Luminol-錳(II)離子-過碘酸鉀多重化學發光系統之動力學探討及應用於羥基抗氧化劑之偵測

Abstract

Luminol 是一種常用的化學發光試劑，通常在鹼性環境與氧化劑及催化劑作用產生最大強度波長在425 nm 的藍光。利用停止流光譜儀(Stopped-flow spectrometry)發現在luminol-KIO4 系統加入錳二價金屬離子(Mn2+)除了增強化學發光訊號，另會產生多重發光的現象。變化不同濃度之luminol、Mn2+、KIO4 以及pH 值後，隨試劑濃度條件改變，可對多重化學發光訊號進行相互消長的調控。利用結合Hill-equation與一級反應速率定律式的半經驗公式可使用十個參數將多重化學發光拆解為三個獨立訊號，相關係數平方最高可達0.99999。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 為產生起始及訊號1化學發光的主因，推測主要是來自•O2-及•OH，加入可消滅•OH 之消滅劑可抑制其化學發光；訊號2與特定的自由基關聯不如前兩者，可能與金屬錯離子催化的關聯較大。主要與過量Mn2+(>100 μM)與配位子(如OH-、O2、luminol)形成錯合物進而形成活性物質有關；加入可消滅單態氧(1O2)之消滅劑(1,4 -diazabicyclo[2,2,2]octane)也可抑制其訊號。 此化學發光系統可應用於靈敏檢測如catechol、dopamine、(+)-enpinephrine等酚類化合物。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。少數抗氧化劑如Ascorbic acid、6,7-dihydroxy-flavone、與7,8–dihydroxy-flavone應用於此系統時會產生讓訊號2延遲的現象，推測是三者分別與金屬離子形成更複雜的錯合物催化化學發光有關。 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。

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 Stopped-flow- spectrometry, we found that addition of Mn2+ metal ion to the luminol-KIO4 solution not just enhanced the CL emission by two orders of magnitude but also produced multiple CL emission. A systematic study on the multiple CL of the luminol-Mn2+-KIO4 system was performed. First, we have tested all possible combinations of luminol, Mn2+, KIO4, and pH. By suitable adjustment of the reagent concentrations at pH above 13.0, multiple CL was observed within 5 sec. Radical-scavenging studies revealed that reactive oxygen species (ROS) such as •O2-, •OH, and 1O2 play crucial roles in CL enhancement. ROS -scavenging studies using selective scavengers for O2- (glutathione), •OH (uric acid), and 1O2 (1,4-diazabicyclo[2,2,2]octane) suggest that the initial and 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. This CL system provides a sensitive way to determine phenolic compounds such as catechol, dopamine, and (+)-enpinephrine. 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. 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 ascorbic acid, 6,7-dihydroxy-falvone, and 7,8–dihydroxy-flavone not only inhibited the CL emission, but also made the time of signal 2 produced delay. In the future, we will try to develop more efficient systems for detecting biomolecules, antioxidants, drugs and metal ions.