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Copyright Brian J. Kirby. With questions, contact Prof. Kirby here. This material may not be distributed without the author's consent. When linking to these pages, please use the URL http://www.kirbyresearch.com/textbook.

This web posting is a draft, abridged version of the Cambridge University Press text. Follow the links to buy at Cambridge or Amazon or Powell's or Barnes and Noble. Contact Prof. Kirby here. Click here for the most recent version of the errata for the print version.

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Jump To: [Kinematics] [Couette/Poiseuille Flow] [Fluid Circuits] [Mixing] [Electrodynamics] [Electroosmosis] [Potential Flow] [Stokes Flow] [Debye Layer] [Zeta Potential] [Species Transport] [Separations] [Particle Electrophoresis] [DNA] [Nanofluidics] [Induced-Charge Effects] [DEP] [Solution Chemistry]

12.5 Protein and peptide separation [microchip separations top]

Proteinmeasurement, quantification, and separation are central to bioanalysis, since proteins are the primary mechanism by which cells perform tasks. A variety of analyses are important for bioanalytical chemistry, including protein separation, protein concentration, and immunoassays. Here we focus on microfluidic separation of proteins. We also include separations of peptides, which are sections of proteins, typically obtained through enzymatic digestion.

We first discuss protein properties that affect the transport issues related to separation, then describe separation modalities, then finally describe how these modalities can be combined to improve separation fidelity.

12.5.1 Protein properties

To understand protein transport, we must note several key properties. Proteins are electrically charged. All amino acids have amine groups (pKa 8) and carboxylic acid groups (pKa 4); in addition to this, many amino acids (e.g., lysine and arginine) have ionizable side groups. Because of this, proteins in general have measurable properties, such as anelectrophoretic mobility that is a function of pHand concentration, as well as anisoelectric point, i.e., a pH at which their electrophoretic mobility is zero. Proteins are roughly spherical and have an inflexible structure in their native state; thus they can be thought of as hard spheres (approximately) with a characteristichydrated radius of order 1-10 nm. Hydrated radius implies the characteristic radius that describes the protein as well as the layer of water molecules that are bound to that protein. Proteins can be denatured for analysis, i.e., their molecular structure can be disrupted, making them behave as long chains rather than fixed, tight spheroids. This is most commonly done with sodium dodecyl sulfate (SDS). SDS-denatured proteins are linear and highly charged, and behave much like DNA (see Chapter 14). In denatured state, proteins have a characteristic length rather than a characteristic radius. In denatured form, proteins have measurable properties such as electrophoretic mobility in bulk liquid (which tends to be roughly the same for all proteins) and in gels (which tends to be a function of protein size, with smaller proteins moving faster). The amino acids that make up proteins are of variable hydrophobicity and charge states, thus proteins tend to show different adsorption properties depending on how hydrophobic or hydrophilic they are or how strongly or weakly charged they are. Each of these properties is related to different protein characteristics, and motivates different separations, listed in the following section.

12.5.2 Protein separationtechniques

Given the properties above, many techniques exist (and have been applied on microfluidic substrates) for separating proteins.

Capillary zoneelectrophoresis

Capillary zone electrophoresis (or just capillary electrophoresis) is the technique described in the earlier sections of this chapter and involves injecting a sample bolus of liquid into a channel across which an electric field is applied. Each species i moves with a net electromigratory velocity given by

microfluidics textbook nanofluidics textbook Brian Kirby Cornell

Because the velocities of different species are different, the elution time t for each species is different. On microchips, samples are typically injected using pinched electrokinetic injections and detected using laser-induced fluorescence or electrochemical detection.

Microchip capillary electrophoresis has generally resulted in higher performance than macroscale CE for two reasons. First, capillary electrophoresis separations work most rapidly when high electric fields are applied; microchips can be used at high electric fields (a) because their lengths are typically short and even modest high-voltage power supplies can produce high electric fields and (b) microchips are much better heat sinks than capillaries and high fields can be applied without heating samples excessively. Second, pinched electrokinetic injectionscan be used to inject small samples (~ 100pL), which can then be separated rapidly at high-resolution.

Liquidchromatography

Liquid chromatography involves injecting a sample bolus of liquid into a channel and moving this bolus through the channel using pressure. The surface of the channel and the mobile phase (i.e., the fluid used to carry the protein sample) are both chosen such that proteins occasionally stick to the surface. Proteins elute (i.e., exit the end of the separation column) at a different time depending on their chemical affinity for the surface. For example, a channel with a hydrophobic wall coating causes more hydrophobic proteins to elute more slowly than hydrophilic.

Typically, chromatography is carried out in channels filled with porous media and thus a high surface area to volume ratio, using macroscopicHPLC systems (Figure 12.11).


microfluidics textbook nanofluidics textbook Brian Kirby CornellFigure 12.11: An agilent HPLC system. From http://www.agilent.com.


These porous materials require high pressure gradients, and these techniques are referred to ashigh-performance liquid chromatography or high-pressure liquid chromatography. Both terms are interchangeable and are abbreviated with HPLC. HPLC has been difficult to integrate into microchip systems, because filling microchips with surface-functionalized porous media can be difficult, high-pressure connections between macroscale devices (e.g., a capillary) and microchipsare difficult, as is flow control and injection at high pressures. Two solutions to this are (1) Hewlett-Packard’s HPLC-Chip and (2) laser-polymerized fluoroacrylate valves (Figure 12.12). Despite these challenges, HPLC separations on microchips have become common.


microfluidics textbook nanofluidics textbook Brian Kirby CornellFigure 12.12: A high-pressure picoliter HPLC injector mated with a photopolymerized reversed-phase HPLC separation column. From [124].



microfluidics textbook nanofluidics textbook Brian Kirby CornellFigure 12.13: Rapid microchip HPLC separations of a set of three proteins.


Isoelectricfocusing

All proteins have an isoelectricpoint or pI, namely the pH at which the protein is uncharged on average. Isoelectric focusing (See Figure 12.14) concentrates and separates proteins by exposing proteins simultaneously to a pH gradient and also an electric field. Proteins migrate based on their charge. Positively-charged proteins migrate toward the cathode, become exposed to higher-pH environments, and become more negatively charged. Negatively-charged proteins migrate toward the anode, become exposed to lower-pH environments, and become more positively charged. The steady-state solution has separate bands of proteins, each immobilized at its pI and containing all of the molecules of that protein from that sample.


microfluidics textbook nanofluidics textbook Brian Kirby CornellFigure 12.14: Isoelectric focusing. (a) a mixture of proteins is exposed to a pH gradient as well as an electric field. (b) the steady-state solution, with bands localized at their pI.


Establishing a pH-gradient requires use of carrierampholytes, which are mixtures of polyelectrolytes that help to establish the pH-gradient.

12.5.3 Isotachophoresisandfield-amplified sample stacking

Both of these techniques are ways to concentrate proteins using complicated buffer systems.

12.5.4 Capillaryelectrochromatography

Electrochromatography entails using electromigration to move analytes through a channel that is filled with a chromatographic material. Analyte migration is affected both by the analyte electrophoretic mobility and the analyte’s affinity for the chromatographic surface.

SDS-PAGE

SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separates proteins based on size in a manner similar to agarose gel separation of DNA. Sodium dodecyl sulfate is a surfactant that adheres to and denatures proteins, leaving them in elongated form with large negative charge owing to the sulfate groups.

Related work on chemical separations from our research group can be found here.

[Return to Table of Contents]



Jump To: [Kinematics] [Couette/Poiseuille Flow] [Fluid Circuits] [Mixing] [Electrodynamics] [Electroosmosis] [Potential Flow] [Stokes Flow] [Debye Layer] [Zeta Potential] [Species Transport] [Separations] [Particle Electrophoresis] [DNA] [Nanofluidics] [Induced-Charge Effects] [DEP] [Solution Chemistry]

Copyright Brian J. Kirby. Please contact Prof. Kirby here with questions or corrections. This material may not be distributed without the author's consent. When linking to these pages, please use the URL http://www.kirbyresearch.com/textbook.

This web posting is a draft, abridged version of the Cambridge University Press text. Follow the links to buy at Cambridge or Amazon or Powell's or Barnes and Noble. Contact Prof. Kirby here. Click here for the most recent version of the errata for the print version.


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