Label-Free Strategy for In-Situ Analysis of Protein Binding Interaction Based on Attenuated Total Reflection Surface Enhanced Infrared Absorption Spectroscopy (ATR-SEIRAS)

A versatile ATR-SEIRAS methodology is described herein for highly sensitive analysis of immunoglobulin (IgG) recognition. This strategy allows in situ tracking of specific protein binding at the liquid–solid interface. Most importantly, interferential signal from environmental molecules (e.g., water, nonspecific binding molecules, and bulk molecules) can be eliminated to negligible levels by using the ATR analysis mode, and the sensitive IR structural information of target proteins is obtained simultaneously. A simplified numerical model has been established to quantitatively describe the kinetics and thermodynamics of protein recognition processes at surfaces. Compared with conventional label-free methods for protein binding study, experimental results obtained from IR spectroscopic information are more reliable. The presented ATR-SEIRAS method is powerful in studying surface limited protein binding reactions.