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標題: | Mn2+-Luminol系統之雙重化學發光性質研究與應用 Dual Chemiluminescence of Mn2+-Luminol System and Its Applications |
作者: | Tsung-Yuan Huang 黃琮元 |
指導教授: | 林萬寅(Wann-Yin Lin) |
關鍵字: | 化發發光,魯米諾,過碘酸鉀,自由基,流動注入分析法,停止流儀, luminol,chemiluminescnec,potassium periodate,free radical,flow-injection analysis,stopped-flow spectrometry, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | Luminol是一種常用的化學發光試劑,通常在鹼性條件下與氧化劑及催化劑作用,產生425 nm左右的放光。利用流動注入分析法(flow-injection analysis)發現在luminol-KIO4系統加入醛類(乙醛、丙醛)、金屬離子(Mn2+、Co2+、Fe3+)、酚類化合物(pyrogallol、gallic acid)後,訊號可增強約2個數量級。選擇性自由基消滅劑之抑制結果顯示,活性含氧物質(ROS): •O2-、•OH、1O2為增強化學發光的主因。此化學發光系統可應用於靈敏檢測如hydroquinone、catechol、dopamine等酚類化合物。
由於流動注入分析法的結果顯示,此系統應該不只一種化學發光路徑,因此我們利用停止流(stopped-flow)光譜儀來偵測化學發光隨時間的變化,以期了解是否有多重化學發光的現象。首先針對含有luminol、Mn2+、KIO4、乙醛及gallic acid之各種組合中化學發光較強者進行測試,發現有幾個組合都具有多重化學發光。我們選擇具有雙重化學發光(dual chemiluminescence)性質的luminol-Mn2+-KIO4系統做系統性的探討。變化不同濃度之luminol、Mn2+、KIO4以及pH值後,發現隨試劑濃度條件改變,2秒內會有兩根化學發光訊號相互消長的現象出現。推測第一根主要是來自•O2-及•OH,加入可消滅•OH之消滅劑(DMSO、CH3OH)可抑制其化學發光;第二根主要與過量Mn2+(>100uM)與配位子(如OH-、O2、luminol)形成錯合物進而形成活性物質有關;加入與Mn2+相同濃度之EDTA可完全抑制第二根訊號,加入可消滅單態氧(1O2)之消滅劑(NaN3、1,4-diazabicyclo[2,2,2]octane)也可抑制其訊號。 藉由改變Mn2+濃度與試劑添加可有效控制兩根發光訊號在luminol-Mn2+-KIO4系統的消長,可將此特性應用於偵測抗氧化劑與金屬離子等環境分析。在0.5 uM抗氧化劑(hydroquinone、catechol、trolox、sesamol、resorcinol)存在下,兩根訊號皆被抑制,尤其對第一根訊號有更靈敏的抑制;在尿酸(uric acid)、pyrogallol、gallic acid、兒茶酚胺(catechoamines)、維他命存在下則可抑制第一根訊號並在某濃度範圍內增強第二根訊號;添加Ni2+、Zn2+離子於本系統中,也可增強luminol之放光強度。未來,將對此系統做更近一步的測試,並將此特性應用於更多生化分子、藥物、抗氧化劑的偵測。 關鍵字: 化發發光、魯米諾、過碘酸鉀、自由基、流動注入分析法、停止流儀 Luminol is a common chemiluminescence (CL) reagent. Luminol-based CL reaction, which emits light at 425 nm, is usually carried out in alkaline solution and in the presence of an oxidant and a catalyst. Using flow-injection analysis, we found that addition of aldehydes (CH3CHO, CH3CH2CHO), metal ions (Mn2+, Fe3+, Co2+) and phenolic compounds (pyrogallol、gallic acid) to the luminol-KIO4 solution enhanced the CL emission by two orders of magnitude. Radical-scavenging studies revealed that reactive oxygen species (ROS) such as •O2-, •OH, and 1O2 play crucial roles in CL enhancement. This CL system provides a sensitive way to determine phenolic compounds such as hydroquinone, catechol, and dopamine. The results from flow-injection analysis suggest that more than one CL pathways are possible. Therefore stopped-flow spectrometer was used to measure the time courses of the CL profiles, trying to detect the existence of multiple CL emission. First, we have tested all possible combinations of luminol, Mn2+, KIO4, acetaldehyde and gallic acid that exhibit intense CL emission. We found that several of the combinations produced multiple CL. We then performed a systematic study on the dual CL of the luminol-Mn2+-KIO4 system. By suitable adjustment of the reagent (luminol, KIO4, Mn2+) concentrations at pH above 13.0, dual CL was observed within 2 sec. ROS-scavenging studies using selective scavengers for O2- (glutathione), •OH (uric acid, DMSO, methanol), and 1O2 (NaN3, 1,4-diazabicyclo[2,2,2]octane) suggest that the first peak might be due to the generation of O2- and •OH through the catalytic effect of Mn2+, while the second peak might be attributed to the production of 1O2 through the Mn2+-complex involving ligands such as IO4-, OH-, O2, and luminol anion. The important features of the dual CL for the luminol-Mn2+-KIO4 system are that the relative intensity of the two peaks can easily be controlled by adjusting the reagent concentrations and that the CL signal with different intensity ratio of the two peaks may respond quite differently to the analytes in terms of selectivity and sensitivity. For example, hydroquinone, catechol, trolox, and sesamol inhibited both peaks, though to different extent. Uric acid, pyrogallol, gallic acid, and catecholamines inhibited the first peak, but further enhanced the second peak. Thus this CL system can provide a large variety of probes by changing the relative intensity of the two CL peaks, thereby improving the selectivity and sensitivity of the analysis. Adding Ni2+、Zn2+ also enhanced the CL emission. In the future, we will try to develop more efficient systems for detecting biomolecules, antioxidants, drugs and metal ions. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44051 |
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