Capillary Gel Electrophoresis

Introduction: Protein Analysis through Electrophoresis

Gel electrophoresis is the separation of particles by their charge and mass through the application of an electric field. Briefly, when the field is applied charged particles migrate towards the cathode or anode, for negatively and positively charged particles respectively, at a speed proportional to their charge density, due to their electrophoretic mobility, and size and shape, due to gel filtration. Smaller molecules have more mobility in the gel than larger molecules, and thus molecules of a similar charge and size separate and move as a band through the gel, effectively separating the proteins. This is usually done in an agarose or polacrylamide gel, in between two glass plates or in a capillary. There are many different advantages to carrying out gel electrophoresis in a capillary as opposed to its' larger scale, making capillary gel electrophoresis (CGE) a useful tool for specific applications. How CGE works is that a small plug of the sample that needs separating is put into one end. Then, the electric field is put on the capillary, and components separate and move through the capillary in plugs.Capillaries used in CGE usually have diameters around 20-100 µm. This narrow diameter makes it such that lateral diffusion effects are negligent, and that high voltages can be used with very little temperature differences within the capillary. These high voltages mean a quick and efficient separation of small samples of product, as opposed to electrophoresis in a gel slab. The conditions for these separations are easy to reproduce, making CGE very useful for analytical applications, before moving it up to the higher scale. CGE, due to the gel that affects the velocity of the particles based off of size, is used in many biological applications for separating protein and DNA molecules, which may have similar charges but different sizes.

Figure 1.

Current Methods

Gel

For proteins, generally the method used for purification is SDS-PAGE. PAGE stands for polyacrylamide gel electrophoresis, which has a pH high enough (around 9) so that proteins generally have negative charges, and move in the same direction towards the cathode, and a set pore size so that the filtration in the gel is characteristic of size. SDS stands for sodium dodecyl sulfate, which is a detergent that denatures proteins so that the separation by size is based purely off of molecular weight, as opposed to other size factors in folded proteins. Agarose gel, which is better for larger molecules, is usually the gel used for separating DNA fragments.

Capillaries

Usually, the material used for capillaries for CGE is fused silica, which is heated and stretched to the desired dimensions to form the capillaries. However, there are challenges faced due to the silica capillaries, due to the negatively charged silanol groups that compose the inner diameter of silica capillaries. of One is electroosmotic flow (EOF). EOF occurs when liquid near a charged surface, such as in electrophoresis, causes bulk liquid flow at that surface. Due to the small inner diameter in CGE, which creates a high surface area to volume ratio, EOF can be a significant effect on fluid flow. EOF increases the velocity of the flow, which could push the protein solution out of the capillary prior to completing separation. The velocity of EOF is determined by this equation:

V=-(εζ/4πη)E

In this equation, ε is the dielectric constant, ζ is zeta potential, η is viscosity, and E is the potential applied with the electric field. Zeta potential, in terms of CGE, is the charge on the inner wall of the capillary due to the makeup of the capillary wall. Zeta potential is proportional to the charge density on the surface, and modifying the surface to reduce this zeta potential is thus a common way of reducing the effects of EOF.

Another problem with the capillary surface is that the proteins that need separating can end up interacting with the negatively charged silica wall, because of its' small diameter. Controlling this is very important, which is why the inner surface of silica capillaries is often coated or treated with base to reduce the effects of EOF and protein interactions with the wall. Treatment of base, as shown in the figure below, leads to hydrolysis of the silanol groups on the surface of the wall, which negates the negative charge of the surface and reduces the effects of EOF in the capillary. Materials that capillaries are generally coated with are polyacrylamide, polyvinyl alcohol, polyethylene glycol, and polyvinylpyrolidone, which all have high pH values. The way coating reduces EOF and protein interaction effects is that they alter the pH of the wall, or shield the negative charges of the wall, for the same reasons of reducing surface interactions.

Microchip Devices for CGE

CGE is done on a miniaturized microchip platform, making benefits from CGE being in a smaller system increase, for a simple and efficient method for analyzing proteins, and other biomedical materials. Column length in microchips is small, so this process can be done quite fast, this experiments can be reproduced quickly with many trials without much variation, with low cost. This miniaturization is important, because many biomedical analyses that use electrophoretic separation are used by the biotechnology industry. Biotechnology, pharmaceutical companies especially,can be pricey, and thus lowering costs of analysis for these uses would be beneficial for that purpose. Generally, PDMS chips on glass have been used for these purposes. This has already become a tool used for pharmaceutical analysis. It can be used to determine drug concentrations in fluids, which is important in knowing the kinetics and toxicity of the drug such that appropriate doses can be determined. PDMS based microfluidic devices are useful because they can be designed such that different channels can be running multiple analytical processes on a single chip. For example, as shown in the figure below, when separating DNA microchips can have a chamber for PCR to amplify the DNA, which can then flow into the separation chamber to separate the DNA strands. However, due to the incompatibilities in the different parts of the process, it has been difficult to get all parts of protein purification and analysis onto a single chip. Once progress is made on this, it could become a very important tool for drug development purposes.

References

1. Nuchtavorn, Nantana, Suntornsuk, W., Lunte, S., Suntornsuk, L. (2015) Recent applications of microchip electrophoresis to biomedical analysis. Journal of Pharmaceutical and Biomedical Analysis. 113, 72-96. http://dx.doi.org/10.1016/j.jpba.2015.03.002

2. Zhu, Zaifang, Lu, J., Liu, S. (2011) Protein Separation by Capillary Electrophoresis: A Review, doi: 10.1016/j.aca.2011.10.022

3. Whatley, Harry, (2001), Basic Principles and Modes of Capillary Electrophoresis, Pathology and Laboratory Medicine Part 1, 21-58, DOI 10.1007/978-1-59259-120-6_2

4. Morbioli, Giogio, Mazzu-Nascimento, T., Aquino, A., Cervantes, C., Carrilho, E.,(2016) Recombinant drugs-on-a-chip: The usage of capillary electrophoresis and trends in miniaturized systems- A review. Analytica Chimica Acta. 935, 44-57. http://dx.doi.org/10.1016/j.aca.2016.06.019

5. Fuji, Teruo (2002) PDMS-based microfludic devices for biomedical applications. Microelectric Engineering, 61-62, 907-914. http://dx.doi.org/10.1016/S0167-9317(02)00494-X