探討T3噬菌體與宿主菌在不同水溶液樣本中的交互作用

Abstract

近年來隨著公共衛生的意識提升，各種食源性病原菌檢測方法推陳出新以滿足人們對於食品安全的需求。對於檢測方法的追求無外乎靈敏度、選擇性、方便性以及低成本，因此許多新興方法會在這些項目上嘗試與傳統方法做出區別，並且期望使檢測不再侷限於實驗室而是普及到一般大眾。在各式新興方法中，電化學生物感測器作為一個普遍被認為快速、靈敏、低成本且易於操作的方法受到了研究者的青睞，被認為在這個領域具有很大的潛力。 一般而言，食源性病原菌的電化學檢測可以偵測到包含病原菌的溶液中與目標菌相關的電流、阻抗等變化，藉此確認病原菌的存在。許多種可以辨認病原菌的生物元件被應用到電化學生物感測器上，其中噬菌體這類病毒因為其天生對細菌的感染特性，在電化學檢測方法上具有得天獨厚的優勢而受到了關注。基於噬菌體的電化學生物感測器橫跨了微生物學、電化學以及材料科學，許多研究偏向導電介面的優化、材料的開發，而較少討論噬菌體在溶液中與宿主菌的交互作用，目前為止幾乎無法在文獻中找到未經電極修飾以及噬菌體附著步驟的方法直接檢測噬菌體感染/裂解宿主菌的研究。因此本篇論文透過從噬菌體的製備到電化學方法（循環伏安法）直接檢測噬菌體與宿主交互作用的嘗試，希望從不同的角度為噬菌體電化學感測器奠定基礎。 這篇論文可以分為噬菌體的製備以及不同水溶液中電化學檢測兩大部分。噬菌體製備中培養了四種噬菌體，最終選擇T3噬菌體作為水溶液樣本中電化學的討論對象；電化學檢測利用循環伏安法來觀察磷酸鹽緩衝液、蘋果汁與綠茶作為樣本討論噬菌體與病原菌在不同的基質下的交互作用的反應電流。此外，還對可能影響到電化學檢測的感染複數、檢測溫度進行了討論。 結果顯示在磷酸鹽緩衝液中能夠看到與細菌濃度（log CFU/mL）呈現負相關的電流反應，檢測效果最符合預期。但是在蘋果汁中的電流反應卻同時與細菌濃度一起上升，得到了與磷酸鹽緩衝液樣本結果相反的線性關係，這跟普遍認為細菌在水溶液中呈現的絕緣性質相反。而綠茶中則觀察到反應電流不會隨著時間有顯著變化，分光光度計的檢測結果也同樣顯示噬菌體在綠茶中不會進入裂解週期，在食品溶液中進行的電化學檢測都有強烈的基質效應。簡而言之，研究結果顯示在不進行電極修飾、訊號放大的情況下，直接使用電化學方法來進行基於噬菌體感染/裂解宿主菌的檢測較不可行。基於噬菌體的電化學檢測方法將噬菌體作為裂解性噬菌體來感測目標菌種，仍需要考量噬菌體在不同基質下的裂解性質的不同，還有電化學方法的優化。

Recently, with the raising awareness of public health, various new detection methods for foodborne pathogens have been introduced to meet the requirement of public health and food safety. People always looked for sensitive, selective, easy-operating, and cost-effective detection methods that differed from traditional methods. Among these emerging methods, an electrochemical biosensor is a potential approach for its numerous advantages. Generally, the electrochemical assay can detect changes in physical properties such as current and impedance related to the pathogens in the solution, thereby confirming the presence of the target bacteria. Various biological elements that can identify pathogenic bacteria have been applied to electrochemical detection. The bacteriophage is a popular biological element due to its infection specificity to bacteria and lysis properties. However, the excellent coordination of several research areas in microbiology, electrochemistry, and materials science ensures the successful development of bacteriophage-based biosensors. Interestingly, although many studies of phage-based electrochemical biosensors have been published, most were applying bacteriophages on electrodes through either covalent or non-covalent bonds for bacterial detection and did not discuss the effect of food matrix on bacteriophages alone. Therefore, this study aims to explore the interactions between phages and host bacteria in different aqueous samples without phage pre-treatment or immobilization. The study contains bacteriophage preparation and analyses of phage-bacteria interactions using cyclic voltammetry, hoping to lay a foundation for developing phage-based electrochemical biosensors. In phage preparation part, four kinds of bacteriophages were cultivated for phage preparation, and T3 phage was finally selected to study phage-bacteria interactions in aqueous samples. The phage-bacteria interactions were first studied by cyclic voltammetry (CV) in three different matrices (PBS buffer, apple juice, and green tea). Other methods, such as growth curve, bacterial lysis patterns, and multiplicity of infection (MOI), were also incorporated to add to the study of phage-bacteria interactions in different matrices. In electrochemical part, the interactions of phage-bacteria in PBS buffer were as expected, the current response of CV demonstrated negative linearity with the bacterial concentration (log CFU/mL). However, the results obtained from the apple juice test showed an opposite linear relationship between the current response and bacterial cell number, which contradicted the generally accepted insulating properties of bacteria in aqueous solutions. In the green tea test, it was observed that the current response of CV did not change significantly along with the increase of bacterial cell number, which the optical density test also demonstrates low phage-induced bacterial lysis in green tea samples. Accordingly, strong matrix effects were observed for phage-bacteria interactions, which greatly hinder the use of free-phage to detect target bacteria. These results indicated that without the modification of phage to the electrode and the amplification of electrical response, the free phage is hard to identify or quantify the target bacteria in aqueous samples. Thereby the lytic properties of phages in different matrices and the optimization of electrochemical methods have to be thoroughly investigated before employing bacteriophage as a virulent phage to sense the target species.