No description available.

No description available.

本論文中我們將介紹一種新穎且便利的利用微波的固相多肽合成方法，以及在此方法協助下對一組混合肽之間相互作用的研究。與傳統的多肽合成相比，微波輻射幫助提高耦合過程的效率，從而大大縮短了合成時間，以及提高了反應效率。家庭用和實驗室用微波爐的比較表明，家用微波爐在實驗室條件下足以勝任微波輔助固相多肽合成。在第二部分，我們利用微波固相多肽合成合成了一組多肽用以探究其之間相互作用。一對在之前文獻報導的，通過庫篩選得到的結合肽被用來與另一組捲曲肽組合構成混合肽，期望得到強而且具特異性的相互作用以用作蛋白的標記。然而文獻報導的這對多肽並未達到預期的相互作用，因此我們需要在後續實驗中重新設計混合肽。 / This thesis summarizes an investigation into a novel solid-phase peptide synthesis protocol assisted by microwave irradiation, as well as an attempt to design hybrid peptides for enhanced binding properties. Compared with conventional peptide synthesis, a brief microwave irradiation during coupling and deprotection was shown to significantly reduce the time for peptide synthesis, at the same time yielding satisfactory product purity. A comparison of a domestic and a laboratory microwave oven indicated that the former could be easily adapted for conducting microwave-assisted solid phase peptide synthesis in a research laboratory, a facile and budget-efficient solution for enhanced efficiency in solid phase synthesis. In the second part of the research, we utilized the developed protocol to synthesize a set of peptides to investigate peptide-peptide interaction. A pair of binding peptides previously identified through library screening and genetic selection reported in literature was fused with designed coiled coil peptides to afford hybrid peptides, which was expected to result in exceptionally strong interaction and enhanced specificity. These hybrid peptide pairs could find applications in protein labeling, immunoblotting, and purification. However, the peptide pair did not demonstrate the claimed binding affinity in the literature. Therefore, the current design is flawed, and we shall re-design hybrid peptides based on other binding pairs in the following research. / Detailed summary in vernacular field only. / Zhang, Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references. / Abstracts also in Chinese. / 摘要 --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.III / ACKNOWLEDGEMENT --- p.V / ABBREVIATION --- p.VI / Chapter Chapter 1 --- Microwave -assisted synthesis of a defensin peptide: a comparison of domestic and laboratory microwaves --- p.1 / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Defensins --- p.1 / Chapter 1.2. --- Solid-phase peptide synthesis (SPPS) --- p.5 / Chapter 1.3. --- Microwave (MW) heating in organic synthesis --- p.6 / Chapter 1.4. --- Microwave (MW) heating assisted solid phase peptide synthesis --- p.7 / Chapter 2. --- METHODS --- p.8 / Chapter 2.1. --- General synthetic approach of SPPS --- p.8 / Chapter 2.2. --- Microwave-assisted SPPS --- p.9 / Chapter 2.3. --- Analysis and characterization of the product --- p.12 / Chapter 3. --- RESULT AND DISCUSSION --- p.12 / Chapter 3.1. --- Exploring the coupling step under microwaving --- p.12 / Chapter 3.2. --- Nαdeprotection under microwave heating --- p.14 / Chapter 3.3. --- Synthesis of the target peptide --- p.16 / Chapter 3.4. --- SPPS in laboratory MW reactor --- p.18 / Chapter 3.5. --- SPPS by standard conventional method --- p.20 / Chapter 3.6. --- Comparison of the three methods --- p.21 / Chapter 3.6.1. --- Product p Product p urity --- p.21 / Chapter 3.6.2. --- Time and cost --- p.24 / Chapter 3.6.3 --- Sc alabiltiy of SPPS --- p.24 / Chapter 4. --- Exploration of the mechanism of microwave-assisted SPPS: thermal or nonthermal effect? --- p.25 / Chapter 5. --- CONCLUSION --- p.29 / REFERENCE --- p.30 / Chapter Chapter 2 --- Hybrid peptides with enhanced affinity and specificity for protein labeling --- p.33 / Chapter 1. --- INTRODUCTION --- p.33 / Chapter 1.1. --- Protein labeling --- p.33 / Chapter 1.2. --- Hybrid peptides sequences --- p.35 / Chapter 2. --- EXPERIMENTAL SECTION --- p.36 / Chapter 2.1. --- HPLC result --- p.37 / REFERENCE --- p.48

Today’s solid phase microextraction (SPME) is a well known technique that combines knowledge from different fields in an attractive, efficient, and economic way. The development of SPME has seen huge growth since its introduction as a new approach to sample preparation in the early 1990s. The applications of SPME are continuously expanding, and one of the most interesting current aspects consists of applying SPME for fast analysis of biological fluids, both in vitro and in vivo. In spite of this great potential, development of new bio-applications is considerably hindered by the lack of suitable SPME products. The goal of this study is to find SPME coatings that can be utilized for in vivo and in vitro extractions, in direct contact with a biological matrix such as blood or tissue. This thesis presents several effective ways of preparing SPME coatings based on biocompatible polymers and silica-based extractive phases, focusing on their biocompatibility as a must. After fabrication, the proposed coatings are tested for biocompatibility and analytical utility. Finally, some practical applications of the new coatings are presented, such as fast drug analysis and determination of drug plasma protein binding. Six test drugs with different physico-chemical properties are chosen for the investigation: diazepam, verapamil, lorazepam, warfarin, nordiazepam, and loperamide. It is shown that the application of these new SPME fibers for biological sample analysis greatly reduces the time required for sample preparation and limits the exposure of the analytical personnel to potentially infectious biofluids.

Today’s solid phase microextraction (SPME) is a well known technique that combines knowledge from different fields in an attractive, efficient, and economic way. The development of SPME has seen huge growth since its introduction as a new approach to sample preparation in the early 1990s. The applications of SPME are continuously expanding, and one of the most interesting current aspects consists of applying SPME for fast analysis of biological fluids, both in vitro and in vivo. In spite of this great potential, development of new bio-applications is considerably hindered by the lack of suitable SPME products. The goal of this study is to find SPME coatings that can be utilized for in vivo and in vitro extractions, in direct contact with a biological matrix such as blood or tissue. This thesis presents several effective ways of preparing SPME coatings based on biocompatible polymers and silica-based extractive phases, focusing on their biocompatibility as a must. After fabrication, the proposed coatings are tested for biocompatibility and analytical utility. Finally, some practical applications of the new coatings are presented, such as fast drug analysis and determination of drug plasma protein binding. Six test drugs with different physico-chemical properties are chosen for the investigation: diazepam, verapamil, lorazepam, warfarin, nordiazepam, and loperamide. It is shown that the application of these new SPME fibers for biological sample analysis greatly reduces the time required for sample preparation and limits the exposure of the analytical personnel to potentially infectious biofluids.

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.

Abstract Matrix solid-phase dispersion and gas chromatography-mass spectrometry were used for the rapid screening of 36 pesticides in agricultural products. Homogenized sample (0.5 g), C8-modified silica (0.5 g) and Na2SO4 (1 g) were mixed and transferred to a cartridge containing activated silica (0.5 g). Best recoveries (>60%) were found using dichloromethane-ethyl acetate (4:1) for elution. Analytical characteristics at spiking levels (10–100 lg/kg) were calculated for each pesticide. Matrix effects were studied by comparing the slopes of the matrix-matched calibration curves. Fruit and vegetable samples from South Africa complied with EU and South African current regulation, except for cypermethrin in green beans.

No description available.

No description available.

The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples. The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.