A label-free untethered approach to single-molecule protein binding kinetics.

Single molecule approaches provide rich real-time dynamics of molecular interactions that are not accessible to ensemble measurements. Previous single molecule studies have relied on labeling and tethering, which alters the natural state of the protein. Here we use the double-nanohole (DNH) optical tweezer approach to measure protein binding kinetics at the single molecule level in a label-free, free-solution (untethered) way. The binding kinetics of human serum albumin (HSA) to tolbutamide and to phenytoin are in quantitative agreement with previous measurements, and our single-molecule approach reveals a biexponential behavior characteristic of a multistep process. The DNH optical tweezer is an inexpensive platform for studying the real-time binding kinetics of protein-small molecule interactions in a label-free, free-solution environment, which will be of interest to future studies including drug discovery.