功能性二氧化鈦氧化鐵磁性奈米粒子應用於致病菌檢測與毒殺之研究

Abstract

細菌感染如果沒有及時處理，可能會造成不可挽救的傷害，抗生素是目前用於治療細菌感染最有效的藥物。然而，具有抗藥性的細菌感染常會造成治療上的困難，因此發展快速篩檢以及替代的治療方法是迫不容緩的，所以在本篇論文中，開發了一種快速診斷細菌感染以及光觸媒殺菌的方法。

在本論文第一部份中，首先合成具有光觸媒性質之銳鈦礦晶型二氧化鈦包覆氧化鐵磁性奈米粒子，並以多巴胺分子螯合在二氧化鈦外以做為架橋分子以利IgG之鍵結 (IgG-Fe3O4@TiO2)。此IgG-Fe3O4@TiO2奈米粒子具有辨識多種表面具有蛋白質A與M蛋白質之金黃色葡萄球菌或是化膿性鏈球菌的能力，一旦此奈米粒子辨識到細菌，即可利用磁性分離將奈米粒子-細菌之共結物分離出來並重新懸浮於培養基中，之後再將此共結物擺放於UV-B燈源下 (λmax~306nm, 0.412 mM/cm2) 照射20分鐘內可導致80 %以上的細菌生長受到抑制。

而另一個部份則利用未經修飾之Fe3O4@TiO2奈米粒子為吸附細菌之探針，利用二氧化鈦和革蘭氏陰性菌外膜之具有多磷酸根的內毒素結構有作用力之特性，可用以抓取細菌，因此在抓取到細菌後在UV-B燈源 (λmax~306nm, 0.412 mM/cm2) 照射五分鐘即能使細菌的存活率低於10 %。

而在快速細菌診斷方面也發展了一個結合Fe3O4@TiO2奈米粒子搭配蛋白質體分析的快速偵測方法。利用革蘭氏陰性菌表面之內毒素與奈米粒子之間的作用力，利用微波輔助加熱的方法可在1分30秒內進行細菌快速萃取之後，並可再利用微波1分鐘進行細菌之酵素消化反應，並搭配基質輔助雷射脫附游離質譜儀之一次質譜與二次質譜做為鑑定菌種之方法。並可經由結合質譜之結果與蛋白質資料庫的搜尋鑑定菌株種類。在本研究中已發現在O157:H7型大腸桿菌 、尿道感染型大腸桿菌 、綠膿桿菌 、志賀氏宋內菌、克雷伯氏肺炎菌等菌株中可做為生物標幟峰之離子，並可在15分鐘內分析臨床尿液樣品 (10 μL) 之進行菌株確認，此方法的偵測極限約在數百隻菌至數千隻菌左右。

Bacterial infections may cause fatal damages if proper treatment is not conducted in time. Antibiotics are effective medicines against pathogenic bacteria. However, the emergence of antibiotic-resistant strains of bacteria results in the difficulties in the treatment of bacterial infections. Thus, it is urgent to develop rapid diagnostic methods and alternative therapy. A rapid detection method and two photo-killing approaches for pathogenic bacteria were proposed in this work. In the first part of this thesis, magnetic Fe3O4/TiO2 core/shell nanoparticles (Fe3O4@TiO2 NPs) consisting of anatase structure titania self-assembled with dopamine molecules was covalently with immunoglobulin G (IgG). The NPs were employed as affinity probes and photokilling agents for pathogenic bacteria such as Staphylococcus aureus and Streptococcus pyogenes, whose surfaces contain the binding proteins of IgG, i.e., protein A and M protein, respectively. Once bacteria were targeted by the NPs, the NP-bacteria were isolated and resuspended in medium solution followed by illumination of UV-B light (λmax~306nm, 0.412 mM/cm2). The results demonstrated that the survival ratio of the bacteria targeted by the NPs under UV light illumination was decreased to <20% within 20 min. An alternative photo-killing approach by using unmodified magnetic Fe3O4/TiO2 core/shell (Fe3O4@TiO2) NPs as affinity probes and photo-killing agents was also proposed in this work. It has been demonstrated that titania shell on the NPs is capable of interacting with lipopolysaccharides (LPS), which contains a number of phosphate functional groups, on the surfaces of Gram-negative bacteria. Thus, Fe3O4@TiO2 NPs are capable of interacting with these bacteria. Similarly, once the NPs targeted the bacteria, the cell growth of the bacteria targeted by the NPs can be effectively inhibited under illumination of UVB light. The results show that the survival ratio of the bacteria targeted by the NPs under UV light illumination within 5 min is decreased to <10 %. In this thesis, a rapid detection methods based on the combination of Fe3O4@TiO2 NPs with proteomics strategies was developed. On the basis of the interactions between the LPS of Gram-negative and the NPs, the bacteria can be effectively concentrated by the NPs within 1.5 min under microwave-heating. The bacteria-NP conjugates were then subjected into a microwave oven for further enzymatic digestion for 1 min. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and MS/MS were then employed for further characterization. Bacteria species can be identified by combining the resultant mass spectra with protein database searches. The potential biomarker ions for Gram-negative bacteria including E. coli O157:H7 、E. coli UTI 、P. aeruginosa 、S. sonnei、K. pneumoniae were discovered through the study. It was also demonstrated that this approach can be used to directly identify trace of bacteria from clinical urine samples (10 μL) within 15 min. The detection limit of this approach for bacteria is as low as several hundred or thousand cells.