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

Sun Young Lee; Eun-Ok Kim; Daehyuk Jang; Soonjae Hwang; Ki-Jong Rhee; Miyong Yun

doi:10.1155/2023/5842652

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

Scientifica (Cairo). 2023 Jul 11:2023:5842652. doi: 10.1155/2023/5842652. eCollection 2023.

Authors

Sun Young Lee^{1

2}, Eun-Ok Kim³, Daehyuk Jang^{1

2}, Soonjae Hwang⁴, Ki-Jong Rhee⁵, Miyong Yun^{1

2}

Affiliations

¹ Lab of Functional Aptamer, Department of Bioindustry and Bioresource Engineering, College of Life Sciences, Sejong University, Seoul, Republic of Korea.
² Resource Upcycling and Discovery Research Institute, Sejong University, Seoul, Republic of Korea.
³ Medical Science Research Center, Korea University College of Medicine, Seoul, Republic of Korea.
⁴ Department of Biochemistry, Lee Gil Ya Cancer and Diabetes Institute, College of Medicine, Gachon University, Incheon, Republic of Korea.
⁵ Department of Biomedical Laboratory Science, College of Software & Digital Healthcare Convergence, Yonsei University MIRAE Campus, Wonju, Gangwon-do, Republic of Korea.

Abstract

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 × 10⁵, 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 × 10⁵ 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 × 10⁵. Taken together, our data suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.