本實驗以溶膠凝膠法 (sol-gel) 之合成方式，摻雜對環境酸鹼值敏感之螢光染劑，螢光異硫氰酸鹽 (fluorescein isothiocyanate, FITC) 製成螢光中孔洞二氧化矽奈米粒子 (pH-sensitive fluorescent dye-doped mesoporous silica nanoparticles, FSNPs)。此中孔洞奈米材料具有高表面積的優勢，可作為良好之藥物載體；本實驗利用β-內醯胺類抗生素，penicillin G作為裝載藥物，當penicillin G被β-內醯胺酶水解為青黴噻唑酸 (penicilloic acid) 使環境之酸性提升，導致螢光中孔洞二氧化矽奈米粒子上的螢光異硫氰酸鹽，在酸性條件下形成不具發光特性之酯類結構而產生螢光猝滅現象 (fluorescence quenching)，並以此方式作為篩檢出抗藥性細菌存在之方法。此方法實際檢測25種臨床樣品最快可於一小時內得到檢測結果，且β-內醯胺酶之活性偵測極限最低可達7.8×10-4 U/mL，展現出此檢測方法應有潛力作為臨床診斷 (point-of care) 的工具，提供醫療人員抗生素藥物之選用參考，並預防抗生素藥物之濫用。

The major mechanism for antibiotic-resistant bacteria is the production of β-lactamases, which can catalyze the hydrolysis of β-Lactam antibiotics into penicilloic acid by breaking down the β-lactam ring, resulting in loss of the antibacterial activity of drug. Thus far, kinds of methods have been applied to detect β-Lactamases activity. But above all, the disadvantages of these techniques are labor intensive, time-consuming manipulation or requiring complex equipment and skilled worker. However, limit their use in clinical applications. As a result, an easy operating assay method, economic, reliable system for detecting β-lactamases can meet clinical testing requirements to achieve early screen and sensitive detection is highly required.

Here we present a simple, rapid, sensitive, universal, inexpensive screening method for the determination of β-lactamases activity of antibiotic-resistant bacteria, by designing a pH-sensitive fluorescent dye-doped mesoporous silica nanoparticles (FSNPs) encapsulated with a β-lactam antibiotic (penicillin G) as a substrate. Hydrolyzing of penicillin G can be catalyzed by β-lactamases to produce penicilloic acid, leading to a pH decrease in the microenvironment of FSNPs, which caused the pH-sensitive green fluorescent dye (fluorescein isothiocyanate, FITC) turned to a non-fluorescent product at the pH around 5.5. Therefore, we can assess the activity of β-lactamase by determining the quenching of fluorescence intensity at a given time. Currently, the most common method used in clinical of antimicrobial susceptibility testing requires 24-72 hours. Compare to the traditional method, our rapid, sensitive optical method can detect a broad spectrum of β-lactamases of clinically relevant samples at less than 1 hour. Moreover, the detection limit of β-lactamase activity was 7.8×10-4 U/mL, which was determined within 8 hours.

目錄
壹、緒論 1
1、前言 1
2、測定β-內醯胺酶之活性方法 1
2.1、抗菌敏感性測試 4
2.2、碘測定法 9
2.3、酸性測定法 9
2.4、β-內醯胺類酶底物顯色基團測定法 10
2.5、β-內醯胺類酶底物修飾螢光基團之螢光測定法 11
2.6、電化學生物感測器測定法 14
2.7、聚合酶連鎖反應測定法 15
2.8、質譜檢測法 16
2.9、金奈米粒子測定法 17
2.10、DNA適體與氧化石墨烯測定法 18
2.11、有機染劑/金屬離子錯合物偵測法 20
3、奈米材料簡介與發展 21
4、二氧化矽奈米材料的特性與應用 21
4.1、二氧化矽奈米材料之製備 22
4.1.1、水解反應 (hydrolysis) 22
4.1.2、縮合反應 (condensation) 23
4.1.3、聚合反應 (polymerization) 23
4.2、中孔洞二氧化矽奈米材料之簡介 24
4.3、界面活性劑之簡介 25
5、抗生素之抑菌機制 27
6、細菌之抗藥性 30
7、抗藥性細菌之介紹 32
7.1、鮑氏不動桿菌 33
7.2、克雷伯氏肺炎菌 33
7.3、大腸桿菌 34
8、研究動機 36
貳、研究內容 37
1、藥品與儀器 37
1.1、藥品 37
1.2、儀器 38
2、實驗方法 39
3、實驗步驟 40
3.1、螢光中孔洞二氧化矽奈米粒子之合成步驟 40
3.2、penicillin G之藥物裝載 42
3.3、螢光中孔洞二氧化矽奈米粒子之量子產率計算 43
3.4、螢光中孔洞二氧化矽奈米粒子之藥物裝載量計算 43
3.5、細菌樣品之製備 44
3.6、以螢光中孔洞二氧化矽奈米粒子檢測臨床細菌樣品 44
3.7、試片擴散法 (Disk diffusion test) 45
3.8、以螢光中孔洞二氧化矽奈米粒子測定β-內醯胺酶之活性 45
3.9、以酸性測定法檢測β-內醯胺酶 45
3.10、以β-內醯胺酶水解penicillin G 46
參、結果與討論 47
1、螢光中孔洞二氧化矽奈米粒子之合成 47
1.1、螢光染劑修飾量優化 47
1.2、螢光染劑修飾方法優化 48
2、螢光中孔洞二氧化矽奈米粒子之發光特性 51
3、以β-內醯胺酶水解penicillin G 53
4、螢光中孔洞二氧化矽奈米粒子之官能基鑑定 56
5、螢光中孔洞二氧化矽奈米粒子之SEM與TEM圖 58
6、以PEN G@FSNPs檢測β-lactamase 60
6.1、penicillin G之濃度選擇 61
6.2、螢光中孔洞二氧矽奈米粒子之penicillin G裝載量測定 62
6.3、決定PEN G@FSNPs檢測β-lactamase方法之用量 63
6.4、比較以藥物載體(PEN G@FSNPs, particle)與非藥物載體(non-particle)方式檢測β-lactamase方法之效果 66
6.5、比較以超音波震盪混合方式對PEN G@FSNPs檢測β-lactamase方法之影響 68
7、以Pen G@FSNPs檢測臨床細菌樣品 70
8、以螢光預測方法快速檢測β-lactamase 77
8.1、螢光預測方法之建立 77
9、比較螢光預測法與傳統酸性測定法檢測β-內醯胺酶之效果 81
10、比較本篇研究之檢測方法與其它相關研究之偵測極限 83
肆、結論 85
伍、參考文獻 87
陸、附錄 97

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