Comparison of different approaches to single particle tracking of enzymes displaying enhanced diffusion

Enzymes have been shown to perform faster diffusion with the presence of their substrate. Recently, we have revealed new insights of this emergent enzyme activity using single particle tracking (SPT). We found that while the overall mobility of enzyme is improved by 2-3 folds at saturated substrate concentration, the mode of diffusion remains Brownian. Meanwhile, in order to achieve long trajectories, a polymer brush coated surface and a large viscous polymer was utilized to slow down the diffusion, raising questions as to the effect of these additives. Here, we investigate the effect of the surface coating by replacing the polymer brush with a lipid bilayer; we also replace the crowding polymers with a smaller viscous molecule, glycerol. We make the same diffusion measurements and compare the results from both methods. We found a faster diffusion together with an artifactual anomalous exponent for enzymes diffusing on lipid bilayer. Also, the presence of high percentage of glycerol, leads to the failure in reproducing enhanced diffusion due to the low enzymatic reaction rate in such a high viscous environment. Our results indicate the critical responsibility of the polymer brush in slowing the diffusion and enabling the direct reporting on single enzyme enhanced diffusion.