奈米粒子結合高分子聚合反應輔助致病菌檢測的研究

Abstract

高分子聚合反應或高分子聚合所聚合成的材料，在過去己被廣泛地應用在分析科學研究上。而在眾多被提出的聚合方法中，以原子轉移自由基聚合 ( ATRP ) 反應最常被學者所使用。而奈米粒子由於具有易於合成，比表面積大，易於修飾等優異的特性，因此常常被當作為製作探針之重要基材。基於以上的特點，本論文中提出以氧化鐵奈米粒子和二氧化矽奈米粒子為基材的致病菌偵測探針來對病菌進行偵測，實驗中以腐生葡萄球菌和尿道感染桿菌作為實驗之樣品菌種。

腐生葡萄球菌是可和IgG的Fc區辨認之細菌，而引發尿道感染之大腸桿菌則無法和IgG的Fc區有任何辨識作用。本論文中的氧化鐵奈米粒子是表面修飾有能辨識致病菌的人類免疫球蛋白G的辨識探針，而二氧化矽奈米粒子則是表面同時修飾有人類免疫球蛋白G和能引發原子轉移自由基聚合的起始劑的親和探針，因此當樣品中存在能被人類免疫球蛋白G辨識的細菌時，兩奈米粒子探針便會和致病菌辨識而形成可以看成為一個整體粒子團 (包含兩種奈米粒子和細菌)之團聚物，而經磁性分離收集後，對於辨識腐生葡萄球菌後的奈米粒子其整體體積會明顯的增大；而對於尿道感染桿菌其體積則沒有任何改變。因此可利用此簡單的方法來偵測水溶液和尿液中是否有目標菌種的存在，之後再進一步利用MALDI-MS和原子轉移自由基聚合反應來對肉眼判別的結果進行確認。

結果證實在本論文中所設計的方法確實可用來辨識腐生葡萄球菌，在緩衝液中肉眼觀測之偵測極限分別為105 cfu/mL，而經由MALDI-MS和原子轉移自由基聚合反應之輔助則可把偵測極限再降至104 cfu/mL。而在二氧化矽奈米粒子表面沒有修飾起始劑的情況下，同樣以此實驗設計對目標菌種進行偵測時，在緩衝液和尿液中肉眼對腐生葡萄球菌的偵測極限分別為105 cfu/mL和107 cfu/mL；而利用MALDI-MS進行分析時在緩衝樣品液和尿液樣品中對腐生葡萄球菌的偵測極限則分別為104 cfu/mL和106 cfu/mL。

Polymerization has been widely applied in the development of analytical methods. Owing to the ease of synthesis, high specific surface area, and ease of modification, nanoparticles have been used as functional probes for interacting target species. Nanoparticles such as silica beads can be generated from polymerization, i.e., Stöber’s method. In this thesis, two-steps of functionalized nanoparticle-based affinity approach for the detection of pathogenic bacteria was proposed. Staphylococcus saprophyticus and Escherichia coli were used as the model bacteria.

It has been known that Fc site of immunoglobulin G (IgG) is capable of interacting with pathogenic bacteria such as S. saprophyticus through the binding proteins on the cell wall. However, there is no binding interaction between the Fc site with E. coli. In this study, two types of affinity probes bound with IgG were generated: magnetic iron oxide nanoparticles generated from co-precipitation and silica nanoparticles generated from polymerization. Magnetic iron oxide nanoparticles were first used to target S. saprophyticus and E. coli followed by magnetic isolation. The aggregated NPs were then interacted with the IgG-bound silica beads (ca. 30 nm). The results show that when the magnetic isolated conjugates contain Fc binding-bacteria, i.e. S. saprophyticus, the volume of the conjugates can be enlarged by the IgG-silica nanoparticles and lead it is possible to be visible by the naked eye. However, when using E. coli as the sample, the volume of the conjugates remains similar to that of the original magnetic iron oxide nanoparticles. The results indicate that this approach can be used to detect the presence of target bacteria from aqueous and urine samples simply by using this two-step functionalized nanoparticle-based affinity approach.

MALDI-MS and further polymerization by ATRP (atom transfer radical polymerization) were also employed for confirmation. The detection limits of this approach for S. saprophyticus in aqueous and urine samples (90 μL) are were as low as 104 cfu/mL and 106 cfu/mL, respectively.