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研究生: 許敦淯
Tun-Yu Hsu
論文名稱: 微生物生物光電感測晶片之開發及化粧品檢測之應用
Development of bio-photoelectric sensing chips for microorganisms and applications in cosmetic testing
指導教授: 陳秀美
Hsiu-Mei Chen
口試委員: 陳秀美
Hsiu-Mei Chen
戴守谷
Shou-Ku Tai
賴惠敏
Hui-Min Lai
廖國宸
Kuo-Chen Liao
葉旻鑫
Min-Hsin Yeh
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 182
中文關鍵詞: 生物光電感測晶片化粧品檢測大腸桿菌綠膿桿菌金黃色葡萄球菌白色念珠菌
外文關鍵詞: Bio-photonic Sensor Chip, Cosmetic testing, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans
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紫膜 (purple membrane, PM)為Halobacterium salinarum細胞膜成分之一,其內部具有細菌視紫質 ( bacteriorhodopsin, BR) 。BR具有高穩定性也是一個光驅動質子泵浦,透過BR光驅動質子泵產生可被偵測的電流訊號,可以作為生物光電傳感器的信號轉換器。
先前已經利用單層紫膜晶片開發出可檢測微生物的紫膜光電感應晶片,具有高檢測靈敏度還有快速檢測的特性。傳統檢測化粧品方法大都具有操作步驟繁瑣、檢測時間長、靈敏度低等缺點。國內外的化粧品規範中,大腸桿菌、綠膿桿菌、金黃色葡萄球菌、白色念珠菌此四株菌被列為不得檢出的菌。本文即針對這四株不得檢出菌開發出高特異性的各種抗體晶片,並進行針對單一菌種、混合菌液、化粧品樣品之分析。結果顯示,所開發出的四種抗體-PM生物光電感測晶片能各個別檢出其目標菌株,最低檢測濃度均為 1 Colony-forming unit (CFU)/mL,且各種抗體檢測晶片也都具有高專一性在混和菌液中可明顯分別目標菌。
對於不同劑型的化粧品(液劑、乳劑、霜劑)的檢體時,四種抗體-PM生物光電感測晶片檢測結果都與平板計數法得出的結果是一致的。化粧品防腐效能的測試中也顯示晶片的檢測結果是符合測試指引的。
最後利用結合奈米金與適體的複合粒子再去標定被抗體晶片所捕捉的菌株,結果發現可以使晶片的檢測靈敏度更加提升,使其最低檢測濃度可低達1 CFU/100 mL。
本研究所開發的四種微生物感測晶片具有直接且快速檢測、高靈敏度的優點,未來可以應用於食品、水質的檢測。


Purple membrane (PM) resides in the cellular membrane of Halobacterium salinarum and contains bacteriorhodopsin (BR). BR has high stability and functions as a light-driven proton pump, which drives photocurrent production, and hence can be applied as the signal transducer of a photoelectric biosensor. In our previous studies, a PM monolayer-coated electrode was devised and applied to detect microorganisms, with the advantages of high sensitivity and rapid detection. For cosmetics, plate counting is the traditional gold-standard way to detect microorganisms, suffering the disadvantages of complex and time-consuming procedures, low sensitivity, and requirement of professional operators. According to domestic and international cosmetic regulations, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans are considered must-not-be-detectable bacteria. In this thesis, four highly sensitive and specific PM-based microbial biosensors are developed for the detection of these four strains respectively, in single-strain, mixed-strain, and cosmetic samples. The results show that each biosensor detects its target strain with a limit of detection of 1 CFU/mL. All four biosensors have high specificity with the capability to distinctly discriminate their own target strain from the other three strains in a mixed solution. The results of microbial tests in liquid, emulsion, and cream forms of cosmetics using biosensors are consistent with those using the plate-count method. The test on the antiseptic efficacy of cosmetics demonstrates the applicability of those four biosensors. Finally, using gold-nanoparticle-labeled aptamers with sequences specific to the target strain, the sensitivity of each biosensor is further enhanced with a limit of detection of 1 CFU/100 mL. This research demonstrates the feasibility of the applicability of PM-based microbial biosensors in microbial detection of cosmetics.

中文摘要 I Abstract II 致謝 III 目錄 IV 表目錄 VI 圖目錄 XVII 附錄目錄 XXV 第一章 緒論 1 第二章 文獻回顧 2 2-1現代化粧品產業 2 2-1-1 化粧品產業現況 2 2-1-2 國內化粧品產業的規範 3 2-1-3 化粧品防腐效能試驗指引 4 2-2 四種不得檢出菌株的檢測方法 5 2-2-1平板計數法 5 2-2-2橫向流動分析 6 2-2-3免疫吸附測定 (ELISA) 7 2-2-4聚合酶鏈反應(PCR) 8 2-2-5環介導等溫擴增技術 ( LAMP ) 9 2-2-6生物傳感器 9 2-2-7我國化粧品法理規定之四種不得檢出菌株的檢測方法優缺點比較統整 12 2-3細菌視紫質 ( bacteriorhodopsin, BR ) 13 2-3-1 H. salinarum與 BR簡介 13 2-3-2 BR結構敘述 14 2-3-3 BR光循環機制 16 2-4 核酸適體(aptamer)介紹 17 2-4-1核酸適體應用於微生物檢測 17 2-4-2 AuNPs-aptamer複合粒子製備方法 20 2-5 紫膜晶片檢測法與平板計數檢測法的優劣勢比較 21 第三章 實驗 22 3-1 實驗目的 22 3-2 實驗流程 23 3-3 實驗藥品 29 3-4 實驗設備 31 3-5 量測系統 33 3-5-1光電訊號檢測系統 33 第四章 實驗結果與討論 34 4-1 使用平板計數法量測四種菌株的菌濃度與OD600nm的檢量線 34 4-2 四種抗體-PM複合晶片的製程探討 36 4-3 以四種抗體-PM複合晶片檢測四種菌株 38 4-4 使用AuNPs-aptamer複合粒子提升紫膜晶片的檢測靈敏度 50 4-4-1 選定目標為四種菌株的核酸適體 50 4-4-2 使用四種抗體-PM晶片並添加AuNPs-aptamer複合粒子檢測四種菌株檢測成果 51 4-5 使用ANOVA分析四種抗體-PM生物光電感測晶片的檢測專一性 57 4-6混合菌株檢測實驗 82 4-6-1 四種抗體-PM晶片檢測混合四種菌株的檢測成果 83 4-6-2 使用Matlab軟體探討混合菌株的權重參數(Wij) 89 第五章 化粧品測試 99 5-1 同步以紫膜晶片與平板計數法檢測乳劑、霜劑、液劑的殺菌效能 99 5-2 同步以紫膜晶片與平板計數法檢測樣品中4種不得檢出菌株實驗 112 5-3 同步以紫膜晶片與平板計數法做產品防腐效能的檢測 118 5-4四種抗體晶片檢測化粧品時零檢出的分界線 129 第六章 結論與建議 140 第七章 參考文獻 141 第八章 附錄 147

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