Studies On New Rapid Microfluidic Based Methods Of Environmental Immune-bioanalysis

With rapid urbanization and economic development, environmental issues have been attracting more and more public attention because of growing concerns of health and safety. The interactions between the human being and natural ecosystem are the research focus, especially in relationship between the microorganisms and public health. Accurate analysis, especially the bioanalytical technique is the essential for any environmental science. The new developments in biotechnology have been more and more widely used in the study of environmental science. Although the new biotechnology analysis method has higher sensitivity and specificity compared to traditional physical and chemical method, it is difficult to conduct on-site testing due to the equipment constraints. In addition, the cost for bioanalysis is still high due to the high expense of biological reagents. Therefore there is a great need to develop a fast, simple, portable, low-cost detection technology for application in actual environmental analysis needs.With the development of microfabrication technology, the microfluidic chips have been applied in many fields, and so far many traditional chemical and biology method can be represented in the microfluidics. Microfluidics has become an attractive research topic due to its less time-consuming, less reagent consumption, easy automation and parallel processing. For standard bioanalysis in microfluidics, the total reaction volume is generally less than1uL, and bioreactions can be completed within less than30minutes, such as widely used ELISA (Enzyme Linked Immunosorbent Assay). Many different types of immunoreaction have been performed successfully in microfluidic chips, with potential application in environmental science.In this thesis, we developed several analytical methods based on microfluidics fro rapid environmental bioanalysis. The analysis targets include small molecules (pesticide), protozoa in water (based on its characteristic biological macromolecules), and pathogen analysis in the air. The research is briefed as following:Glyphosate (N-(phosphonomethyl)glycine, C3H8NO5P)) is a non-selective herbicide widely used in agriculture and forestry for aquatic weed control. In the Guidelines for Drinking-water Quality, the health-based value of glyphosate is0.9mg/litre. Herein we developed a new microfluidic device for selective and rapid detection of glyphosate. The reaction mechanism is based on Enzyme-linked immunosorbent assay (ELISA). This device showed good selectivity to glyphosate, and the detection limit is below0.1ppb. Compared to the conventional ELISA kit, our device can save3/4time and reduce4orders reagent consumption.The protozoa detection was added into the Guidelines for Drinking-water Quality, because of the hazard to people health. For the protozoa analysis, microfluidic devices were also successfully designed and fabricated for rapid detection of the Giardia lamblia cyst, Cryptosporidium oocyst and Entamoeba cyst in water. Immunoassay was also developed for serodiagnosis, through detection of Entamoeba histolytica antibody detection in serum samples for patients with amebiasis, very promising to develop a fast protozoa detection platform.The cysts and oocysts capture is the initial and key step for the cysts and oocysts detection in water. In the proposed microfluidic devices, the cysts and oocysts were first intercepted and captured by the microcolumns inside the device. The fluorescent-linked immunoassay inside the microfluidic device were utilized for the detection of the cysts and oocysts through a CCD camera. We also develop a microfluidic assay for the characteristic protein detection of Entamoeba histolytica. The test results were consistent with the convention ELISA results, with1/3time consuming and much less reagent consumption. This device also has the potential application in the rapid detection of other biological macromolecules and microorganisms.The detection of pathogenic microorganism in the air plays an important role for public health. Highly efficient collection and enrichment is always the key for the rapid analysis of the airborne microbe detection. Herein we successfully developed microfluidic devices which are capable of fast and efficient aerosol bacteria collection, enrichment and detection. The device is validated with Escherichia coli and Staphylococcus Aureus enrichment and detection, and our results showed that the enrichment efficiency can reach close to100%. The enrichment efficiency of the microfluidic chip is4-5times higher than the sedimentation method, and the Staphylococcus Aureus detection limit is below10cells. The device is fabricated from plastics, simple, cheap and disposable, perfect for field application.Above all, we successfully designed and fabricated series of microfluidic chips for rapid detection of glyphosate, Giardia lamblia cyst, Cryptosporidium oocyst, Entamoeba cyst as well as microfluidic devices for capture, enrichment and detection of airborne pathogens. The microfluidic devices showed improved efficiency, higher speed, high sensitivity, high specification, low consumption compared with traditional techniques. After further optimized, all the microfluidic chips can be connected with the other detection module and control module, and applied in field environmental tasks.