Thermally Assisted Acoustofluidic Separation Based on Membrane Protein Content

Abstract

The over- and under-expression of certain proteins in extracellular vesicles has been observed in many physiological and pathological conditions; however, a simple method to sort vesicles based on contrast in protein content is yet to be developed. We herein present a nonaffinity-based method for rapid and inexpensive isolation of lipid vesicles based on their membrane protein content. Based on a composition-specific thermophysical property change of vesicles at different protein contents, an acoustic property change that enabled an acoustophoretic separation was observed. This change was demonstrated in a thermally modulated acoustofluidic device in the form of a shift in vesicle migration from the nodal plane to antinodal plane at a specific temperature known as the acoustic contrast temperature (T Φ). Using phosphatidylcholine vesicles containing the membrane proteins gramicidin D, alamethicin, and melittin at molar contents ranging from 0.001% to 10%, we observed that increasing the membrane protein content brought about conformational changes in the membrane which afforded the vesicles distinctive acoustic properties. Then, by establishing an acoustic contrast temperature window, vesicles with the same protein but different molar content were successfully separated. The efficiency of the separation was studied for various vesicle mixtures and a separation efficiency as high as 97% was accomplished. In order to confirm the technique’s applicability for biological samples, sheep red blood cells with various melittin peptide contents similarly demonstrated the depressing effects of melittin on membrane bending modulus and depressed the T Φ of the cells. This method holds promise for a myriad of applications in the biomedical field, especially in bioanalytical research.

Trends and challenges in microfluidic methods for protein manipulation—A review

“…Reprinted with permission from Springer Nature. (C) Device schematic and experimental micrographs of acoustophoretic separation of vesicles based on their protein content [148]. Reprinted with permission from the American Chemical Society.…”

“…Mirtaheri et al. [148] took advantage of the properties of gramicidin, alamethicin, and melittin to assist in the separation of lipid vesicles with an average diameter of 1.41 µm. The separation was based on the protein content of the vesicles and the temperature dependence of the proteins, which leads to modifying their acoustic contrast when exposed to a change in temperature (Figure 3Ci).…”

“…Results demonstrated that the device can perform as well as standard centrifugation. Moreover, the content of albumin was even lower (2 g/L) to that achieved by centrifugation (3 g/L) with the added advantages of requiring far less space, no special and experimental micrographs of acoustophoretic separation of vesicles based on their protein content [148]. Reprinted with permission from the American Chemical Society.…”

Designer Exosomes: Smart Nano-Communication Tools for Translational Medicine

“…A step higher strategy such as double conjugation of tetraspanins rich exosome with luciferase and lipophilic fluorescent dyes could yield better results. Another avenue is acousto-fluidics; combining acoustics material in biocompatible fluids with nano-drug-loaded exosomes may serve as promising tools in bio-medical sensor detection in protein separation [ 82 ]. The functional efficacy of exosome-based delivery of drugs depends on the nature of the producer cells.…”

The Effect of Proteome and Lipidome on the Behavior of Membrane Bound Systems in Thermally-Assisted Acoustophoresis

“…Based on a composition-specific thermophysical property change of vesicles at various protein contents, an acoustic property change that enabled an acoustophoretic separation was observed. 187 This change was demonstrated in a thermallymodulated acoustofluidic device in the form of a shift in vesicle migration from the nodal plane to antinodal plane at a specific temperature known as the acoustic contrast temperature (TΦ). In this section, we examine the influence of membrane protein content (molar ratio) of particularly channel-forming proteins, on the acoustophoretic behavior of vesicles.…”