Department of Chemistry, Alfaisal University, Al Zahrawi Street, Al Maather, AlTakhassusi Rd, Riyadh, 11533, Saudi Arabia.
King Faisal Specialist Hospital and Research Center, Zahrawi Street, Al Maather, Riyadh, 12713, Saudi Arabia.
Mikrochim Acta. 2019 Nov 21;186(12):828. doi: 10.1007/s00604-019-3817-7.
The autosomal recessive-hyper immunoglobulin E syndromes (AR-HIES) are inherited inborn primary immunodeficiency disorders caused mainly by mutations in the dedicator of cytokinesis 8 (DOCK8) gene. A method is described for the selection of DNA aptamers against DOCK8 protein. The selection was performed by using a gold electrode as the solid matrix for immobilization of DOCK8. This enables voltammetric monitoring of the bound DNA after each selection cycle. After eight rounds of selection, high affinity DNA aptamers for DOCK8 were identified with dissociation constants (Kds) ranging from 3.3 to 66 nM. The aptamer which a K of 8.8 nM was used in an aptasensor. A gold electrode was modified by self-assembly of the thiolated aptamer, and the response to the DOCK8 protein was detected by monitoring the change in the electron transfer resistance using the ferro/ferricyanide system as a redox probe. The aptasensor works in the 100 pg.mL to 100 ng.mL DOCK8 concentration range, has a detection limit of 81 pg.mL and good selectivity over other proteins in the serum. Graphical abstractSchematic representation of an electrochemical screening protocol for the selection of DNA aptamer against dedicator of cytokinesis 8 protein using electrode as solid support for target immobilization.
常染色体隐性遗传性高免疫球蛋白 E 综合征（AR-HIES）是一种主要由细胞分裂蛋白 8（DOCK8）基因突变引起的遗传性原发性免疫缺陷病。本文描述了一种针对 DOCK8 蛋白的 DNA 适体筛选方法。该方法通过使用金电极作为 DOCK8 的固定基质来进行筛选。这使得在每个选择循环后都可以对结合的 DNA 进行伏安监测。经过八轮选择，确定了与 DOCK8 具有高亲和力的 DNA 适体，解离常数（Kd）范围为 3.3 至 66 nM。使用 Kd 为 8.8 nM 的适体在适体传感器中进行检测。金电极通过硫醇化适体的自组装进行修饰，并通过使用铁氰化钾/亚铁氰化钾体系作为氧化还原探针监测电子转移电阻的变化来检测 DOCK8 蛋白的响应。该适体传感器在 100 pg.mL 至 100 ng.mL 的 DOCK8 浓度范围内工作，检测限为 81 pg.mL，对血清中的其他蛋白质具有良好的选择性。
