Dielectrophoretic separation of randomly shaped protein particles
Introduction
Bio-derived materials are an enduring feature of the human experience. Many bio-derived materials, such as silk, collagen, and wool, are made of proteins with repetitive amino acid sequences [1]. Tandem repeat sequence segments, or motifs, play key roles as the building blocks of structures and functions in these protein materials [2], [3]. Tandem repeat protein evolved via gene duplication, which is a major mechanism for new structures and functions. The potential for programmable biosynthetic materials has attracted increasing attention due in part to the discovery of these motifs. By combining the concept of biomimicry and theoretical foundations of block copolymers, these structural motifs have been exploited to design protein-based materials with remarkable new properties. Many reports have shown that these new materials can have interesting and useful mechanical, optical, and thermal properties [4], [5], [6], [7], [8], [9], [10], [11], [12]. Moreover, tandem repeat proteins govern the assembly of composites materials such as graphene oxide and MXenes (Ti3C2Tx) for 2D layered structures as well as self-healing characteristics of conducting polymers for applications in flexible electronics and biocompatible electronics [13], [14], [15].
Given the recent development of programmable protein materials, several applications to artificial drug-delivery systems have been reported [16], [17]. Crucial requirements for carrier materials in drug-delivery applications include high and constant loading of therapeutic agents, maintenance of physical structures and physicochemical properties [18] in the patient body during drug delivery, and harmless degradation after the delivery process is finished. To meet these requirements, a variety of separation methods have been used to isolate protein particles that have the desired properties. Centrifugation has been used with the sucrose-gradient technique to separate produced protein particles from non-compacted particles by sedimentation velocity [19]. Gel filtration chromatography is used to separate proteins by size and under native or denaturing conditions using porous gels [20]. In addition, various chromatography methods that utilize molecular interaction, affinity, and ion exchange are widely being used [21]. However, these approaches still remain challenging in the separation of proteins with the consistency of both size and morphology from all others.
Dielectrophoresis (DEP), an electrical particle-manipulation technique, has been utilized for a variety of biomedical applications such as particle separation and patterning [22], [23], [24], [25], intermolecular force spectroscopy [26], [27], localized heat generation [28], and drug delivery and discovery [29], [30], [31]. When dielectric particles are introduced into a non-uniform electric field, these particles experience electrical attraction or repulsive force according to the physical/electrical properties of the particles and media, and distribution of the electric field gradient, which is called the DEP force. Notably, DEP has been widely used for the manipulation of particles in various biological and biomedical applications because it offers advantages such as easy operation, high-throughput efficiency, tighter control, and less damage to the particles, compared with traditional techniques such as filtration, centrifugation, and electrophoresis [32], [33].
Here, we demonstrate dielectrophoretic separation of designer protein particles based on size and morphology within a microfluidic DEP trapping device in a single operation. The capability of the protein particles to deliver aqueous solvents was tested using a mixture of water and urea as a model. The protein powder was produced using recombinant expression in E. coli bacteria followed by single step purification. From the powder, protein aggregates with a distribution of sizes and morphologies were synthesized by precipitation from a solution of hexafluoroisopropanol (HFIP), one of the few solvents that can dissolve this type of protein. To facilitate the separation of this diverse mixture, we fabricated an array of circular openings on a chromium layered glass substrate with an indium-tin-oxide (ITO) counter electrode to apply an omni-directional DEP force to the center of a circular trap. The system allows the selective isolation of specific size of spherical protein particles from the diverse mixture.
Section snippets
Gene editing, protein expression and extraction
Construction of repetitive DNA constructs, their expression in E. coli and purification were well described in our previous work [6]. Briefly, unit gene (n = 1) fragments including cloning sequences were purchased from Genewiz (Fig. 1a). Double-stranded templates for protected digestion of rolling circle amplification (PD-RCA) were prepared by ScaI digestion, followed by blunt-end ligation. PD-RCA reaction was performed for 24 h (18 h of RCA and 6 h of PD), and then agarose-gel-purified
Protein particle characterization
To characterize the morphology and determine the size of the protein particles, the prepared stock dispersion (Fig. 3a) was examined by SEM and DLS measurement. As shown in Fig. 3b, the morphology of the protein particles investigated by SEM shows irregular shapes and dispersed sizes. We subsequently analyzed the size distribution of individual protein particles using DLS measurement as shown in Fig. 3c, and diameters from 1 µm to 26 µm were obtained. The average diameter was 2.91 µm with a PDI
Conclusion
In this study, we produced protein particles and selectively collected spherical 2 and 4-µm particles from a mixture with diverse sizes and shapes using a DEP trapping system. In addition, we validated the capability of these size- and shape-separated protein particles to store and release a solute in aqueous solution. The results show the capability of rapid one-step separation of protein particles by DEP.
The high swelling ratio of the size- and shape-selected protein particles suggests that
CRediT authorship contribution statement
Tae Joon Kwak: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Writing - original draft, Visualization. Huihun Jung: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization. Benjamin D. Allen: Conceptualization, Resources, Writing - original draft, Supervision. Melik C. Demirel: Conceptualization, Resources, Writing - original draft, Supervision, Funding acquisition. Woo-Jin Chang:
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MCD HJ and BA have published patents in tandem repeat protein design and synthesis. Other authors declare no competing interests.
Acknowledgments
We acknowledge the use of instrumentation at the Advanced Analysis Facility of the University of Wisconsin-Milwaukee. The authors also acknowledge Georgije Stanisic’s data analysis help. The authors are also grateful for the Distinguished Graduate Student Fellowship and Distinguished Dissertation Fellowship provided to T.J.K. by the University of Wisconsin-Milwaukee. MCD, HJ, BA are funded by the Army Research Office (grant no. W911NF-16-1-0019 and W911NF-18-1-0261) as well as Huck Endowment of
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- 1
This study was mostly done while T.J.K. was working at University of Wisconsin-Milwaukee.
- 2
T.J.K. and H.J. contributed equally to this work.