunoassay is in urgent priority. In another hand, magnetic microspheres have showed great possibilities in biomolecules separation, purification and drug delivery for their unique feature of response to magnetic field.This study is committed to develop a lateral flow immunoassay using magnetic microspheres as the label. The main works and results are as follows:(1) Water soluble magnetic microspheres were synthesized by a solvothermal reduction method. Field Emission Scanning Electron Microscope image showed that these microspheres with a mean diameter of 250 nm are nanoparticle aggregates with size of 15nm to 30nm, and according to vibrating sample magnetometer, these aggregates are superparamagnetic material whose saturation magnetization value were 56.15emu/g. Fourier Transform Infare spectrophotometer has confirmed the existence of surface carboxyl. The aqueous condition of these microspheres were carried out by dynamic light scattering, result showed they are rather stable in aqueous. The morphology depending on reaction time and iron concentration has also been investigated respectively. It was found that microspheres grew while increasing the iron concentration, and when lasted the reaction time, the size of the small particles increased.(2) In order to make the immune magnetic microspheres, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC) and N-Hydroxysuccinimide(NHS) were used to covalently couple the monoclonal antibodies with the surface carboxyl of microspheres. Optimal coupling condition was determined through univariate analysis mothed. To activate the surface carboxyl on microspheres, microspheres(counted by iron), EDC and NHS was mixed in pure water for 30min, while the mass ratio of each was kept at 1:0.5:0.3. To couple the antibodies with the microspheres, 0.02M borate buffer with pH value of 6.6 was used as the coupling buffer, and the coupling period lasted for 1h. Transform Infare spectrophotometer confirmed that the antibodies has been successfully anchored on microspheres. Dynamic light scattering test showed that microspheres maintain high water stability after coupling.(3) the as made immune magnetic microspheres were then used as be label to make lateral flow immunoassay test strips to detect human chorionic gonadotropin. In this Experiment, sensitivity of two different flow-speed membranes from Millipore corporation was compared, and as a consequence, slower the flow-speed was, higher the sensitivity. Prussian Blue stain was used to enhance the test strip sensitivity for the very first time, and results showed positive respond. Lateral flow immunoassay test strips for Cardiac troponin I were made by the same mothed and process, results of which also showed relative high sensitivity.According to experiments above, magnetic microspheres synthesized by solvothermal reduction method here are well suitable for lateral flow immunoassay as labeling material. The process of this kind of lateral flow immunoassay test strips can be greatly simplified. Using Prussian blue stain as the enhance agent for test strips has showed positive result. Finally, this mothed was proved to be generally practicable in a extent, which shows great potential for manufacturing. |
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