Protein-aptamer binding studies using microchip affinity capillary electrophoresis

Abstract

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.

Figure 1

Voltage configuration. (a) Sample loading, 10 s. (b) Injection, 1 or 2 s. (c) Dispensing/separation, 30 s. The unit of voltage is kV. For hydrodynamic injection, all voltages in (b) were floated for 20 s. BR, buffer reservoir; SR, sample reservoir; BW, buffer waste reservoir; SW, sample waste reservoir; F, floating; and G, ground.

Figure 2

Comparison of injection methods. The separation buffer consisted of 25 mM Tris, 192 mM glycine and 5.0 mM KCl (pH 8.4). Sample solution contained 500 nM aptamer and 300 nM thrombin prepared in the separation buffer and incubated for >1 h at room temperature in the dark. Groups (i), (ii) and (iii) were obtained with floating, gated, and pinched injections, respectively. Peaks 1 and 2 are the free and the bound aptamers, respectively. Internal standard (IS) was 400 nM rhodamine B.

Figure 3

(a) Separation of L- and G-aptamers based on the separation length. The run buffer was Tris/glycine (25/192 mM, pH 8.4) with 10 mM KCl; (b) Complex peak versus the detection length. Electropherograms i-v were obtained at 0.5, 1.0, 2.0, 3.0 and 5.0 cm, respectively, from the intersection. Other conditions are the same as in Fig. 2. Peaks 1 and 2 are the free and bound aptamers, respectively.

Figure 4

KCl effect on thrombin-aptamer complex formation and microchip CE separation. Separation buffer contained 25 mM Tris, 192 mM glycine and various concentrations of KCl (pH 8.4). Sample solution contained 500 nM aptamer and 300 nM thrombin prepared in the separation buffer. Peaks 1 and 2 are the bound and free aptamers, respectively.

Figure 5

Thrombin adsorption effect on complex peaks. (a) The sample contained 200 nM aptamer and 1280 nM thrombin. Peaks were detected at 1.0 cm. (b) Comparison of the first and the eighth injections. The sample contained 500 nM aptamer and 300 nM thrombin. Repeated injections were detected at 5.0 cm. Other conditions are the same as in Fig. 2.

Figure 6

Peak area assignment. Parts 1, 2, and 3 are complex, dissociated aptamer, and free aptamer, respectively. Fronting was added to part 1 and tailing was added to free aptamer. The electropherogram was obtained using hydrodynamic injection with the sample containing 250 nM thrombin and 500 nM aptamer. Other conditions are the same as in Fig. 2.