使用[Os(bpy)(phen-benz-COOH)]/Nafion/石墨烯薄膜的电化学发光阵列检测双链DNA中的氧化损伤
Electrochemiluminescent Array to Detect Oxidative Damage in ds-DNA Using [Os(bpy)(phen-benz-COOH)]/Nafion/Graphene Films.
作者信息
Bist Itti, Song Boya, Mosa Islam M, Keyes Tia E, Martin Aaron, Forster Robert J, Rusling James F
机构信息
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States; Department of Chemistry, Tanta University, Tanta 31527, Egypt.
出版信息
ACS Sens. 2016;1(3):272-278. doi: 10.1021/acssensors.5b00189. Epub 2016 Jan 8.
Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os(bpy)(phen-benz-COOH)] (OsNG, {bpy= 2,2'-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl) benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton's reagent or by ROS formation during redox cycles involving NADPH, Cu, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of Cu, NADPH, and model metabolites.
活性氧（ROS）将DNA中的鸟苷氧化形成8-氧代-7,8-二氢-2'-脱氧鸟苷（8-oxodG），这是一种氧化应激的生物标志物。在此，我们描述了一种新型的64孔电化学发光（ECL）阵列，能够在不进行水解的情况下灵敏地多重检测双链DNA中的8-oxodG。将含有ECL染料[Os(bpy)(phen-benz-COOH)]（OsNG，{bpy = 2,2'-联吡啶，phen-benz-COOH = (4-(1,10-菲咯啉-6-基)苯甲酸)}）的Nafion和还原氧化石墨烯薄膜组装到热解石墨晶片上的微孔中，通过电化学发光（ECL）检测寡核苷酸中的8-oxodG。监测了芬顿试剂引起的DNA氧化或涉及NADPH、Cu和模型代谢物的氧化还原循环过程中ROS形成引起的DNA氧化。氧化DNA样品的超高效液相色谱-串联质谱（UPLC-MS/MS）用于校准。流体阵列的检测限为每670个完整核碱基中有一个8-oxodG，即0.15%。该方法灵敏度足以评估由Cu、NADPH和模型代谢物的生物相关ROS生成反应引起的DNA氧化。
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