| Molecular imprinting technology(MIT), which imitates the specific binding mechanism of antigen and antibody based on intermolecular forces, can be used to separate specific compound from the complex samples. MIT has a great potential to be used in the field of biology and chemistry, and attracts wide attention in the world. With the continuous development of science and technology, single discipline is often limited for its development, and double discipline, even multidisciplinary is the developing trend now. In this thesis, MIT, membrane technology and photonic crystal technology were combined, with some representative molecular as the template, to prepare molecularly imprinted membrane, which provides foundation for the biochemical sensor chip research and development.Chapter 1 presents a detailed introduction to the basic principles and preparation methods of MIT, and the advantages and disadvantages of those methods were analyzed. And then the types, development of molecularly imprinted membranes in recent years, and the difference between common membranes and molecularly imprinted membranes were introduced. Finally, the basic principles and preparation methods of photonic crystal material was introduced in the end of this chapter and the significance of the interdisciplinary of molecular imprinting technology, membrane technology and photonic crystal technology was discussed.Chapter 2, lysozyme(Lys) was used as the template, and acrylamide was employed as the functional monomer to prepare the polyacrylamide molecularly imprinted polymer membranes(MIP) and non-molecularly imprinted polymer membranes(NIP) as the reference. The polymerization conditions and elution conditions were optimized, and elution condition was determined to use sodium dodecyl sulfate(SDS) and acetic to remove the template Lys. In the adsorption experiments, the adsorption of MIP was compared in the different pH conditions; results showed that the PBS buffer solution(pH 6.0, 25mM) was the best condition for Lys adsorption. Further adsorption kinetics tests show that MIP could reach adsorption equilibrium in 30 mins. Scatchard equation was used to analyze the absorption data, it was found that the MIP had homogeneity of binding site, and the theoretical maximum adsorption capacity was 8.2 mg/g. Selective adsorption experiments demonstrate that the adsorption capacity to Lys was much better than other proteins, and the NIP didn’t have significantly change for all proteins. MIP or NIP was added into the mixed protein solution for competitive adsorption experiments, SDS-PAGE and HPLC were used to analyze the protein solution. The results showed that the concentration of Lys was significantly lower after adsorbed by MIP(the peak area of Lys decreased to 19%), concentrations of bovine serum albumin(BSA) and ovalbumin(OVA) decreased less than Lys(72% and 92%). Reproducible experimental results showed that MIP had good repeat performance. In a word, the MIP had a good prospect for rapid test of low-abundance Lys.Chapter 3, the monodisperse polystyrene microspheres were firstly used as the porogen to prepare the molecularly imprinted polymer(PS-MIP). The addition of monodisperse polystyrene microspheres molded the pores of submicron on the surface of the PS-MIP to improve the specific surface area. This would help to improve the dynamic performance of the PS-MIP, and more binding sites were expected to be exposed to improve the absorbability. In this study, THF was used to remove the polystyrene and study showed that the use of THF would not have asignificant effect on the adsorption properties of PS-MIP. The dosage of the polystyrene in the polymerization system was optimized with the concentration of polystyrene 0.1%(IF=6.0) for the best imprinting effect of PS-MIP. In the adsorption experiments, comparison was made with different pH conditions for the adsorption property of PS-MIP, study showed that the PBS buffer solution(pH 6.0, 25mM) was the best condition for Lys adsorption. The adsorption kinetics research showed that PS-MIP could reach adsorption equilibrium in 15 mins. And absorption data were analyzed with Scatchard model, it was found that the PS-MIP had two kinds of binding sites, and the theoretical maximum adsorption capacity was 13.0 mg/g. In the selective adsorption experiments, the adsorption capacity to Lys was much better than other proteins, the PS-NIP didn’t have significantly different for all proteins. In the competitive adsorption experiments, PS-MIP or PS-NIP was added into the mixed protein solution, and SDS-PAGE and HPLC were used to analyze the protein solution. Results showed that the concentration of Lys was significantly lower after adopted by PS-MIP(the peak area of Lys decreased to 6.4%), the concentration of bovine serum albumin(BSA) and ovalbumin(OVA) decreased less than Lys(85% and 97%). Compared with MIP prepared in the second chapter, PS-MIP had the higher selectivity and the greater adsorption capacity. Therefore, PS-MIP had a greater advantage than former MIP for rapid test of Lys.In the last chapter, the Levodropropizine(LDPZ) imprinted photonic crystal polymer membranes(MIPC) were firstly prepared with methacrylic acid(MAA) as the functional monomer, 2-hydroxyethyl methacrylate(HEMA) as an auxiliary functional monomer. In this study, MIT, membrane technology and photonic crystal technology were combined, which is expected to extend the application of molecularly imprinted photonic crystal polymer to the alkaline compound. MIPC could transform the recognition of LDPZ to adsorption peak shift or diffraction peak shift due to the feature of minor volume change of the imprinted polymers after adsorption. In this study the polymerization system was optimized, the ratio of template, functional monomer, auxiliary monomers and cross-linking agent was determined to enable good adsorption selectivity and swelling properties for MIPC. Then monodisperse silica particles with the size of about 240 nm were synthesized for the preparation of the photonic crystal membrane by the method of colloidal self-assembly. And the imprinting system was infiltrated into the photonic crystal membrane and polymerized with heating, then hydrofluoric acid was used to remove the silica to obtain the MIPC. Further adsorption experiments were carried out, results showed that MIPC had the best adsorption effect to LDPZ in the PBS buffer solution(pH 6.0, 25mM), adsorption equilibrium time was 30 minutes. In the selective adsorption experiments, MIPC showed the larger peak shift when rebound the LDPZ than other analogues, which demonstrated that MIPC had good selectivity for LDPZ. Reproducible experimental results showed that MIPC had good repeat performance. Therefore, the MIPC had a good prospect for rapid test of LDPZ.In a summary, the monodisperse polystyrene microspheres were firstly introduced as the porogen to prepare the molecularly imprinted polymer(PS-MIP). As compared with non-porogen molecularly imprinted polymer(MIP), the addition of porogen can improve the imprinting effect, thus providing a reference for protein detection in biosensor assay. Later the Levodropropizine(LDPZ) imprinted photonic crystal polymer membranes(MIPC) were firstly prepared with the MIT, membrane technology and photonic crystal technology combined. MIPC could transform the recognition of LDPZ to adsorption peak shift or diffraction peak shift due to the feature of minor volume change of the imprinted polymers after adsorption, which can be used as indicator paper or sensor chip for rapid and sensitive detection of LDPZ. |
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