Determination of binding constants by equilibrium titration with circulating sample in a surface plasmon resonance...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biophysics

Background:

• Surface plasmon resonance (SPR) biosensors are valuable tools for studying molecular interactions.
• Traditional SPR methods can be limited by mass transport artifacts and the need for surface regeneration.
• Accurate determination of binding constants and kinetics is crucial for understanding biological systems.

Purpose of the Study:

• To develop an improved SPR-based method for measuring binding constants and kinetics of macromolecular interactions.
• To overcome limitations of existing SPR techniques, such as mass transport artifacts and immobilization-related issues.
• To enable precise characterization of molecular interactions with high affinity or slow dissociation rates.

Main Methods:

• Utilized a commercial SPR biosensor (BIACORE X) in a closed-loop system with continuous sample circulation.
• Employed stepwise titration to obtain binding isotherms and measure binding constants under equilibrium conditions.
• Developed a solution competition titration method to circumvent immobilization artifacts and measure binding in solution.
• Integrated external pump delivery with microfluidic competitor injection for kinetic analysis.

Main Results:

• Achieved accurate measurement of binding constants free from mass transport artifacts and without requiring surface regeneration.
• Demonstrated the ability to measure interactions with high affinity or slow dissociation rates due to extended experimental times and high baseline stability.
• Successfully employed solution competition titration to determine binding constants in solution, avoiding immobilization artifacts.
• Obtained kinetic information on complex dissociation by minimizing rebinding through rapid competitor injection.

Conclusions:

• The developed SPR biosensor methodology provides a robust and versatile approach for quantitative analysis of molecular interactions.
• This method enhances the accuracy and applicability of SPR for determining binding constants and kinetic parameters.
• The technique offers significant advantages for studying systems with challenging binding characteristics, advancing biochemical and biophysical research.