Localized Generation of Attoliter Protein Solution Droplets by Electrofocused Liquid−Liquid Separation

Abstract

We develop a simple technique for the generation of droplets of a high-concentration protein solution of attoliter volume and a diameter of >or=500 nm, containing several thousand protein molecules, and their accurate deposition on micron-size electrodes, standalone or in an array. The technique is based on liquid-liquid phase separation in the homogeneous region of the phase diagram, induced by a nonuniform weak electric field, which also accurately directs the generated droplets toward the electrode. We show that the protein molecules are not chemically modified and retain their integrity, conformation, and activity post deposition. The technique is applicable to the manufacture of various types of protein/microelectrode arrays and is tunable, scalable, and applicable to a broad range of proteins.

Ostwald-Like Ripening of the Anomalous Mesoscopic Clusters in Protein Solutions

“…(Note that the dense liquid is metastable with respect to the crystals, which would form under these conditions after about 2 days; the phase lines of the solution-crystal equilibrium are not shown in Figure a and can be seen in, e.g., ref ). The fast nucleation and growth of the droplets of stable dense liquid is consistent with previous microscopy observations. , After 15 min at 9 °C, temperature was raised to 12 °C, which is above the L-L binodal. Figure e demonstrates that the dense liquid droplets forms at 10 and 9 °C rapidly dissociate.…”

“…The fast nucleation and growth of the droplets of stable dense liquid is consistent with previous microscopy observations. 42,44 After 15 min at 9 °C, temperature was raised to 12 °C, which is above the L-L binodal. Figure 5e demonstrates that the dense liquid droplets forms at 10 and 9 °C rapidly dissociate.…”

The influence of low frequency of external electric field on nucleation enhancement of hen egg-white lysozyme (HEWL)

“…The waveform generator (MAX038, MAXIM, USA) was used to provide a 2.4 V/cm external electric field with adjustable frequency in the desired range of 1-1000 Hz. Noted is that, according to Shah et al [35], under this condition, electrolysis of water is negligible and the integrity of protein can be preserved. A Netchi-Denchi temperature controller and Peltier elements were used to maintain temperature of the crystallization cell with an accuracy of 0.1 1C throughout experiments.…”

“…On the theoretical side, it has been reported that two major intermolecular interactions, double-layer force and van der Waal force, are both dependent on the external electric field frequency [26,28]. Furthermore, Shah et al have shown the binodal temperature of liquidliquid separation of lysozyme has arisen but of peroxidase has reduced when an external electric field applied [29], which directly reflects the change of the intermolecular interactions between protein molecules [30].…”