| Atto- and femto-liter droplets of protein solution containing of the order of 1000 protein molecules are of interest for emerging bio-sensor, bio-fuel cell, and other technologies. Since existing methods cannot produce protein arrays of such sizes, this work proposes to utilize the separation of the protein solution into two liquid phases that can be controlled through the solution temperature. The separation of a protein solution into two liquid phases is also of interest because it is a fundamental biological phenomenon and its characteristic structures and dynamics may underlie fundamental biological time- and length-scales. The protein dense liquid phases may act as precursors for the formation of ordered solid phases that underlie several diseases: sickle cell anemia, eye cataract, and Alzheimer's.; As a first step in the studies of liquid-liquid (L-L) phase separation with proteins, the structures and dynamics of the emerging dense liquid phase are investigated. Depending on the quench depth, the evolution of the new-phase may follow either of two mechanisms: nucleation or spinodal decomposition. For these investigations lysozyme is used as a model protein for which the phase diagram is known. It is shown that the evolution of the structure during nucleation is similar to that during spinodal decomposition and reveals no singularity predicted upon crossing the metastability boundary, in agreement with predictions of non-mean-field theories. More importantly, these are the first experimental results that show that the spinodal is not defined by a sharp line in the phase diagram but rather by a diffuse area below the coexistence line.; These results also show that the generation of a predetermined number of droplets of a chosen size can be reproducibly accomplished at low undercooling levels in the nucleation regime. However, the nucleation of these droplets occurs at random locations, while the applications outlined above require deposition of the droplets over, for instance, electrodes. Towards such refinement of the droplet generation technique, it is shown that the generation of droplets of the dense protein phase can be induced over micrometer-size electrodes by applying a non-uniform electric field with a moderate potential of ∼1.4 V. The applied potential redistributes the counterions causing its concentration to increase locally above the electrodes, thereby facilitating L-L phase separation. Tests show that the protein molecules retain their integrity, configuration, and activity post deposition. The proposed technique for protein/microelectrode arrays is reversible, scalable, applicable to a range of proteins, and does not involve chemical modification of the protein or substrate. |
