Electrogenerated chemluminescence.: 72.: Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using Tris(2,2′-bipyridyl)ruthenium(II) labels
Electrogenerated chemluminescence.: 72.: Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using Tris(2,2′-bipyridyl)ruthenium(II) labels
复制标题
DOI：
10.1021/ac034596v
复制
发表时间：
2003-11-01
影响因子：
7.4
通讯作者：
Bard, AJ
中科院分区：
文献类型：
--
作者：
Miao, WJ;Bard, AJ
采用三（2,2’-联吡啶基）钌（II） (Ru(bpy)（3)(2+)）标记固定在Au（111）电极上，以三正丙胺（TPrA）为反应物的阳极电致化学发光（ECL）技术测定DNA和c反应蛋白（CRP）。在1-乙基-3-（3-二甲氨基丙基）carbodiimide hydrochloride （EDAC）存在的情况下，将从炭黑芽孢杆菌中提取的23-mer合成单链DNA （ss）在5'端有氨基修饰基团的共价连接到预涂有3-巯基丙烷酸（3- mpa）自组装巯基单层的Au（111）底物上，然后与带有Ru(bpy)(3)(2+) ECL标记的目标ssDNA杂交。同样，在EDAC和n -羟基琥珀酰亚胺存在的情况下，生物素化的抗crp物种被有效地固定在Au（111）底物上，通过亲和素与Au（111）上的3-MPA和16-巯基十六酸混合巯基单层反应共价连接的亲和素层。然后将Ru(bpy)(3)（2+）标记的CRP和anti-CRP偶联到表面层。通过将上述修饰电极浸入含有tpra的电解质溶液中，产生ECL响应。采用乙醇胺阻断游离-COOH基团、牛血清白蛋白阻断针孔、EDTA/NaCl/Tris缓冲液洗涤、惰性气体喷涂等一系列电极处理，减少了标记物的非特异性吸附。ECL峰强度在1 ~ 24杯/mL范围内与分析物CRP浓度成线性关系。采用基于该技术的标准加入法测定了两份未知人血浆/血清标本的CRP浓度。
Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 mug/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.