Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin.

ABSTRACT

We used the methods of electrochemical indicators and the quartz crystal microbalance (QCM) for detection of thrombin-aptamer interactions. We analyzed how the method of immobilization of aptamer to a solid support, the aptamer configuration as well as variation in ionic strength and pH will affect the binding of thrombin to the aptamer. The immobilization of aptamer by means of avidin-biotin technology revealed best results in sensitivity in comparison with immobilization utilizing dendrimers of first generation and in comparison with chemisorption of aptamer to a gold surface. Linear and molecular beacon aptamers of similar structure of binding site revealed similar binding properties to thrombin. Increased concentration of NaCl resulted in weakening of the binding of thrombin to the aptamers, probably due to shielding effect of Na(+) ions. The binding of the thrombin to the aptamer depends on electrolyte pH, which is presumably connected with maintaining the three dimensional aptamer configuration, optimal for binding the protein.