Mid‐scale free‐flow electrophoresis with gravity‐induced uniform flow of background buffer in chamber for the separation of cells and proteins

Abstract

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

Preparative free‐flow electrophoresis, a versatile technology complementing gradient centrifugation in the isolation of highly purified cell organelles

“…Connecting both devices to the inlets and outlets of the separation chamber, respectively, allowed an equalized buffer flow with minimized pulse of the peristaltic pump by exploiting the accessory gravitational forces in the system (see illustrations in for details). Based on this system, the group invented a mid‐scale FFE (7 × 4 cm separation chamber), which requires less buffer and sample flow than the classic FFE systems but still have the potential for extensive analyte sampling, required for a substantial biochemical or cell biological characterization of the samples . As a further improvement a thermoelectric cooling system combined with a ceramic base plate of the separation chamber was added to the preparative system .…”

Microalgae separation by inertia‐enhanced pinched flow fractionation

“…Dong et al developed a free-flow electrophoresis technique and separated Escherichia coli and Staphylococcus aureus effectively. This work provided a new idea for cell separation [7]. Similarly, He et al separated complete mitochondria from mitochondria resuspension buffer using the method of free-flow isoelectric focusing.…”

“…developed a free‐flow electrophoresis technique and separated Escherichia coli and Staphylococcus aureus effectively. This work provided a new idea for cell separation [7]. Similarly, He et al.…”

Purification of low‐abundance lysozyme in egg white via free‐flow electrophoresis with gel‐filtration chromatography

“…FFE has many merits: (i) continuous solute separation; (ii) high recovery due to absorbance matrix-free; (iii) high preservation of bioactivity due to mild aqueous circumstance fitting to requirement of enzyme and cell purification; (iv) low cost due to cheap buffers used as separation media; and (v) better resolution achieved in space [14,15]. Owing to these merits, FFE has been used for separations of numerous solutes, e.g., protein [16,17], organelles [18], DNA [19], chiral drug [8,20], cell membranes [21,22], malaria parasites, and viruses [23][24][25][26][27][28]. These target solutes generally existed as highcontent target solute in its sample matrix [16][17][18][19][20][21][22][23][24][25][26][27][28].…”