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研究生:張家銘
研究生(外文):Jia-Ming Chang
論文名稱:雙配體金/銀奈米團簇作為「開-關-開」螢光奈米探針用於靈敏檢測魚精蛋白和胰蛋白酶
論文名稱(外文):Biligand gold/silver nanoclusters as an “on-off-on” fluorescent nanoprobe for sensitive detection of protamine and trypsin
指導教授:邱泰嘉、胡焯淳
指導教授(外文):Cho-Chun Hu,Tai-Chia Chiu
學位類別:碩士
校院名稱:國立臺東大學
系所名稱:應用科學系
學門:自然科學學門
學類:其他自然科學學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:中文
論文頁數:77
中文關鍵詞:奈米團簇魚精蛋白感測器胰蛋白酶胰蛋白酶抑制劑
外文關鍵詞:nanoclustersprotaminesensortrypsintrypsin inhibitor
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  • 被引用被引用:0
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在 本項研究 中 我們利用卵清蛋白( Ovalbumin, OVA 與 6-氮雜 -2-硫胸腺
嘧啶( 6-aza-2-thiothymine, ATT)作為還原劑 和保護劑 來合成 OVA-ATT-AuAgNCs。魚精蛋白 會 與帶負電的 OVA-ATT-AuAgNCs產生 靜電吸引力 使
OVA-ATT-AuAgNCs聚集誘導 放 射增強 AIEE 。添加魚精蛋白後 觀察到電
位從 -27.27 mV增加到 -16.2 mV,並且 可以從 DLS觀察到 平均粒徑變大 。胰蛋
白酶 會 水解魚精蛋白中的精胺酸殘基導致 OVA-ATT-AuAgNCs的 AIEE降低。
螢光感測器可準確檢測「 開 」模型中的魚精蛋白和「 關 」模型中的胰蛋白酶。
在最佳實驗條件下,魚精蛋白在 1-10 μg/mL範圍內實現了最佳 的 線性範圍 。 胰
蛋白酶在 0.01-0.1 μg/mL範圍內 有 最佳 的 線性 關係 。 偵測極限分別為 0.76 μg/mL與 0.0058 μg/mL。 在 真實 血清樣本中,魚精蛋 白 和胰蛋白酶 量測出的 加標回收
率範圍為 在 92.1 %至 102.1 %,證明 OVA-ATT-AuAgNCs是可以應用於真實樣
品中 。此外,也研究胰蛋白酶對其自身活性的 抑制 作用。胰蛋白酶抑制劑 的 半
數最大抑制濃 度 IC50 與其他文獻比較也有較低的數值 。 顯示 此方法可以擴
展到篩選其他蛋白酶抑制劑並了解這些抑制劑的作用機制,這有助於它們作為
消化性 新藥的潛在應用。 此材料也可以製作成水凝膠,並應用於溫度的 測定
當作一種隱形的溫度記錄器。
We synthesized OVA-ATT-AuAgNCs using ovalbumin (OVA) and 6-aza-2-thiothymine (ATT) as a reducing and stabilizing agent. The electrostatic attraction between protamine and negatively charged OVA-ATT-AuAgNCs induced aggregation-induced emission enhancement (AIEE). Upon the addition of protamine, the observed Zeta potential increased from -27.27 mV to -16.2 mV.Trypsin hydrolysis of arginine residues in protamine resulted in a reduction in the aggregation-induced emission enhancement (AIEE) of OVA-ATT-AuAgNCs. The fluorescence sensor accurately detected protamine in the "off-on" model and trypsin in the "on-off" model. Under optimal experimental conditions, protamine exhibited the most effective linear range between 1-10 μg/mL, whereas trypsin demonstrated the best linear range between 0.01-0.1 μg/mL. In real serum samples, the measured recovery rates for protamine and trypsin ranged from 92.1 % to 102.1 %. Additionally, the inhibitory effect of trypsin on its own activity was investigated. The half-maximal inhibitory concentrations (IC50) of trypsin inhibitors and benzamidine were 4.22 μg/mL and 3.11 μg/mL, respectively. This method can be expanded to screen other protease inhibitors and to understand their mechanisms of action, thereby contributing to their potential applications as new drugs. The material can also be formed into hydrogels and utilized as an inconspicuous temperature recorder for temperature sensing applications.
目錄
摘要
iii
Abstract iv
圖目錄
vi
表目錄
ix
第一章
緒論 1
1.1 金屬奈米團簇 1
卵清蛋白(
Ovalbumin, OVA 1
1.2 檢測物介紹 2
1.2.1 魚精蛋白( Protamine 2
1.2.2. 胰蛋白酶( Trypsin 3
1.2.3. 胰蛋白酶抑制劑( Trypsin Inhibitor 4
1.3 文獻回顧 6
1.4 研究動機與目的 13
第二章
材料與方法 15
2.1 化學藥品 15
2.2 儀器與表徵 15
2.3 OVA-ATT-AuAgNCs合成 16
2.4 檢測金屬離子與胺基酸 16
2.5 檢測魚精蛋白 16
2.6 胰蛋白酶的檢測 16
2.7 真實樣品的檢測 17
2.8 胰蛋白酶抑制劑的測量 17
2.9 水凝膠的製作與溫度記錄器的應用 17
第三章
結果與討論 18
3.1 OVA-ATT-AuAgNCs材料基本性質 18
3.1.1 OVA-ATT-AuAgNCs合成 18
3.1.2 OVA-ATT-AuAgNCs表徵 25
3.2 OVA-ATT-AuAgNCs材料之應用 40
3.2.1 OVA-ATT-AuAgNCs用於檢測魚精蛋白 40
3.2.2 使用 OVA-ATT-AuAgNCs檢測胰蛋白酶 44
3.3.3 胰蛋白酶抑制劑的檢測 51
3.2.4 OVA-ATT-AuAgNCs作為隱形溫度計的應用 55
3.3 魚精蛋白與胰蛋白酶的檢測機制 61
第四章
結論 64
第五章
參考文獻 65
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