臺灣博碩士論文加值系統

(一) 利用聚乙烯吡咯烷酮包覆普魯士藍奈米材料作為消光材料用以
偵測含磷酸根之生物化學物螢光感測器
普魯士藍為一在十八世紀時發現的配位聚合物，並被做為染料使用。在我們開發的螢光檢測系統中，我們利用利用聚乙烯吡咯烷酮包覆普魯士藍奈米材料作為良好的消光材料，並使用BODIPY-ATP 做為檢測系統中的螢光染料。普魯士藍奈米材料與BODIPY-ATP 兩者鍵結產生消光，藉由添加含磷酸根之生物化學物質與BODIPY-ATP 競爭，便能偵測含磷酸根之生物化學物質，如：三磷酸腺苷(ATP)經鹼性磷酸酶(ALP)水解後產生之磷酸根(Pi)或是焦磷酸根(PPi)，也可以透過此種方式進行鹼性磷酸酶之偵測。在焦磷酸根檢測部分，我們設計之檢測系統的線性區間為0.08 至1 μM；在鹼性磷酸酶檢測部分，我們所設計之系統的線性區間為0.01 至1 U/L，而後我們進行了酵素結合免疫吸附分析法，利用IgG 抗體綁上鹼性磷酸酶的方式檢測IgG抗原，所得到偵測線性區間為0.05 到1 ng/mL。

(二)以第六型溶菌酶穩定結構之金奈米簇作為螢光探測器來偵測鹼
性磷酸酶
金奈米簇是由數個至數十個金原子組成之材料，尺寸大小小於3 nm，
能階分布屬於離散能階，因此具有螢光之特性。在我們所設計的螢光感測系統中，我們利用第六型溶菌酶穩定結構之8 個原子金奈米簇作為螢光訊號來源，利用碘分子與碘離子作為消光材料，其消光方式為碘分子及碘離子可以將金奈米簇蝕刻，使其螢光淬熄，透過抗壞血酸還原碘酸鉀(KIO3)形成碘分子的方式，我們可使用抗壞血酸磷酸鈉透過鹼性磷酸酶(ALP)水解形成抗壞血酸的方式來定量鹼性磷酸酶。在抗壞血酸檢測部分，我們設計之檢測系統的線性區間為0.02 至0.6 mM；在鹼性磷酸酶檢測部分，我們所設計之系統的線性區間為0.05 至125 U/L，而後我們進行了酵素結合免疫吸附分析法，利用IgG 抗體綁上鹼性磷酸酶的方式檢測IgG 抗原，所得到偵測線性區間
為2 至50 ng/mL。

(一) PVP-capped Prussian Blue Nanomaterials Act as Quencher for Phosphate Group Biochemicals Fluorescence Sensor
Prussian blue nanoparticles (PBNPs) is an ancient dye that is found in 18 century, a well-known coordination polymer (CP) with the formation Fe4[Fe(CN)6]3. In this study, we developed a sensor that polyvinylpyrrolidone (PVP)-capped PBNMs act as an efficient quencher and boron-dipyrromethene conjugated adenosine triphosphate (BODIPY-ATP) as fluorescein dye for sensing the analytes with phosphate groups such as pyrophosphate ion (PPi) and adenosine triphosphate (ATP). PPi is one of the product in ATP hydrolysis, the size of PPi is much smaller than BODIPY-ATP, it could compete with BODIPY-ATP to chelate on the surface of PBNPs. The fluorescence increases linearly with the PPi concentration increasing range from 0.08 μM to 1 μM, with the limit of detection is 0.03 μM. Thus, we could set up a turn-on fluorescence sensor to detect phosphate group biochemicals and phosphatase, we could detect alkaline phosphatase (ALP) in the system we proposed by using the ALP-catalyzed hydrolysis of BODIPY-ATP, the product will be BODIPY conjugated adenosine and phosphate ions, the fluorescence signal could be read because of BODIPY conjugated adenosine. The linear range of the ALP concentrations is 0.01 U/L to 1 U/L, with the limit of detection is 0.003 U/L. And we use this system for ELISA detection of IgG, with the linear range 0.05 ng/mL to1 ng/mL, the limit of detection is 0.02 ng/mL.
 
(二) Turn-Off Fluorescent Strategy for Sensing Alkaline Phosphatase Based on Iodine-Mediated Etching of Lysozyme Protected Gold Nanoclusters
Gold nanoclusters has fluorescence due to it’s particle size is under 3 nm that made the energy level is fitting the Fermi level, the energy level of gold nanoclusters is not continuous. In this study, we developed a sensor that iodine molecules (I2) and iodide ions (I-) act as an efficient quencher and lysozyme type vi – stablilized Au8 (Lyz@Au8) nanoclusters as fluorophore for sensing various species. For instance, ascorbic acid (AA) is one of the product form ascorbic acid-phosphate (AAP) hydrolysis by alkaline phosphatase (ALP). That ascorbic acid could let potassium iodate reducing to be iodine molecules and it could etching gold nanoclusters. The fluorescence decreasing linearly with the AA concentration increasing range from 0.02 mM to 0.6 mM, with the limit of detection is 0.007 mM. Thus we could design a turn-off fluorescence sensor to detect alkaline phosphatase by using the ALP-catalyzed hydrolysis of AAP, the product will be AA and phosphate ions, the fluorescence signal could be read because of Lyz@Au8. The linear range of the ALP concentrations is 0.05 U/L to 125 U/L, with the limit of detection is 0.02 U/L. And we use this system for ELISA detection of IgG, with the linear range 2 ng/mL to50 ng/mL, the limit of detection is 0.7 ng/mL.

英文摘要 I
中文摘要 III
第一章 1
1. 前言 2
1.1.1 金屬有機框架材料簡介 2
1.1.2 普魯士藍 2
1.2 螢光消光機制種類與文獻探討 3
1.3 鹼性磷酸酶簡介 11
1.4 酵素免疫分析法 12
1.5 研究動機 13
2. 實驗部分 14
2.1 實驗藥品 14
2.2 實驗儀器 19
2.3 藥品配製與合成 22
2.4 實驗過程 26
3. 實驗結果與討論 30
3.1 比較以鐵(III)與鐵(II)離子合成之PVP-PB NMs差異 30
3.2 探討PVP-Fe(III) PB NMs之基本性質 38
3.3 PVP-Fe(III) PB NMs與BODIPY-ATP偵測系統最佳化 41
3.4 PVP-Fe(III) PB NMs與BODIPY-ATP之消光機制探討 48
3.5 以PVP-Fe(III) PB NMs與BODIPY-ATP定量焦磷酸鈉鹽 54
3.6 以PVP-Fe(III) PB NMs與BODIPY-ATP定量鹼性磷酸酶 59
3.7 以PVP-Fe(III) PB NMs與BODIPY-ATP之ELISA檢測 63
4. 結論 69
5. 參考文獻 70
第二章 75
1. 前言 76
1.1.1 金奈米簇 76
1.1.2 金奈米簇合成方法 76
1.2 尺寸效應 78
1.3 研究動機 79
2. 實驗部分 80
2.1 實驗藥品 80
2.2 實驗儀器 85
2.3 藥品配製與合成 86
2.4 實驗過程 89
3. 實驗結果與討論 94
3.1 比較以不同pH值合成之第六型溶菌酶穩定結構之金奈米簇抗蝕刻性質 94
3.2 Lyz@ Au8對碘離子蝕刻偵測系統最佳化 99
3.3 Lyz@ Au8對碘離子蝕刻偵測系統定量抗壞血酸 106
3.4 Lyz@ Au8對碘離子蝕刻偵測系統定量鹼性磷酸酶 111
3.5 Lyz@ Au8對碘離子蝕刻偵測系統之ELISA偵測 116
4. 結論 123
5. 參考文獻 124

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