Preparation And Application Of Novel Environment Functional Materials (IMAC/MIP)

There are a lot of analytes existing in nature with low abundance, including biomacromolecule and small organic pollutants. Biomacromolecules are very important in physiological functions of life, and to analyse organic pollutants providing security for health of flora and fauna. It is difficult to directly analyse both of these targets due to their very low concentrations. Enrichment procedures thus should be put into first place. Aiming at pre-concentrated phosphopeptide and pollutants, novel immobilized metal ion affinity chromatography (IMAC) and molecularly imprinted polymer (MIP) have been systematically and intensively developed. The main findings of this thesis were as follows:(1) A novel IMAC material for phosphopeptide enrichment was prepared based on modified cellulose and was applied in rice phosphoproteome analysis. Firstly, cellulose was modified with phosphoric acid via esterification, and then Ti4+was chelated onto the phosphorylated cellulose. The synthesized materials were ultrafine powders and had good dispersibility in acidic buffer, and as supporting matrix, phosphorylated cellulose exhibited good biocompatibility and chemical stability. Enrichment conditions were optimized and the optimum loading buffer was40%acetonitrile (ACN) with6%trifluoroacetic acid (TFA). Finally, the Ti4+-phosphate functionalized cellulose was submitted to phosphopeptides enrichment prior to mass spectrometry (MS). For a-casein lysates,14phosphopeptides were detected with high intensities even though the sample concentration was as low as2pmol. Besides,15phosphopeptides were still identified by using the digest mixture of α-casein and bovine serum albumin with molar ratio of1:100, which demonstrated high specificity and sensitivity for phosphopeptides enrichment.19phosphoproteins were identified from200μg of salt-free rice leaf protein lysates, while30phosphoproteins were identified from salt-stressed rice leaf protein lysates, and most of these proteins were related to the biological processes in response to abiotic stimulus.(2) Herein, we report the development of a novel MIP-based method for the recognition of tyrosine phosphopeptides in aqueous media using an epitope approach. Phenylphosphonic acid, which has been regarded the "epitope" of phosphotyrosine, was used as a template, and commercially available zinc acrylate was used as a functional monomer to prepare the MIP. The one-pot synthetic process was simple, efficient, and the resulting MIPs were low-cost, robust, and recyclable. The MIP demonstrated significant higher levels of adsorption capacity and selectivity for phenylphosphonic acid than the non-imprinting polymer (NIP) over its structural analog benzoic acid. The MIP was also used as a molecular receptor to recognize tyrosine phosphopeptides in aqueous media, and showed a clear preference for tyrosine phosphopeptides over interfering serine peptides compared to TiO2. These results revealed the feasibility of the use of MIPs to effectively mimic the epitope approach, and provided a promising alternative to the immunoaffinity techniques commonly used for capturing tyrosine phosphopeptides.(3) A novel zwitterion acting as both functional monomer and crosslinker, with protein-resistant ability concomitantly was synthesized for preparing water compatible diclofenac imprinted polymers. This new imprinted polymer showed high imprinting efficiency for template and strong anti-protein adsorption in aqueous medium.(4) Herein, we developed a simple one-pot method for the synthesis of covalent imprinted polymers using mannose as the model template and3-acrylamidophenylboronic acid as the functional monomer. Unlike traditional covalent imprinting, precipitation polymerization was conducted in situ after esterification of3-acrylamidophenylboronic acid with an excess mannose in water. This one-pot synthetic process was simple and efficient. Experiment results of UB NMR spectra and FT-IR spectra have proved the feasibility of this method. The performance of the resulting MIPs was first evaluated by rebinding mannose, and then a competition experiment was conducted to assess the specificity for mannose when mixed with xylose and cellobiose. The imprinting factor of the MIPs for mannose was up to2.04, compared with just0.24and0.12for xylose and cellobiose, respectively. Furthermore, the MEPs showed fast kinetic binding for template (within5min) in water phase.