To monitor the enrichment of aptamers after selection, the FAM-labeled ssDNA pool was incubated with 5610 4 AGR2-GST-beads or GST-beads in binding buffer (200 mL) at 37uC for 45 min. Beads were washed three times with 200 mL binding buffer by means of filtration, and suspended in binding buffer (250 mL). The fluorescence intensity of the resulting beads was monitored with a FACSAria cytometer (Becton Dickinson Immuno cytometry systems) by counting 10000 events. The binding affinities of aptamers were determined by incubating AGR2-GST-beads (5610 4 ) with various concentrations of FAMlabeled aptamers (pre-heat-treated) in binding buffer (200 mL) at 37uC for 45 min in the dark. Beads were then washed three times with the binding buffer, then resuspended in binding buffer (250 mL) and subjected to flow cytometry analysis. The FAMlabeled unselected ssDNA library was used as negative control for the nonspecific binding evaluation. All binding experiments were repeated two to four times. The mean fluorescence intensity of target protein labeled by aptamers was used to evaluate binding affinity by subtracting the mean fluorescence intensity of nonspecific binding produced by unselected ssDNA library. The dissociation constants (K d ) of the fluorescent ligands were obtained by fitting the dependence of fluorescence intensity of specific binding on the concentration of the ligands to the equation (1): Y = B max X/(K d +X) using SigmaPlot software.