Bulk-Effect-Free Method for Binding Kinetic Measurements Enabling Small-Molecule Affinity Characterization

[Image: see text] Optical technologies for label-free detection are an attractive solution for monitoring molecular binding kinetics; however, these techniques measure the changes in the refractive index, making it difficult to distinguish surface binding from a change in the refractive index of the analyte solution in the proximity of the sensor surface. The solution refractive index changes, due to solvents, temperature changes, or pH variations, can create an unwanted background signal known as the bulk effect. Technologies such as biolayer interferometry and surface plasmon resonance offer no bulk-effect compensation, or they alternatively offer a reference channel to correct in postprocessing. Here, we present a virtually bulk-effect-free method, without a reference channel or any computational correction, for measuring kinetic binding using the interferometric reflectance imaging sensor (IRIS), an optical label-free biomolecular interaction analysis tool. Dynamic spectral illumination engineering, through tailored LED contributions, is combined with the IRIS technology to minimize the bulk effect, with the potential to enable kinetic measurements of a broader range of analytes. We demonstrate that the deviation in the reflectivity signal is reduced to ∼8 × 10(–6) for a solution change from phosphate-buffered saline (PBS) (n = 1.335) to 1% dimethyl sulfoxide (DMSO) in PBS (n = 1.336). As a proof of concept, we applied the method to a biotin–streptavidin interaction, where biotin (MW = 244.3 Da) was dissolved at a final concentration of 1 μM in a 1% solution of DMSO in PBS and flowed over immobilized streptavidin. Clear binding results were obtained without a reference channel or any computational correction.