适体和蛋白质解离常数检测方法比较分析

Comparative analysis of detection methods for dissociation constant of aptamer and protein

对基于石英晶体微天平(QCM)生物传感器的两种检测核酸适体与蛋白质解离常数方法进行了比较,提出来一种更加精确合理的检测流程。以凝血酶和凝血酶适体TBA15为模型,耗散型石英晶体微天平(QCM-D)为传感器,实时检测末端修饰巯基适体的固定、表面封闭剂对非特异性结合位点封闭以及两种不同的蛋白进样方式引起的频率响应,实验数据拟合得到解离常数。不同蛋白的进样方式得到的解离常数不同,非特异性位点的封闭也同样影响解离常数的检测。从低浓度到高浓度依次通入固定体积蛋白的进样方式,实验重复性高且消耗样品量小于1μg,是较为理想的检测方式。

Compare the two detection methods of dissociated constant of aptamer and protein based on quartz crystal microbalance（ QCM） biosensor,a protocol with high accuracy and reasonability is introduced. Thrombin and thrombin aptamer named TBA15 are used as models. The immobilization of thiolated aptamer,the block of the non-specific site and the signal response induced by two different sampling methods are monitored by QCM with dissipation（ QCM-D） biosensor in real time and dissociated constant are calculated by data fitting. Dissociated constant are different when different sampling methods are used,and different block agent applied,blocking of nonspecific site. The sampling method with fixed bulk of protein from low concentration to high concentration are proved ideal method with high repeatability and fewer regents needed which is less than 1 μg.