Square-wave voltammetry assays for glycoproteins on nanoporous gold.
Square-wave voltammetry assays for glycoproteins on nanoporous gold.
复制标题
DOI：
10.1016/j.jelechem.2014.01.009
复制
发表时间：
2014-03-15
期刊：
影响因子：
--
通讯作者：
Stine KJ
中科院分区：
文献类型：
--
作者：
Pandey B;Bhattarai JK;Pornsuriyasak P;Fujikawa K;Catania R;Demchenko AV;Stine KJ
电化学酶联凝集素吸附测定 (ELLA) 是使用纳米孔金 (NPG) 作为蛋白质固定的固体支持物和作为电化学测定碱性磷酸酶 (ALP) 与磷酸对氨基苯酯 (p-APP) 之间反应产物（即对氨基苯酚 (p-AP)）之间的电极而开发的。糖蛋白或刀豆球蛋白 A (Con A) 和 ALP 缀合物共价固定到 NPG 上的硫辛酸自组装单层上。 Con A – ALP（或大豆凝集素 – ALP）缀合物与共价固定在 NPG 上的糖蛋白结合，随后与 p-APP 底物一起孵育，可产生方波伏安图，其峰差电流随糖蛋白的特性而变化。 NPG 呈递共价结合的糖蛋白，用作溶液中糖蛋白（转铁蛋白和 IgG）竞争性电化学测定的基础。使用固定化 Con A-ALP 缀合物证明了基于酶-底物反应的空间位阻的动力学 ELLA，从而降低了糖蛋白结合后的酶促反应速率。使用固定化的 Con A-ALP 缀合物，发现免疫球蛋白 G (IgG) 的结合亲和力为 105 nM，发现转铁蛋白的结合亲和力为 650 nM。在动力学和竞争性 ELLA 中，当存在 5 mg mL−1 BSA 作为模型血清蛋白时，观察到干扰最小。使用甲基 D-甘露糖苷对 TSF 和 IgG 与 Con A-ALP 的结合进行抑制研究；发现 IC50 值分别为 90 μM 和 286 μM。使用溶液耗尽和 BCA 蛋白质浓度测定来估计蛋白质的表面覆盖率。
Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay.
登录
查看更多内容
影响因子：
3.4
作者：
Dammer, U;Hegner, M;Guntherodt, HJ
通讯作者：
Guntherodt, HJ
用于细胞表面碳水化合物电化学测定的碳水化合物单层策略
影响因子：
15
作者：
Ding, Lin;Cheng, Wei;Ju, Huangxian
通讯作者：
Ju, Huangxian
影响因子：
6.6
作者：
Borkowska, Z;Tymosiak-Zielinska, A;Nowakowski, R
通讯作者：
Nowakowski, R
影响因子：
7.4
作者：
DeLouise, LA;Miller, BL
通讯作者：
Miller, BL
用酶功能化的基于凝集素的纳米探针，用于高灵敏电化学监测活细胞动态碳水化合物表达
影响因子：
7.4
作者：
Ding, Lin;Ji, Quanjiang;Ju, Huangxian
通讯作者：
Ju, Huangxian