Microscale electrokinetic assessments of proteins employing insulating structures

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Microscale electrokinetic systems have offer attractive characteristics for the analysis of proteins; in particular, systems that rely on both, linear and nonlinear electrokinetic effects, have proven to be effective platforms for sensing, sorting and purifying protein particles. The present contribution is focused on the assessment of protein samples (protein solutions, exosomes, protein nanocrystals) in devices that contains insulating structures for the generation of nonlinear EK fields. Such devices combine several electrokinetic phenomena that enable effective particle manipulation, especially when suspending medium and applied potential characteristics are carefully fine-tuned. An analysis of the latest findings on protein manipulation in microscale electrokinetic devices is presented and discussed, highlighting the valuable findings obtained by several leading research groups.

Introduction

Microscale electrokinetics (EK) methods have emerged as powerful techniques for analytical applications. The use of electric fields as stimulus to manipulate particles and fluids results in device simplicity an ease of integration. There is a wealth of knowledge on the manipulation of proteins by electrophoresis (EP) based methods, which rely on exploiting the electrical charge of the protein molecules; many of these systems also feature electroosmotic (EO) flow for driving the sample [1]. Highly selective systems can be achieved by also exploiting particle polarization effects. Dielectrophoresis (DEP) is referred as the motion of particles (charged and non-charged), due to polarization effects under the effects of nonuniform electric fields. DEP has grown significantly during the last decade due to its high flexibility and versatility. Dielectrophoretic-based devices can operate with DC and AC electric fields, as DEP does not depend on the field direction. Furthermore, DEP can be employed for both continuous sorting [2,3] and enrichment [4,5] of particles of interest [6]. Moreover, DEP has the unique and significant characteristic of being able to sort, separate and enrich particles across multiple scales; from macromolecules to parasites [6,7, 8, 9, 10]. Few techniques offer this exceptional capability, as traditional separation techniques like chromatography are more suitable for nano-scale particles such a proteins; while newer separation methods such as flow cytometry are mainly applicable to micron-sized particles.

DEP was discovered by Pohl in 1951, however, an important occurred four decades later, thanks to the advances in microfabrication techniques [6]. A plethora of DEP designs and systems have been developed since Pohl’s discover. A large majority of these systems can be as: electrode-based DEP (eDEP) or insulator-based DEP (iDEP). The former type of systems usually features arrays of microelectrodes, while the latter contains insulating structures that distort the distribution of an applied electric field. Figure 1 illustrates the concept for both types of systems. Particles can exhibit positive DEP (pDEP) or negative DEP (nDEP), depending on the relative polarizability of the particle when compared to the immersion medium. Positive DEP is when particles have a higher polarizability than the medium and migrate towards the regions with higher field gradient; nDEP is the opposite effect resulting in particle migration away from these regions.

The present review is focused on the recent advances of microscale EK methods that combine DEP, EP and EO for the characterization and enrichment of protein particles in systems that feature insulating structures. An analysis of the latest findings on protein manipulation with EK by several research groups is presented and organized by type device and operating conditions. The conclusions provide a summary and suggest future research directions.

Section snippets

First reports on DEP of protein molecules

Important pioneering studies on protein manipulation with DEP were carried out almost simultaneously during the mid to late 90’s and early 2000’s by two the Washizu and the Morgan groups [14••,15]. The late development of DEP of macromolecules was due to the belief that DEP of submicron particles was not possible, due to the effects of Brownian motion. The Washizu group achieved in 1994 the manipulation of protein particles with DEP, demonstrating that it was possible to generate DEP forces

Microposts systems

More recently, there has been an important increase on the number of studies focused on DEP of protein [14••,15,16]. An important portion of these reports employ iDEP channels with insulating posts (Figure 2a) under the regimes of trapping iDEP and streaming iDEP. Streaming or filamentary iDEP is when particle migration is influenced by both linear EK (EP and EO) and DEP effects; resulting in particles flowing in streamlines. Trapping iDEP is when DEP overcomes all other forces and particles

Nano-constrictions systems

Advances in microfabrication technologies have enabled the creation of nano-constrictions in iDEP devices. Several groups have taken advantage of the capability of high electric field gradient enhancement provided by submicron structures. The Chou and Swami groups have reported several studies that exploit nano-constrictions for the enriching biomarkers; in particular they employed high conductivity media and AC fields, which distinguishes their work from the previous studies cited here [5,26,27

Sorting iDEP systems

An important characteristic of iDEP systems is their ability to work across the regimes of streaming and trapping DEP [6,13]. Streaming iDEP allows for rapid particle sorting, in particular with multiple outlets. The Ros group also published on the sorting of protein nanocrystals with one-constriction iDEP systems [36,37]. Assessment of protein structure via protein crystallography requires a narrow size distribution of the crystals being analyzed, the Ros group designed a robust system for

Concluding remarks and future perspectives

Microscale systems that combine several electrokinetic phenomena (EP, EO and DEP) offer an attractive option for the analysis of proteins. The reports cited in this contribution illustrate the flexibility of these systems for assessing a wide array of proteins particles, ranging from protein solutions, to protein containing exosomes and proteins nanocrystals. The types of systems reviewed here spanned from traditional microchannel with micron-sized insulating posts, to micropipette-based system

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

The author acknowledges the financial support provided by the National Science Foundation (grant number CBET- 1705895).

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