Enhancement of Ligand Immobilization on Gold Surface by Optimizing Monolayer Concentration and Pre‐Concentration Studies for Surface Plasmon Resonance Analysis of Rutin‐Lysozyme Interaction

Abstract

Surface plasmon resonance (SPR) is a versatile tool for real time monitoring of molecular interactions, providing biophysical data such as affinity and kinetics. Rutin is found in various plants and fruits and is utilized in several industries. Because of its therapeutic and industrial importance, the binding of rutin to one of the major proteins found in blood and saliva, lysozyme (LZM), was investigated to determine the affinity constants of the rutin‐LZM interaction. Chicken egg white lysozyme (CEWLZM) was selected as a model ligand. 11‐mercaptoundecanoic acid (MUA) was chosen for the self‐assembled monolayer (SAM) design on the gold chip surface. After optimizing MUA concentration and pre‐concentration studies to enhance ligand immobilization, optimum MUA concentration in SAM formation, ideal running and coupling buffers, flow rate and ligand concentration were determined to perform SPR analysis. Various concentrations of rutin (20–300 μM) were injected into the SPR for kinetic analysis. The association (ka), dissociation (kd) rate constants, and equilibrium constant (KD) were calculated from the kinetics of the binding curves. SPR binding results indicated that the KD value of rutin‐CEWLZM binding was determined as 80.07±1.22 μM. This study demonstrates the binding affinity of rutin to CEWLZM, offering potential insights for medical and pharmaceutical sciences.