A protocol for studying the interaction between small-molecular drug and DNA using microdialysis sampling integrated with chemiluminescent detection

Abstract

It is of great significance to understand how drug molecules interact with DNA, which is one of the most important aspects of biological investigations in drug discovery at molecular level. Herein, with the model of ractopamine and calf thymus DNA (ct-DNA), a protocol using microdialysis (MD) sampling integrated with flow injection (FI)-chemiluminescent (CL) detection was developed for studying the interaction between small-molecular drug and DNA. After incubating ractopamine with ct-DNA, unbound ractopamine was on-line sampled using a MD probe, followed by being introduced into a FI-CL system for quantitation. The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe, and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters. The MD probe exhibited a mean recovery of 27.3% for ractopamine sampling under the optimal conditions. The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8 × 106 M−1 and 3.9 × 106 M−1, respectively, showing negligible difference. Ractopamine was estimated to have only one type of binding site on ct-DNA. The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.