巨噬細胞(macrophage)與嗜中性白血球(neutrophil)於人體免疫反應及發炎反應時會產生活性氧(reactive oxygen species, ROS)抵禦外來感染源，活性氧包含了羥基(•OH)、超氧陰離子(O2•-)與過氧化氫(H2O2)。人體活性氧過多時，會導致癌症;活性氧過少時，會導致慢性肉芽腫(CGD)及許多免疫疾病，甚至敗血病死亡。目前臨床上是利用硝基四氮唑藍(nitroblue tetrazolium, NBT)活性染料與吞噬細胞所釋出之超氧陰離子反應，產生二甲基(diformazan)，藉由光譜儀(spectrophotometer)檢測。但是因為光譜儀較昂貴及需要專業人員的操作，本實驗開發微孔電極檢測細胞來達到價格較低及簡單操作之目的。又因為超氧陰離子是不穩定的物質，所以本實驗電位設於+0.7 V (vs. Ag/AgCl)檢測穩定且具有電化學特性之過氧化氫。首先，設電位為-0.2 V於微孔金電極上電鍍奈米白金，因為電鍍白金會使電極表面呈黑色，為使得顯微鏡下的細胞於微孔中能分辨，所選用電鍍參數為30秒。利用開發之微孔電極檢測過氧化氫，最低極限可至5 pM，可檢測單顆RAW 264.7細胞於微孔中所釋出低濃度之過氧化氫。於微孔電極檢測RAW 264.7細胞實驗中，每次捕捉1~4個於微孔中，PMA (phorbol 12-myristate 13-acetate)刺激20~150秒後可觀察突波電流，計算累積突波電流之總電量，每次實驗細胞產生約0.15 pC~39 pC，換算後約有0.77×10-3~200 fmol的過氧化氫在電極上被檢測到。另一方面，將微孔高度由25 μm降低為10 μm，由於PMA擴散時間的縮短，細胞之訊號提早被偵測到。本研究利用微流體晶片檢測RAW 264.7細胞，未來可應用於臨床巨噬細胞之免疫反應。

Macrophages and neutrophils could produce reactive oxygen species (ROS) to fend off infection when the body could be inflamed. ROS is included hydroxyl radical (‧ OH), superoxide anion (O2•-) and hydrogen peroxide (H2O2). When there are too much ROS, cancer will occur. When there aren't enough ROS, chronic granulomatous disease (CGD), the immune disease and septicemia will occur. Clinical method for H2O2 detection is using nitroblue tetrazolium (NBT) with O2•- to produce diformazan, which can be detected by spectrophotometer. However, a spectrophotometer is expensive and could only operate by professionals. In this study, we developed the microwell electrode to achieve lower prices and simple operation. O2•- is unstable, so we detected H2O2 which is stable and show electrochemical characteristics. H2O2 could be detected at +0.7 V (vs. Ag/AgCl) in this study. First, the platinum (Pt) was deposited on the surface of the microwell gold electrode at -0.2 V for 30 s. The limit was 5 pM for H2O2 by the microwell electrode. For the detection of H2O2 released from RAW 264.7 cells, the electricity calculated from areas under each current peak were between 0.15 pC~39 pC and the amount of H2O2 released were 0.77×10-3~200 fmol. The signals appeared at 0.5~3 min after stimulation. This study used microwell electrode to detect the H2O2 released by RAW 264.7 cells. On the other hand, the height of microwell was reduced from 25 μm to 10 μm. The current response of the cell was occurred early. In the future, the microwell electrode will be used to detect real samples.

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