Emulsion-Based Technique To Measure Protein Crystal Nucleation Rates of Lysozyme

We measured the nucleation rates of lysozyme protein crystals using microfluidically produced emulsion drops containing supersaturated protein solution. The technique involves quenching several thousand independent nanoliter drops by rapidly lowering the temperature and then counting the number of drops that have not nucleated as a function of time at constant temperature. We fit the number distribution to a theoretical model developed by Pound and La Mer (J. Am. Chem. Soc. 1952, 74, 2323−2332) for heterogeneous nucleation and extract two nucleation rates and the number of nucleation sites per drop. We describe the technique in detail and present our analysis of the measured nucleation rates within the context of Classical Nucleation Theory, which adequately describes our observations. Of the two nucleation rates, one is a slow rate that varies with temperature and one is a fast rate independent of temperature. The nucleation barrier and kinetic prefactors are obtained for each rate. Notably, there is no detectable barrier for the fast rate. Both rates are inconsistent with the process of homogeneous nucleation and are consistent with heterogeneous nucleation.