Method to Determine the Optimal Aptamer-to-Bead Ratio by Using Flow Cytometry

Research on the effective attachment of aptamers to beads, which is essential for using aptamers, has made relatively little progress. Here, we demonstrate a new method based on flow cytometry to determine the optimal aptamer-to-bead ratio for aptamer immobilization. The fluorescence intensity increased with a gradual two-fold increase in the aptamer fluorescence concentration, peaked at an aptamer-to-bead ratio of 2.56 x 10(5), and tended to decrease at higher ratios. A similar pattern was observed in an additional analysis using fluorescence microscopy. However, measurement of the free aptamer concentration after the aptamer-bead conjugation reaction revealed a large aptamer loss compared to the 1.28 x 10(5) aptamer-bead ratio. In addition, the binding efficiency of the aptamer/bead to the target was highest at the aptamer-to-bead ratio of 1.28 x 10(5). Taken together, our data suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.

Automated summary

Research on the effective attachment of aptamers to beads has made little progress. This study demonstrates a new method using flow cytometry to determine the optimal aptamer-to-bead ratio for immobilization. The proposed method shows increased fluorescence intensity with increasing aptamer concentration, peaking at a ratio of 2.56 x 10(5), and decreasing at higher ratios. Analysis using fluorescence microscopy yielded similar results. However, there was a significant loss of aptamer compared to a ratio of 1.28 x 10(5) after the conjugation reaction, and the binding efficiency was highest at the ratio of 1.28 x 10(5). These findings suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.