金奈米粒子親和性探針及抑菌試劑之開發

Abstract

金黃色葡萄球菌是一種常見的致病菌，會造成食物中毒、中毒性休克綜合症以及紅斑疹等等。傳統偵測細菌的方法至少需要數個小時才能完成細菌的分析辨識，且所需的試劑如抗體等等通常相當昂貴。除此之外，由於抗生素的濫用已造成細菌突變成具有抗藥性的細菌，使得細菌感染的治療更加困難。在本論文研究中，我們直接使用具有辨識功能性的探針分子做為還原劑，藉由一鍋反應合成出具有功能性的金奈米粒子。利用此合成出的金奈米粒子為親合探針藉此發展快速的細菌偵測方法以及抑菌療法。在本論文的第一部分，我們使用了對金黃色葡萄球菌具選擇性的胜肽片段為還原劑，合成出表面包覆有此功能胜肽片段的金奈米粒子。利用此被胜肽修飾之金奈米粒子為金黃色葡萄球菌的辨識探針，藉由金奈米粒子的定域化表面電漿共振特性，發展出對金黃色葡萄球菌具感測能力的分析方法。即以可見光-紫外光吸收光譜儀做為偵測器，基於金奈米粒子在吸收光譜上吸收峰的位移，發展出可偵測金黃色葡萄球菌的快速分析方法，此方法只需約十五分鐘即可完成分析。而本論文的第二部分，則是發展出以具多角形的功能性金奈米粒子結合光熱效應做為抑制致病菌如抗萬古黴素糞腸球菌的方法。多角形金奈米粒子是利用萬古黴素為還原劑，以一鍋方法合成出表面修飾有萬古黴素的多角形金奈米粒子。實驗結果顯示包覆在金奈米上的萬古黴素仍然保有它的抗菌活性，而萬古黴素則佔此功能金奈米粒子總重量比例的四十七左右，在加有功能金奈米粒子(9.77 g/mL)的抗萬古黴素糞腸球菌細菌樣品中，細菌幾乎能被百分之百抑制。然而，當在相同樣品投以相似量的萬古黴素時，抑菌量只有百分之十三左右。此外，此功能性金奈米粒子在近紅外光區段具有吸收波峰以及光熱效能。因此，當使用近紅外光雷射照射含此功能性金奈米粒子(44 g/mL)的小體積(0.1 mL)樣品五分鐘，樣品溶液溫度可快速提升約攝氏十五度。當結合此功能性金奈米粒子的抗菌活性以及光熱特性於抑菌實驗時，在同樣的抑菌效果下，此功能性金奈米粒子上的萬古黴素劑量比單用萬古黴素的劑量低了約九倍左右。此外，實驗結果也顯示此功能性金奈米粒子的細胞毒性相當低。雖然本研究所發展的方法已證實對抗藥性腸球菌具有很好的抑制效果，且所需萬古黴素劑量比一般用量還低甚多。然而，進一步的活體實驗是有必要的，才能進一步判斷此抑菌療法的可用性。

Pathogenic bacteria can contaminate food and cause illness. Staphylococcus aureus, which can cause food poisoning, toxic shock syndrome, and erythematous rash, is one of the common pathogenic bacteria. Traditional analytical methods require at least several hours in characterization of target bacteria, whereas the required reagents such as antibodies are usually expensive. In addition, extensive use of antibiotics has led the emergence of antibiotic-resistant bacterial strains. In this dissertation, functional gold nanoparticles (Au NPs) were generated through facile one-pot reactions, and their functional probes were directly used as reducing and capping agents. The generated functional Au NPs were aimed to be used as affinity probes for development of rapid analytical methods and effective antibacterial therapy. In the first part of this dissertation, functional Au NPs were generated and capped by a peptide with the sequence of DVFLGDVFLGDEC (DD), which possesses selective affinity toward S. aureus. The generated AuNPs@DD were used as sensing probes for S. aureus in localized surface plasmon resonance (LSPR) analysis. Ultraviolet-visible absorption spectroscopy was used as the detection tool. On the basis of the LSPR shift in the absorption band derived from AuNPs@DD, a rapid sensing method for S. aureus was explored. It only took ~15 min to conduct one analysis. In the second part of this work, a polygonal AuNP-based photothermal approach for killing pathogenic bacteria such as vancomycin-resistant Enterococcus faecalis (VRE) was also developed. Polygonal Au NPs immobilized with vancomycin (AuNPs@Van) were generated through a one-pot synthesis method. The results demonstrated that vancomycin on AuNPs@Van still retained its antibiotic activity. The generated AuNPs@Van were consisted of ~47% (w/w) of vancomycin. We demonstrated that VRE in a bacterial sample containing AuNPs@Van (9.77 g/mL) can be used to inhibit ~100% of the growth of VRE, while a similar amount of free-form vancomycin can only be used to inhibit ~13% of bacterial growth. Moreover, the polygonal AuNPs@Van possess an absorption band in the NIR region and photothermal capability. Under irradiation of a near infrared (NIR) laser for ~5 min, the temperature of a low volume of sample (0.1 mL) containing AuNPs@Van (44 g/mL) can be rapidly raised for ~15 oC. When combining the antibacterial property of AuNPs@Van with the photothermal approach, the dosage of vancomycin in AuNPs@Van was 9–fold lower than that when free-form vancomycin was used as the antibacterial agent for obtaining similar antibacterial results. In addition, the cell toxicity of the generated AuNPs@Van was quite low. Although we have demonstrated the effectiveness of this approach toward VRE and the effective dosage of vancomycin on AuNPs@Van is much lower than that of free-form vancomycin, in vivo test is required to further clarify the usefulness of this approach.