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研究生:黃弘均
研究生(外文):Hung Chun Huang
論文名稱:新型尼古丁類農藥快速檢驗試劑
論文名稱(外文):Neonicotinoid Insecticides Rapid Test
指導教授:李文仁李文仁引用關係蘇昭瑾
口試委員:姚清發余琬琴
口試日期:2016-07-22
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:有機高分子研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
畢業學年度:104
語文別:中文
中文關鍵詞:膠體金側流試驗亞滅培生化法新型尼古丁類農藥
外文關鍵詞:Colloidal goldLateral flow testAcetamipridBiochemical methodNeonicotinoid insecticide
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目前我國對於農藥的檢驗方式大致可分為兩種,包含了生化法及化學法。化學法具有準確度高、偵測藥物種類多的好處,但檢驗成本較高、花費時間較久而不符合現行生鮮蔬果檢驗的時效性;生化法部份,目前以農試所推廣的乙醯膽鹼酯酶酵素法為主,能對有機磷及氨基甲酸鹽類兩大類農藥進行快速檢驗,其他種類農藥則暫無符合市場性的快速檢驗方式。因此發展出其它種類農藥的快速檢測平台,將可對我國食品安全控管有實質性的助益。
本研究透過對新型尼古丁類農藥結構的分析,找出共同的結構單元,接著以其中的一種農藥:亞滅培作為結構設計標的,進一步設計出一系列的半抗原結構。搭配載體蛋白的接合,成功製作出抗原及免疫原材料。透過動物實驗進行血清感度測試,找出符合的抗體原料,伴隨現今已發展完善的側流試驗方法,並利用抗原抗體間高度特異性的辨識能力,甚至於搭配膠體金明顯的呈色,逐步的發展出一快速檢測新型尼古丁類農藥平台,將推廣給各大通路及食品廠,達到初級篩驗管控,使未來農藥殘留把關能更趨完善。
At present, the test methods can be broadly divided into two types in detecting pesticides, including biochemical method and chemical method. The advantages of chemical method are high accuracy and broader drug detecting, but it has expensive cost and waste more time to put into practice for inspection of fresh fruits and vegetables without complying with the current timeliness. At about biochemical method, the Taiwan Agricultural Research Institute have promoted acetylcholinesterase enzyme -based method so far, and it can detect the groups of Organophosphate and Carbamate rapidly since no other types of pesticides are in line with the market of rapid test methods. Therefore, the development of rapid testing platform for other types of pesticides will have substantial benefit to our food security control.
In this study, through the analysis of the structure of the neonicotinoid pesticides and find out the similar structure unit, then take one of the groups called Acetamiprid as the subject of structure design which create a series of hapten structures further. With the binding of carrier proteins, we make the antigen and immunogen materials in success. In order to collect the adequate antibody materials, we have serum sensitivity tests by using animal experiments, with the method of well developed lateral flow test and the high specificity between antibody and antigen even the clear color of colloidal golds, we develop a rapid detection of neonicotinoid pesticide platform gradually.For the purpose of primary screening test by promote and access the kit to major plants and channels, it will make the pesticides check more perfect in the future.
摘 要 i
Abstract ii
誌 謝 iv
目 錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第二章 文獻探討 3
2.1 農藥簡介 3
2.2 化學農藥 3
2.2.1 殺蟲劑 3
2.2.1.1 氨基甲酸鹽類 3
2.2.1.2 有機磷劑 4
2.2.1.3 有機氯劑 4
2.2.1.4 合成除蟲菊精類 4
2.2.1.5 新尼古丁類 4
2.2.1.6 昆蟲生長調節劑 4
2.2.1.7 抗生素類 5
2.2.1.8 能量代謝抑制劑 5
2.2.2 殺真菌劑 5
2.2.3 殺細菌劑 6
2.2.4 除草劑 6
2.2.5 殺鼠劑 9
2.2.6 植物生長調節劑 9
2.3 新尼古丁類農藥 9
2.3.1 發展歷史 10
2.3.2 結構探討 10
2.3.3 作用機制 11
2.3.4 市場資訊 12
2.3.4.1 台灣市場殘留情形 12
2.3.4.2 相關免疫檢驗試劑的應用 13
2.3.5 亞滅培殘留規範 15
2.3.6 新型尼古丁類農藥相關爭議 15
2.4 小分子藥物半抗原 17
2.4.1 半抗原設計 17
2.4.2 半抗原修飾製備 17
2.4.3 聯結結構的選擇 18
2.4.4 載體蛋白的選擇 18
2.4.5 半抗原與載體蛋白的結合方法 19
2.5 側流系統技術平台 21
2.5.1 平台優缺點 21
2.5.2 平台元件組成 21
2.5.3 側流試驗的不同形式 24
2.5.3.1 競爭法型試驗 24
2.5.3.2 三明治型試驗 25
2.5.4 側流試驗的不同應用 26
2.5.5 側流試驗的市場與分布 28
第三章 實驗 29
3.1 實驗材料 29
3.1.1 實驗藥品 29
3.1.2 實驗動物 30
3.1.3 輔助實驗材料 31
3.1.4 實驗儀器 31
3.2 實驗流程與合成方法 32
3.2.1 新型尼古丁類農藥結構分析 33
3.2.2 半抗原及抗原設計與合成 36
3.2.2.1 第一型抗原設計 36
3.2.2.2 第二型抗原設計 37
3.2.2.3 第三型抗原設計 39
3.2.3 半抗原與抗原鑑定 40
3.3 動物實驗測試 40
3.3.1 免疫試驗及血清收集 40
3.3.2 血清試驗 41
3.3.2.1 抗原盤鍍製 41
3.3.2.2 血清篩選 42
3.3.2.3 靈敏度測試 43
3.4 抗體原料純化與收集 44
3.4.1抗體純化 44
3.4.2 抗體濃縮與定量 45
3.5 平台製作 45
3.5.1結合墊片製作 46
3.5.1.1膠體金製作 46
3.5.1.2 抗體與膠體金結合反應 47
3.5.1.3 結合墊片的配方挑選 48
3.5.2 NC膜元件的製作 48
3.5.3 樣品墊片的製作 49
3.5.4 吸水層墊片的製作 49
3.5.5 各元件組裝 49
3.5.6 平台與樣品測試 49
3.5.7 平台安定性追蹤 50
第四章 結果與討論 51
4.1 第一型半抗原及抗原合成反應 51
4.1.1 反應第一步Scheme 51
4.1.2 反應第二步Scheme 52
4.1.3 反應第三步Scheme 53
4.2 第二型半抗原及抗原合成反應 55
4.2.1 反應第一步Scheme 55
4.2.2 第二型半抗原反應檢討 57
4.3 第三型半抗原及抗原合成反應 57
4.3.1 反應第一步Scheme 57
4.3.2 第三型半抗原反應檢討 60
4.4 動物實驗結果追蹤 60
4.4.1 初期免疫試驗反應 61
4.4.2 第一次強化免疫試驗 65
4.4.3 第二次強化免疫試驗 67
4.4.4 第三次強化免疫試驗 68
4.4.5 不同藥物的靈敏度試驗 70
第五章 結論 72
第六章 未來展望與建議 73
參考文獻 74
附錄A 光譜圖收錄 78
附錄B新尼古丁類農藥容許殘留限量標準 82
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