應用微波輔助磁性奈米粒子親和質譜法及功能化金奈米簇合成法之開發

Abstract

氧化鐵磁性奈米粒子具有多功能性，因此很適合於分析方法的發展。本論文是以官能性氧化鐵磁性奈米粒子為核心結合質譜法發展新的分析技術，並利用微波加熱方式輔助親和質譜法之發展及功能性金奈米原子簇之合成。 本論文的第一部份是開發磁性氧化鐵奈米粒子為輔助樣品脫附游離的基質及親和生化分子之探針，此方法不需要考慮傳統MALDI中基質與樣品互溶性及共結晶化的問題，也不會有訊號集中點的問題，質量偵測上限可達16 k Da左右。可以利用外加磁場吸引這些可當作親和目標物之磁性粒子而達到快速分離的目的並直接進行質譜分析，能夠避免沖提分析物的步驟而造成樣品損失的問題。 在蛋白質體學的研究中，傳統上常需要將大的蛋白質利用酵素經數小時後才能使其消化為各種不同大小的胜□片段，以利於質譜分析及生物資料庫的鑑定及比對，但長時間的酵素消化過程將不利於大量樣品的分析。另外，以質譜法分析複雜的生物樣品時，時常需要藉由適當的分離純化目標物的前處理方法，以有效地降低樣品的複雜程度並去除樣品中所含有的干擾物。近年來微波加熱已成功地應用於加速蛋白質酵素消化反應及分子間的親和反應，因此本論文的第二部分、第三部份及第四部份皆是以官能性氧化鐵磁性奈米粒子能夠吸收微波的特性結合親和質譜法及微波加熱的技術來發展新的分析法，包括：以表面為疏水性的磁性奈米粒子吸附聚核□酸以及利用包覆氧化鋅的磁性奈米粒子吸附親和磷酸化胜□及蛋白質，此過程皆能夠以微波輔助萃取的方法大幅改善傳統上純化分離需消耗長時間的缺點，並從複雜的樣品中純化濃縮出目標物。也驗證氧化鐵磁性粒子的吸收微波之特性可以加速微波輔助蛋白質酵素消化反應，可大幅縮短反應時間至1分鐘內。 在本論文的最後一部份則是利用微波可快速加熱的特性，以蛋白質為反應物合成出 有螢光特性之金奈米原子簇，其中以溶菌□所合成出的AuNCs不但具有螢光性且仍具有生物活性且能夠用以抑制如抗萬古黴素腸球菌及泛抗藥性鮑氏不動桿菌等致病菌的生長。

Owing to the features of a high surface-to-volume ratio, magnetic property, absorption capacity in the broad range of electromagnetic spectrum, and ease of modification, iron oxide magnetic nanoparticles (Fe3O4 MNPs) can be readily designed to have multiple functions for suiting the use in the development of analytical methods. Additionally, Au NCs with good water solubility, excellent stability, and low toxicity have been discovered to be suitable for use in analytical and biomedical researches. In this thesis, two major topics based on the fabrication and the use of Fe3O4 MNPs and gold nanoclusters for method developments and applications were explored.

In the first part of this thesis, an affinity based mass spectrometry using Fe3O4 MNPs as the assisting materials for surface-assisted laser desorption ionization mass spectrometry (SALDI MS) and affinity probes for biomolecules was proposed. After the target species trapped by the Fe3O4 MNPs, the NP-target species conjugates can be rapidly isolated by external magnetic field followed by addition of citrate salts. The mixtures were then directly subjected into a mass spectrometer for SALDI MS analysis. Therefore, the sample loss can be avoided. Additionally, this approach can avoid the problems such as sweet spots that are generally observed in conventional matrix-assisted laser desorption/ionization (MALDI) MS analysis. The upper detectable mass is approximately 16 kDa.

Next, microwave-assisted extraction of DNA and phosphopeptides and microwave-assisted protein enzymatic digestion were developed in this work using functionalized Fe3O4 MNPs as the affinity probes and microwave-heating absorbers. This work demonstrated that the presence of Fe3O4 MNPs in solutions under microwave-heating can accelerate the heating rate. On the basis of quick heating rate, traces of DNA can be effectively enriched by the C18-funcationzliazed Fe3O4 MNPs from aqueous solutions under microwave-heating within 30 sec. Furthermore, the results demonstrated that this approach is suited for enriching DNA from saturated aqueous sodium chloride. Additionally, traces of phosphopeptides from complex samples were also demonstrated to be readily enriched by Fe3O4/ZnO core/shell (Fe3O4@ZnO) MNPs under microwave-heating for 30 sec. In addition, protein enzymatic digestion was accelerated in the presence of functionalized Fe3O4 MNPs under microwave-heating for 60 sec. The feasibility of using trypsin-bound Fe3O4 MNPs was also demonstrated to be applicable in this approach.

In the last part of this work, protein directed synthesis of Au NCs was studied by examining several proteins including bovine serum albumin (BSA), lysozyme, immunoglobulin G (IgG), concanavalin A (con A), cytochrome C, myoglobin, and ovalbumin as the reducing agents. The results show that the presence of cysteine in the protein sequences critical for the generation of Au NCs. Furthermore, lysozyme directed generation of Au NCs (Au@lysozyme NCs) remain their antibiotic activities. To reduce the reaction time, microwave-assisted synthesis of AuNCs by reacting proteins with tetrachloroauric(III) acid under microwave-heating within a short of period (5 min × 8) was proposed. The generated Au@lysozyme NCs also remain their antibiotic activities. It has been demonstrated that the NCs can be used to effectively inhibit the cell growth of pathogenic bacteria including antibiotic-resistant bacteria including vancomycin-resistant Enterococcus faecalis and pan-drug resistant Acinetobacter baumannii. Furthermore, the minimum inhibition concentrations of the proteins on Au@lysozyme NCs for these bacteria were much lower than those when free lysozyme was used.