功能化奈米粒子之合成與細菌光熱抑制與檢測之研究

Abstract

由於抗生素的濫用，導致抗藥性細菌問題日趨嚴重，因此針對細菌感染疾病發展新的抗生素藥物或是治療方法是科學家們必須面對的議題。而利用在細胞組織中穿透率較高的近紅外光雷射結合具有光熱轉換能力的奈米材料進行光熱抑菌是近年來發展相當迅速的新技術。此外，發展能夠快速檢驗菌種的技術也是相當必要的，藉由迅速獲得感染的細菌種類以對症下藥，同時也可以避免為了治療細菌感染而濫用藥效過強抗生素的情形。 本論文第一部分嘗試利用氧化鋁磁性奈米粒子探針結合近紅外光雷射進行光熱抑菌的研究，由於氧化鋁磁性奈米粒子同時具有辨識細菌表面、吸收近紅外光雷射轉換為熱能以及具有磁性等特性，因此當此探針辨識到細菌後可以利用外加磁場將其聚集，再施以近紅外光雷射照射。經實驗結果顯示，當加入80 μg的探針於菌液(60 μL)中，分別辨識溶液中的泛抗藥性鮑氏不動桿菌、抗萬古黴素腸球菌以及出血性大腸桿菌後，以外加磁場聚集再用波長808 nm的近紅外光雷射照射10分鐘，這些細菌的存活率會各降低至0.8%、3.1%以及0.2%，具有良好的抑菌能力。 論文的第二部分是分別以氧化鋁磁性奈米粒子以及修飾有免疫球蛋白G的金奈米粒子做為偵測辨識金黃色葡萄球菌以及抗甲苯青黴素金黃色葡萄球菌的探針。利用氧化鋁磁性奈米粒子輔助微波加速萃取及酵素消化的技術結合質譜法可以快速的得到來自細菌的胜肽片斷訊號，並且將二次質譜圖送入資料庫中比對可得到更詳細的資訊；此外搭配修飾有免疫球蛋白G的金奈米粒子對金黃色葡萄球菌以及抗甲苯青黴素金黃色葡萄球菌表面作用能力的差異，藉由金奈米粒子探針與細菌溶液混合反應後，再經由離心的步驟觀察溶液顏色以及吸收值強度的改變，可以進行初步的辨識。 論文的最後一個部分則是嘗試以茶葉溶液合成具有近紅外吸收性質的多邊形金奈米粒子，並且可以藉由改變反應中添加的茶葉溶液體積調控近紅外光區吸收峰的吸收波長，由實驗結果証實以綠茶以及紅茶溶液合成的金奈米粒子具有不錯的光熱轉換能力，在波長808 nm的近紅外光雷射距離樣品底部4公分照射6分鐘後，溶液溫度可分別上升17 ℃以及23 ℃，而且合成的金奈米粒子對細胞的毒害低，具有應用於生物體中光熱治療的潛力。

Because of the overuse of antibiotics, the emergence of antibiotic-resistance bacteria becomes a serious problem. Therefore, it is urgent to develop new types of antibiotics and therapeutics for bacterial infections. Lately, a new photothermal-based therapeutics using near infrared (NIR) laser with the capability for penetrating tissue in a deeper depth combined with the use of functional nanomaterials capable of absorbing NIR light has been demonstrated. Additionally, it is also significant to develop a rapid detection method for identifying bacterial strains from bacterial infection samples to prescribe proper antibiotics for medical treatment and to avoid the overuse of potent antibiotics.

In the first part of this dissertation, a photothermal based therapeutics using alumina coated magnetic iron oxide nanoparticles (Fe3O4@Al2O3 MNPs) as affinity probes and photothermal substrate combined with the irradiation of NIR laser was developed. Owing to the features of the capability of recognizing bacteria, absorbing NIR light, and magnetic property, Fe3O4@Al2O3 MNPs (80 μg) were used to probe target bacteria (∼2×105 cells, 60 μL) from sample solutions followed by magnetic aggregation and illumination of NIR light (808 nm) for 10 min. The results showed that the target bacteria including pan-drug resistant Acinetobacter baumannii (PDRAB), vancomycin-resistant Enterococcus faecalis, and Escherichia coli O157 H7 treated using this approach have survival fractions of 0.8%, 3.1%, and 0.2%, respectively.

Next, Fe3O4@Al2O3 MNPs and gold nanoparticles (Au NPs) immobilized with IgG (Au@IgG) were explored as affinity probes for S. aureus and methicillin-resistant S. aureus (MRSA). The results present that the bacteria can be rapidly trapped by the Fe3O4@Al2O3 MNPs followed by tryptic digestion under microwave-heating within a few min. The resultant samples were characterized using matrix-assisted laser desorption/ ionization mass spectrometry (MALDI MS) and MALDI MS/MS. In addition, owing to the affinity of Au@IgG toward S. aureus and MRSA in a different degree, the results imply it may be possible to distinguish S. aureus from MRSA using Au@IgG as the detection probes.

In the last part of this work, a new type of photothermal Au NPs with polygonal shapes and the capacity of absorbing NIR light was explored using tea extractants as reducing agents. The maximum absorption band of the as-prepared Au NPs in the NIR region can be adjusted by optimizing the ratio of the reactants. The results demonstrated that the as-prepared Au NPs have desirable photothermal conversion capabilities. The temperature of sample solutions (0.2 mL) containing the Au NPs generated from green tea and black tea can be elevated to 17 ℃ and 23 ℃, respectively, under illumination of a NIR laser (808 nm) with a irradiation distance of 4 cm for 6 min. The cell biocompability examined using MTT assay also show the toxicity of the as-prepared Au NPs is quite low. The results imply the potential of using the as-prepared Au NPs for photothermal therapeutics.