Preparation, Evaluation And Application Of Coliphage F2 Molecularly Imprinted Membranes

Microorganism pollution is one important factor that affects water quality. Enteroviruses are widely existed and can not be removed efficiently by common sterile techniques in water treatment, such as chlorine, ozone, and ultraviolet rays. Food poisons caused by eating seafoods cultured in human enterovirus (for example Small round structured virus) were occurred frequently in Japan, which further indicated that virus can not be removed validly by normal water treatments. Therefore, the enterovirus is indexes of water quality (including drinking, recreation water and groundwater water) in developed countries. Althouth no virus index is used in China water quality standard, the increase impact of animal virus to human health requires effective virus monitoring.Virus can only self-replicates in sensitive cells. In present, virus detection usually includes four steps:separation, concentration, amplification and measurement. Firstly, by adjusting pH of water to a suitable level, the water was applied to non-specific adsorption columns to adsorb charged virus in water. Then the adsorbed viruses are eluted and precipitation from elution by adjusting pH of elution. Later, the virus is suspended in small volume of PBS, removing bacteria in the elution by microporous membranes. Finally, the virus was added to host cells and cultured for a certain period (1-3 days). Plaque Forming Units is used to calculate the titer of virus. Lacking specific adsorption and concentration methods lead to coexists of many other microorganisms with the same charged of virus at the same time. With the development of molecular biology technology, many techniques such as polymerase chain reaction (PCR) and immunofluorescence are utilized for virus identification and quantification. These methods may shorten detect time, but it still need time-consuming and complicated separation and enrichment processes. Specific and efficient ways for separate and enrich virus in water is in great need.In electrospinning, the electrospun polymers solution is suffered from interaction among Coulomb repulsion, electrostatic force and surface tension in the presence of the high voltage electric field. It firstly forms Taylor cone at the spinneret exit in the eligible condition, and then jetting splits after reach to critical voltage. The jet is stretches and elongates repeatedly, eventually dries and solidifies nano-filament deposited on the cathode collector after solvent evaporation. Different microorganisms (including bacterial, virus and cells) were immobilized in electrospun fibers by electrospinning. Therefore, we suggested that by eluting immobilized virus in the electrospun fibers may form specific spatial structure of the target virus, which may be used as artifical antibody of the target virus. This kind of artificial antibody may separate and enrich trace target virus in water selectively and effectively.Because there are different kinds of microorganisms in water, indicators are normally used. For example, Total Coliforms and Fecal Coliforms are two indicators of enteric bacterium in current water quality standards. Enterovirus also need suitable indicator virus. Coliphages, one kind of bacterial virus without human pathogenicity, are ubiquity in sewage and present similar quantities of enterovirus. The resistance of Coliphage f2 to natural environment and water treatment are stronger than bacterial, nearly as the same as that of animal virus. International Organization for Standardization (ISO) and United State Environmental Protection Agency (EPA) recommends Coliphage as indicator of enterovirus. Among these Coliphages, F+RNA Coliphages are the most close to enterovirus in physical characteristics. F+RNA Coliphages are innocuous to human, which already generally use by many studies on evaluating safety, tracking source of viruses, analyzing the efficiency of sewage treatment and so on. Coliphages f2 is the widely exist F+RNA Coliphages in water. In this study, Coliphages f2 is chosen as target virus. Artificial antibody of Coliphages f2 was prepared by electrospinning. Then the adsorption characteristic and practical application of the artifical antibody were further evaluated.The whole research has the following three parts: Part I:Amplification and Purification of Coliphage f2Objective:To obtain high concentration of Coliphage f2Method:Phage f2 were cultured in Escherichia coli 285 in nutrient broth. The propagated phages were purified by PEG-precipitation and chloroform extract. The titer was quantified by double layer plaque titration method.Results:The titer of propagated phages f2 was about 2.45x1010pfu/mL. After purification, the concentration of phage reached to about 6.55×1014pfu/mL.Conclusion:PEG-precipitation and chloroform extract can be used to prepare high purity of Coliphage f2.Part II:Preparation and Evaluation of Electrospun Coliphage f2 Molecularly Imprinted MembranesObjective:To evaluate the adsorption characters of prepared Coliphage f2 molecularly imprinted membranes.Methods:Morphology of molecularly imprinted membranes prepared under different electrospinning situation was observed by scanning electron microscope. Adsorption capacity and selectivity was evaluated by static adsorption experiments.Results:①Electrospinning conditions may influence the morphology of molecularly imprinted membranes. The best electrospun conditions are:0.5gPVA dissolved in 5mL purified water,3mL Methanol and 2mL SM solution or Coliphage f2,The reception distance between needle of spinning outlet and collector was 15.7cm. The high voltage was 15-18kv. Well-distributed, thin, and smooth fibers without pearl-like were observed. The average diameter was metered about 300nm. The speed of electrospinning was 1mL/2h.②Addition of methanol in electrospinning solution did not reduce the phage activity significant (decreased only about 5%).③10%SDS-10%AcOH elution with ultrasound can remove f2 coliophage effectively (about 82.19%), better than 10%SDS-10%AcOH elution combined with oscillation, or single oscillation④Static adsorption experiments represented that adsorption rate (the concentration of phage after/before adsorption) of MIP increased from 60%(104pfu/mL) to 79%(108pfu/mL), NIP accordingly increased from 20% to 50% in 4℃。MIP adsorption rate increased from 71.43%(103pfu/mL) to 83.44% (1012pfu/mL), NIP accordingly increased from 53.57% to 65.63% in 37℃. The adsorption rates of MIP to two kinds of phages with different structures (Coliphage M13 and 285P) were 60%,53.97%, NIP was 57.33% and 63.97% in 37℃respectively. It indicated that MIP could effectively recognize and separate target Coliphage f2. Recoveries of SM solution (79%) were far greater than that of nutrient broth (50%) or 3%beef extract-0.05M glycine (48%).Conclusion:①The electrospinning parameters had to be optimized during electrospinning. There was a spinnability threshold of needle diamater, voltage and collection distance. The addition of organic solvent was contributed to inhibit droplet and improve stability during the process of electrospinning.②Addition of methanol in electrospinning solution did not reduce the phage activity significant③The electrospinned molecularly imprinted membranes could recognize and rebine f2 coliophage selectively and effectively. The adsorption capacity was higher in 37℃than 4℃due to Brownian motion, which behaved briskly in high temperature.④SM solution was better than other eluent to elute f2 coliopahges from imprinted membranes.PartⅢ:PartⅢ:The Application of Coliphage f2 Molecularly Imprinted Membranes in different Water SamplesObjective:To evaluate the application of Coliphage f2 imprinted membranes in different water sample.Methods:Recoveries of spiked lake and pond water using molecularly imprinted membranes or commercial nitrocellulose (0.22μm) were detected.Results:The recoveries of MIP to school pond and Beihu Lake were 74.80% and 68.94%; for NIP were 51.50% and 41.85%; for nitrocellulose membrane were 52.45% and 43.73% respectively.Conclusion:MIP can recognize, separate and enrich Coliphage f2 in different water samples selectively and effectively The main conclusion and the new ideas:1. The main conclusion(1) High purity of Coliphage f2, which were quantified measured by double layer plaque titration method.(2) Coliphage f2 imprinted membranes were prepared by electrospinning. Static adsorption showed the imprinted membranes can separate and enrich Coliphage f2 selectively and effectively(3) The imprinted membranes may enrich f2 Coliphage in different water effectively.2. The new ideas(1) Prepared Coliphage f2 molecularly imprinted membranes by electrospinning method.(2) The Coliphage f2 molecularly imprinted membranes can separate and enrich Coliphage f2 selectivily and effectively in different water samples.