本論文將一氧化氮的感測分子1,2-Diaminoanthraquinone(DAQ)參入水凝膠材料Poly2-hydroxyethyl methacrylate(polyHEMA)中，製成於520 nm 波段有吸收峰值的感測薄膜。當膜的厚度為0.7 mm 時，主要的感測目標為一氧化氮(NO)的末端產物亞硝酸鹽(NO2-)，而非一氧化氮本身。但當膜的厚度減少到430μm 時，它在520
nm 波段的吸收值能夠在通入NO 氣體的2 分鐘內就快速的下降，因為NO 能夠在小於它的生命期的時間內就擴散進入膜裡面。而這樣快速的反應在pH=7±0.3以及生物緩衝液DMEM 的環境中依然是可行的。接著我們將此薄膜製成光波導並連接著兩根光纖，其中波導的寬度為2.5 mm，兩根光纖的間距為1.2 mm。在兩根光纖之間光的傳輸主要是在520
nm 這波段。當收光端光纖的直徑為600μm時，此種量測結構在通入NO 氣體15 分鐘之後就看得到反應。這是一個微型的關鍵生物信號因子NO 的整合型即時感測元件，而非間接性的去量測NO 所衰變成的末端產物。

The nitric oxide (NO) probe 1,2-Diaminoanthraquinone (DAQ) is dispersed in hydrogel Poly 2-hydroxyethyl methacrylate (polyHEMA) to form a sensing film based on 520 nm absorption band. When the film is about 0.7 mm thick it responds only to the end product nitrite (NO2-) only but not NO. When the thickness is reduced to 430 μm its 520 nm absorption is reduced rapidly after 2 minutes of NO bubbling due to the NO diffusion inside the sensing film within its short lifetime. Such rapid NO sensing works at pH=70.3 and in cell culture medium DMEM. The sensing film is made as a waveguide, and it connects two optical fibers. For fiber core of 600 μm this setup responds to NO bubbling after 15 minutes. This is a compact solid-state device for real-time direct monitoring of the key biochemical messenger NO rather than indirectly through its decay products.

摘要.........................................................................................Ⅰ
Abstract..................................................................................Ⅱ
目錄.........................................................................................Ⅲ
圖目錄.....................................................................................Ⅴ
表目錄.....................................................................................Ⅷ
Chapter1 緒論..........................................................................1
1-1 前言....................................................................................................................1
1-2 一氧化氮（NO）與亞硝酸鹽（NO2-）介紹......................................1
1-2-1 一氧化氮（NO）...................................................................................1
1-2-2 亞硝酸鹽（NO2-）.................................................................................9
1-3 研究動機........................................................................................................10
1-4 論文架構........................................................................................................12
Chapter2 實驗原理.................................................................13
2-1 光吸收理論...................................................................................................13
2-2 NO probe的反應原理................................................................................17
Chapter3 實驗材料、製程與量測............................................20
3-1 材料介紹........................................................................................................20
3-2 感測薄膜製程..............................................................................................26
3-1-1 pHEMA平板厚膜製作.................................................................26
3-1-2 pHEMA平板薄膜製作.................................................................27
3-1-3 與光纖耦合pHEMA平板薄膜製作........................................28
3-3 儀器介紹........................................................................................................29
3-4 量測系統介紹..............................................................................................32
3-4-1 吸收光譜儀量測.............................................................................32
3-4-2 雷射耦合光纖量測系統...............................................................32
Chapter4 實驗設計與結果.....................................................34
4-1 DAQ感測分子.............................................................................................34
4-2 pHEMA平板膜在中性環境對NO的感測.........................................35
4-3 pHEMA平板薄膜在生物緩衝液中對NO的感測..........................42
4-4 光波導感測膜對NO的感測...................................................................44
4-5 一氧化氮定量實驗（Nitrite assay）.......................................................49
Chapter5 結論........................................................................51
參考文獻.................................................................................52
圖目錄
圖1-2.1 Furchgott的三明治實驗........................................................................................4
圖1-2.2 血管內皮層組織的NO的作用機制.................................................................5
圖1-2.3 Ignarro’s Spectrophotometry..............................................................................6
圖1-2.4 一氧化氮在體內所負責的八大主要工作.....................................................7
圖2-1.1 原子吸收光譜.......................................................................................................13
圖2-1.2 電子能量躍遷圖...................................................................................................14
圖2-1.3 分子之吸收光譜圖..............................................................................................14
圖2-1.4 溶液中之吸收光譜..............................................................................................15
圖2-1.5 比爾定律吸收光路徑示意圖...........................................................................16
圖2-1.6 造成吸收度非線性的因素...............................................................................17
圖2-2.1 三種常見的o-phenylenediamine NO probe.................................................18
圖2-2.2 DAF-2和一氧化氮反應變成DAF-2 triazole（DAF-2T）............................18
圖2-3.3 DAQ和NO/NO2-反應後變為DAQ-TZ.............................................................19
圖2-2.4 DAQ溶液通入NO前後的吸收光譜變化圖..................................................19
圖3-1.1 DAQ結構圖.............................................................................................................20
圖3-1.5 DMF結構圖.............................................................................................................22
圖3-1.6 DMSO結構圖..........................................................................................................22
圖3-1.7 NaNO2結構圖..........................................................................................................26
圖3-2.1 pHEMA平板厚膜製作示意圖..........................................................................27
圖3-2.2 耦合雙光纖的pHEMA平板薄膜....................................................................28
圖3-3.1 Agilent Cary50 Scan UV-Visible Spectrophotometer.................................29
圖3-3.2 Hitachi F-4500 Fluorescence Spectrophotometer.........................................30
圖3-3.3 StellarNet EPP2000 Fiber Optic Spectrometer.............................................30
圖3-3.4 532 nm雷射系統....................................................................................................31
圖3-3.5 精密光纖耦合器...................................................................................................31
圖3-4.1 吸收光譜儀量測光路徑圖...............................................................................32
圖3-4.2 雷射耦合光纖量測系統....................................................................................33
圖3-4.3 （a）2 ml鐵氟龍水槽 （b）3 ml結合壓克力與鐵氟龍模具之水槽.......33
圖4-1.1 DAQ溶液在NO bubble後吸收光譜的變化..................................................34
圖4-2.2 pHEMA平板厚膜在中性環境中測量NO.....................................................36
圖4-2.3 pH buffer solution在不同浸泡膜的時間的吸收光譜...............................37
圖4-2.4 pH buffer solution隨著NO bubble的pH值變化..........................................38
圖4-2.5 DAQ薄膜在中性環境中分別感測(a)亞硝酸鹽和(b)一氧化氮.............39
圖4-2.6 取出圖4-2.5在525 nm的吸收值.......................................................................40
圖4-2.7 pH buffer solution隨著NO bubble的吸收值變化.......................................41
圖4-2.8 (a)在DI water中進行對DAQ薄膜通入NO的測試 (b)DI water隨著通入NO的pH值變化................................................................................................................42
圖4-3.1 DAQ薄膜在DMEM中和NO的反應................................................................43
圖4-4.1 以DAQ薄膜耦合雙光纖系統量測NO之光譜圖.......................................44
圖4-4.2 厚度為429 μm的膜進行雙光纖量測之時間速率圖............................45
圖4-4.3 膜厚429 μm收光光纖直徑改為400 μm所進行之光纖系統量測................................................................................................................................................46
圖4-4.4 膜厚706μm，收光光纖直徑為600 μm所進行之光纖系統量測.....47
圖4-4.5 改通入N2於光纖量測系統之測試.................................................................48
圖4-5 通入不同時間NO氣體所對應到的NO2-濃度（此濃度為稀釋了16倍）.............................................................................................................................................50














表目錄
表1-2-1 1998年諾貝爾得主....................................................................2
表3-1 DMEM成份表..............................................................................23

[1] c. Wikipedia, Wikipedia, The Free Encyclopedia.
[2] Furchgott, R.F. and J.V. Zawadzki, The Obligatory Role of Endothelial-Cells in the Relaxation of Arterial Smooth-Muscle by Acetylcholine. Nature, 1980. 288(5789):p. 373-376.
[3] Lai, Y.C., et al., Nitrite-Mediated S-Nitrosylation of Caspase-3 Prevents
Hypoxia-Induced Endothelial Barrier Dysfunction. Circulation Research, 2011.
109(12): p. 1375-1386.
[4] Kikuchi, K., et al., Detection of Nitric-Oxide Production from a Perfused Organ by
a Luminol-H2o2 System. Analytical Chemistry, 1993. 65(13): p. 1794-1799.
[5] Marin, M.J., et al., Fluorescence of 1,2-Diaminoanthraquinone and its Nitric Oxide
Reaction Product within Macrophage Cells. Chembiochem, 2011. 12(16): p.
2471-2477.
[6] Galindo, F., et al., Spectroscopic studies of 1,2-diaminoanthraquinone (DAQ) as a
fluorescent probe for the imaging of nitric oxide in living cells. Photochemical &
Photobiological Sciences, 2008. 7(1): p. 126-130.
[7] Dacres, H. and R. Narayanaswamy, Evaluation of 1,2-diaminoanthraquinone
(DAA)as a potential reagent system for detection of NO. Microchimica Acta, 2005.
152(1-2): p. 35-45.
[8] Nims, R.W., et al., Colorimetric assays for nitric oxide and nitrogen oxide species
formed from nitric oxide stock solutions and donor compounds. Nitric Oxide, Pt a
- Sources and Detection of No; No Synthase, 1996. 268: p. 93-105.
[9] Chao, Y.C., et al., Real-time and indicator-free detection of aqueous nitric oxide
with hydrogel film. Applied Physics Letters, 2010. 96(22).
[10] Wun-Yan Hong, Tsung-Wei Yang, Chun-Ming Wang, Jia-Hao Syu, Yu-Chao Lin,Hsin-Fei Meng*, May-Jywan Tsai, Henrich Cheng, Hsiao-Wen Zan*, Sheng-Fu
Horng, Conversion of absorption to fluorescence probe in solid-state
sensor for nitric oxide and nitrite. Organic Electronics, 2013. 14: p. 1136-1141.
[11] Obi-Egbedi, N.O., et al., 1,2-Diaminoanthraquinone as Corrosion Inhibitor for
Mild Steel in Hydrochloric Acid: Weight Loss and Quantum Chemical Study. International Journal of Electrochemical Science, 2011. 6(4): p. 913-930.
[12] Privat, C., et al., Nitric oxide production by endothelial cells: Comparison of
three methods of quantification. Life Sciences, 1997. 61(12): p. 1193-1202.
[13] Bru, M., et al., Cross-linked poly(2-hydroxyethylmethacrylate) films doped with
1,2-diaminoanthraquinone (DAQ) as efficient materials for the colorimetric
sensing of nitric oxide and nitrite anion. Tetrahedron Letters, 2006. 47(11): p.
1787-1791.
[14] Burguete, M.I., et al., Improved polyHEMA-DAQ films for the optical analysis of
nitrite. European Polymer Journal, 2009. 45(5): p. 1516-1523.
Gries, P., Berichte der deutschen chemischen Gesellschaft. 1879. 12: p. 426-428.
[15] P. Griess, Berichte der deutschen chemischen Gesellschaft 1879, 12, 426-428.