# Proteomics/Protein Separations- Electrophoresis/Introduction to Electrophoresis

### Presentation

 Introduction to Electrophoresis

 « Proteomics Introduction to Electrophoresis » Previous Chapter: Protein Separations - Chromatography Gel Electrophoresis

Introduction to Electrophoresis

## Definitions

e•lec•tro•pho•re•sis (ĭ-lĕk'trō-fə-rē'sĭs) n. [1]
1) The migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes. Also called cataphoresis.
2) A method of separating substances, especially proteins, and analyzing molecular structure based on the rate of movement of each component in a colloidal suspension while under the influence of an electric field.

an•a•lyte (a-nə-līt) n. [2]
A chemical substance that is the subject of chemical analysis.

## Electrophoresis Theory

Separation by electrophoresis depends on differences in the migration velocity of ions or solutes through a given medium in an electric field. The electrophoretic migration velocity of an analyte is:

${\displaystyle v_{p}=\mu _{p}E}$

where E is the electric field strength and ${\displaystyle \mu _{p}}$ is the electrophoretic mobility.

The electrophoretic mobility is inversely proportional to frictional forces in the buffer, and directly proportional to the ionic charge of the analyte. The forces of friction against an analyte are dependent on the analyte's size and the viscosity (η) of the medium. Analytes with different frictional forces or different charges will separate from one another when they move through a buffer. At a given pH, the electrophoretic mobility of an analyte is:

${\displaystyle \mu _{p}={\frac {z}{6\pi \eta r}}}$

where r is the radius of the analyte and z is the net charge of the analyte.

Differences in the charge to size ratio of analytes causes differences in electrophoretic mobility. Small, highly charged analytes have greater mobility, whereas large, less charged analytes have lower mobility. Electrophoretic mobility is an indication of an analyte's migration velocity in a given medium. The net force acting on an analyte is the balance of two forces: the electrical force acting in favor of motion, and the frictional force acting against motion. These two forces remain steady during electrophoresis. Therefore, electrophoretic mobility is a constant for a given analyte under a given set of conditions.[3]

## Applications of Electrophoresis

Electrophoresis has a wide variety of applications in proteomics, forensics, molecular biology, genetics, biochemistry, and microbiology.

One of the most common uses of electrophoresis is to analyze differential expression of genes. Healthy and diseased cells can be identified by differences in the electrophoretic patterns of their proteins. Proteins themselves can also be characterized in this way, and some sense of their structure can be derived from the masses of fragments inside the gel. [4]

There are many different types of electrophoresis, and each can be used for something different. Two-dimensional (2-D) electrophoresis, for example, has the ability to discern many more proteins than most of its contemporaries. Many of these methods will be discussed in detail throughout this chapter.

## References

1. ^ The American Heritage Dictionary of the English Language, Fourth Edition. http://www.bartleby.com/61/
2. ^ The Merriam-Webster Online Dictionary. http://www.m-w.com
3. ^ Mans, Andreas et al. Bioanalytical Chemistry. Imperial College Press, 2004.
4. ^ Twyman, Richard. "Two-dimensional polyacrylamide gel electrophoresis." http://genome.wellcome.ac.uk/doc_wtd021045.html

Next page >

## Microfluidic Electrophoresis

### What Will Proteomics Gain from Microfluidic?

Proteomics contributes significantly to the discovery of proteins and their functions that influence the behaviors of an organism. Recent studies have focused on its roles to explore the proteins in a single cell and tissue level as they represent a fingerprint for each individual especially in terms of how a disease exhibits.[1] Due to a limited amount of sample from a single cell or tissue, the study of proteome in these levels becomes a big challenge for a regular benchtop instrument. To facilitate this level of study, a microfluidic technology is introduced and developed into an essential tool for proteomics.

In addition to the capability to handle small sample, the microfluidic technology plays an important role in miniaturizing the entire system. As a result, a better performance in terms of less material consumption, faster processing time, more automated, and lower cost can be achieved. Another advantage in such microscale is that the mixing between the sample and reagents becomes more effective.[2] A certain chemical process that usually takes hours can be completed in minutes. This benefit allows the microfluidic-based immunoassay to be used to monitor the progress of disease.[1] And, with a reduction in cost, the microfluidic device becomes a perfect candidate for many point-of-care applications.[3]

One of the most intriguing features provided by the microfluidic technology is its highly integrated capability with other systems especially with mass spectrometry.[4] Multiplexing of various assays is another example. This multiplexing potential makes microfluidic-based devices a high throughput solution in (bio)chemistry and biomedicine.[5]

### Electrophoresis in Microfluidic Microsystems

Incorporating biotechnology with microfluidic makes a manipulation of very small volume of biological fluid not only feasible, but also effective especially by means of electrophoresis. Recent research and development efforts have been focused on inventing an electrophoretic microsystem that is fully automated, easy to customize for a specific need, and provides the results consistent with the gold standard. This microfluidic microsystem is usually referred to as a lab-on-a-chip. Among the microfluidic microsystems used in the analytical (bio)chemistry, the most widely used methods to control the transport of biomolecules or analytes are either gel or capillary electrophoresis.

Even though both techniques utilize the fact that biomolecules such as proteins, peptides, and DNA become charged in a buffer, the microfluidic gel electrophoresis operates differently from its capillary counterpart in terms of fluid dynamic. In microfluidic gel electrophoresis, the presence of porous gel medium prevents a bulk flow in a microfluidic channel.[3] On the other hand, the bulk flow becomes an engineering factor that influences the electrokinetics of the analytes as the presence of electroosmotic flow needs to be considered as well in microfluidic capillary electrophoresis.[6] [7] In the analytical viewpoint, their electrophoretic separation methods are also different. The gel-based electrophoretic separation of biomolecules is based on the difference in size or molecular weight, while the capillary-based electrophoresis separation operates by taking the advantage of the difference in charge-to-mass ratio among biomolecules.

In comparison to the gold-standard methods, the analytical results obtained from the microfluidic gel and capillary electrophoresis are consistent with those from the traditional slab gel [8] and capillary electrophoresis [5], respectively. In the design and application viewpoints, however, there are some advantages and disadvantages of these two methods needed to be considered. In terms of engineering design, the microfluidic gel electrophoretic system is much easier to customize for specific applications and multiplexing since no influence from the bulk flow needs to be considered. This makes it more straightforward to integrate extra features like sample preprocessing into the microfluidic gel electrophoresis.[3] Nevertheless, since the gel-sieving medium is not required in the capillary-based microsystem, the reusability of the microfluidic capillary electrophoretic device is much higher than the gel-based counterpart. In terms of bio(analytical) applications, the microfluidic capillary electrophoresis has a drawback such that it operates poorly to analyze the charged particles of similar charge-to-mass ratios.[3] It is worth noting that some microfluidic capillary electrophoretic system is capable of direct interface with mass spectrometry.[5]

In this chapter, the device fabrication process, basic principle of operation, and some clinical applications of both microfluidic gel and capillary electrophoresis are described in detail. Even though this chapter is dedicated mainly to the microfluidic electrophoresis, the integration of additional features like sample preprocessing, detection, and quantification processes are also included. Unquestionably, this integration is made possible by the microfluidic technology.

### Microfluidic Gel Electrophoresis

Utilizing microfluidic technology in gel electrophoresis provides several advantages to the study of proteome in many ways that cannot be achieved by the conventional methods. Faster processing time, more sensitive detection, more automated operation, and highly integrated system are the major benefits of using microfluidic. In addition, the microfluidic technology allows gel electrophoretic system to be easily customized for a specific application. For example, a microfluidic gel electrophoretic system can be designed such that off-chip processing can be eliminated. In fact, it can be integrated into the microfluidic-based system. The integration of sample preparation is one of the practical examples that not only simplify the experiment protocol, but also improve the detection sensitivity.[1]

This section is dedicated to a customized microfluidic gel electrophoresis for immunoassay applications.[3] The important aspects such as the fabrication process, principle of operation, and clinical applications will be discussed in detail.

#### I. Fabrication Process

Gel electrophoresis can be customized for a specific analytical study such as an immunoassay by using microfluidic technology. The customized fabrication process of microfluidic gel electrophoretic immunoassay to be described below is based on the device used by Herr et al.[1] [3] In their study, an antibody was used as a reporter to detect the presence of a particular protein as an antigen, potentially a disease biomarker. A step-by-step fabrication process is described as follows.

Step 1: Fabrication of size-exclusion membrane [3]

Step 1: Fabrication of Size-Exclusion Membrane

Materials:

1. Degassed 22% (15.7:1) acrylamide/bis-acrylamide (6% bis-acrylamide cross-linker)
2. 0.2% (w/v) VA-086 [9] (photoinitiator)
3. 1X Tris/glycine buffer

The first component of the microfluidic gel electrophoretic immunoassay to be fabricated is a size-exclusion membrane. This membrane is used to enhance analyte concentration, thus improving sensitivity of detection. A polyacrylamide gel is utilized to fabricate this membrane. It is designed such that the polyacrylamide gel has a pore size small enough for selectively allowing analyte molecules smaller than 10 kDa to pass through. The fabrication process begins with patterning a glass substrate to create microfluidic channels and chambers. This process is carried out by using regular photolithography and wet etch. Holes are drilled on the top glass cover. Both glass substrate and cover are then bonded together by anodic bonding. After the microfluidic structure is created, a solution of degassed 22% (15.7:1) acrylamide/bis-acrylamide (6% bis-acrylamide cross-linker) and 0.2% (w/v) VA-086 is introduced into the channel, as shown in the diagram. A syringe can be used to load the solution into the channel. The gel precursor solution is left for equilibration for approximately 30 minutes.

The size-exclusion membrane is fabricated using laser photo-polymerization. A 355-nm UV laser sheet is used to pattern the polyacrylamide gel at the specified location, shown in the diagram, to create the membrane profile. The polyacrylamide gel membrane is exposed to the laser until polymerized, which takes approximately 15 seconds. The remaining gel solution is vacuumed out, and the channels are then cleaned by rinsing with buffer.

Step 2: Fabrication of separation channel [3]

Step 2: Fabrication of Separation Channel

Materials:

1. Degassed 8% (37.5:1) acrylamide/bis-acrylamide (2.6% bis-acrylamide cross-linker)
2. 0.2% (w/v) VA-086 [9] (photoinitiator)
3. 1X Tris/glycine buffer

After the size-exclusion membrane is fabricated, the next step is to construct a separation channel. This separation channel is the place where protein separation takes place. It contains a medium porosity polyacrylamide gel. Like the membrane, the separation gel is fabricated by photo-polymerization. To fabricate the separation channel, the separation gel precursor solution is carefully loaded into the microfluidic channel by a syringe. The gel solution is composed of the degassed 8% (37.5:1) acrylamide/bis-acrylamide (2.6% bis-acrylamide cross-linker), 0.2% (w/v) VA-086 photoinitiator, and 1X Tris/glycine buffer. The gel loading direction is indicated by the arrow shown in the diagram. The gel is loaded up to the specified location to define the separation channel.

Gel uniformity is a very important factor for the repeatability in the analysis. To guarantee uniformity, all microfluidic channels on the separation side of the membrane must be filled with gel before polymerization. Therefore, a gel plug is created to prevent the gel leakage during the subsequent gel loading. As shown in the diagram, the gel plug is fabricated by photo-polymerization such that the area not to be polymerized is protected by a dark-field mask. Usually, the photo-polymerization process takes about 10 minutes using a 100-Watt UV source.

File:UCEI Step 3.png

Materials:

1. Degassed 3.5% (37.5:1) acrylamide/bis-acrylamide (2.6% bis-acrylamide cross-linker)
2. 0.2% (w/v) VA-086 [9] (photoinitiator)
3. 1X Tris/glycine buffer

In this step, the remaining microfluidic channels (without gel) on the separation side of the membrane are carefully filled with gel precursor solution as indicated by the arrow shown in the diagram. The solution contains the degassed 3.5% (37.5:1) acrylamide/bis-acrylamide (2.6% bis-acrylamide cross-linker), 0.2% (w/v) VA-086 photoinitiator, and 1X Tris/glycine buffer. This gel solution will generate a polyacrylamide gel with large pore size and define the loading channels.

After both separation and loading channels are filled with gel, the whole microfluidic device is exposed to UV for 15 minutes. As a result, the separation and loading gels are polymerized and define separation and loading channels, respectively. This microfluidic device is now ready to use.

Complete microfluidic gel electrophoretic device [3]

Complete Microfluidic Device

The diagram shows the complete microfluidic gel electrophoretic device after fabrication. The device contains polyacrylamide gels with three different pore sizes. The gel with largest pore size is used in the loading channels to facilitate the electrophoresis of the sample and reagents by preventing bulk flow. The gel with intermediate pore size is used in the separation channel for protein separation. Finally, the gel with smallest pore size is used as a size-exclusion membrane for the enrichment process. The detail descriptions about their functions will be covered in the section entitled Principle of Operation.

In the device design, all the loading channels are connected to the loading areas, which are holes drilled at the beginning (before anodic bonding). The sample and reagents are loaded into there. In addition, some holes are used as reservoirs for waste collection. These are the places to which the electric current flows. It is worth noting that in addition to being the loading areas, they are also used as the insertion points for electrodes, which are connected to a programmable supply voltage source.

When not in use, this microfluidic gel electrophoresis device must keep submerging in the buffer and store at 4°C.

#### II. Principle of Operation

Based on the same principle of electrokinetic transport of charged molecules, the microfluidic gel electrophoresis works similarly to the regular slab gel electrophoresis, but with much faster processing, more sensitive detection, and highly automated-integrated systems. In this section, the fundamental operation of the microfluidic gel electrophoresis will be discussed using the microfluidic structure described in the previous section.

The microfluidic gel electrophoretic system to be discussed consists of a structure containing microfluidic channels/reservoirs, power supply, and fluorescence detection system. The channels and reservoirs used to transport the analyte are designed such that they are filled with large pore size polyacrylamide gel. This large pore size gel facilitates the electrokinetic transport of the analyte by preventing the bulk flow.[3] The power supply is designed so that it is programmable. A pair of electrodes and their polarities can be assigned instantaneously. This provides a much better control over the electrokinetic process only attainable in microfluidic environment. Also integrated into the system is the fluorescence detection capability. It is used to detect the analyte of interest and quantify its concentration.

In this section, a step-by-step operation procedure including the principle behind each step will be discussed, assuming that the target analytes are negatively charged. Note that the discussion will mainly focus on the immunoassay application, which is more generalized to the study by Herr et al.[3] A little modification can be made to the system to be used in other applications.

As a tool for immunoassay study, a reporter specific to a particular protein of interest is used. Acting like a receptor with high affinity for a target protein, an antibody can be used as the reporter for the detection of protein or antigen being investigated. Without loss of generality, the term reporter will be used throughout this section.

To begin using the device, the microfluidic channels need to be filled with buffer solution. Then, the reporter solution of known concentration together with sample solution is loaded into the designated reservoir. Typically for the microfluidic device, the volume required per analysis is roughly in the order of a few tens of microliters. For the purpose of detection and quantification, the reporter is usually labeled with fluorescence tag. The detection and quantification methods will be discussed in detail later.

After the fluorescently labeled reporter is loaded, a pair of electrodes connected to the reporter and sample waste reservoirs are activated as shown in the diagram. The electric potential is then applied across both electrodes. As the reporter molecules become charged in the buffer, they are electrokinetically moved toward the sample waste reservoir as indicated by the red arrows. The reporter molecules, however, are blocked by the size-exclusion membrane, which allows only particles of size less than 10 kDa to pass through. In this loading step, only the ionic buffer can penetrate the membrane. At the end of this first electrophoresis, the increasing number of the fluorescently labeled reporter molecules gathers at the membrane on the gel side as shown in the inset.

The next process is to load the sample electrokinetically to combine with the reporter, which already gathered at the membrane. Like the reporter, the analyte molecules become charged in the buffer. To begin the electrophoresis, the electrode contacted to the reporter reservoir is deactivated. The electrode contacted to the sample reservoir is switched on instead. The potential across these two electrodes causes the charged molecules of proteins in the sample to move electrokinetically toward the sample waste reservoir as indicated by red arrows.

Once the charged molecules of the analyte arrive the membrane, some of them whose size less than 10 kDa pass through the membrane and are collected in the sample waste reservoir. Only the larger molecules including the protein of interest stay on the gel size of the membrane as shown in the inset. This process helps increase the concentration of the analyte, improving the probability of binding between the reporter and target protein. However, the non-target proteins become more concentrated as well and might cause a reduction in signal-to-noise ratio level. Using the reporter of high binding specificity can alleviate this problem. It is worth noting that the sensitivity and dynamic range of the microfluidic gel electrophoretic immunoassay depend on this enrichment process.

It needs to be designed carefully so that the electrophoresis occurs long enough so that more reporters and the target proteins are bound together. Normally, the concentration of the reporter used is much higher than that of the target protein. As a result, all the target protein molecules would be more likely to be detected by the reporter molecules. At the end of this second electrophoresis, the reporter and its complex including the remaining non-target proteins stay on the gel side of the membrane.

Switching electrodes to avoid running electric current through the membrane during electrophoretic separation [3]

Avoid Running Electric Current Through Membrane During Separation

It has been reported that the gel electrophoretic separation experienced irreproducible results when running the electric current through the membrane.[3] Therefore, it is necessary to avoid applying the electric potential across the membrane especially during the separation process. One possible solution is to program the power supply such that the electrodes are switched before the target analytes enter the separation gel. The diagram shows the switching process that satisfies this requirement.

According to the diagram, the electric potential is initially applied across the membrane just to transport the analytes away from the membrane. Before the analytes enter the separation gel, the electrode at the upper left buffer reservoir is deactivated. At the same time, a new electrode with the same polarity at the upper right buffer reservoir is turned on. It can be seen that the flow of negatively charged analytes remains in the same course toward the buffer waste reservoir.

It is worth noting that there are other combinations of electrodes that can be used to perform this step as well. The concept is simply not to allow electrophoresis to occur across the membrane during the separation step. At the end of the third electrophoresis, all charged molecules are ready for the gel electrophoretic separation.

Electrophoretic separation of unbound reporter and its complex detectable by laser-induced fluorescence [3]

Gel Separation

In this step, the reporter molecules and their complex are separated by means of gel electrophoresis. The process continues by electrokinetically carrying the charged molecules into the separation gel. Since the reporter molecules and their complex have similar charge-to-mass ratio, the separation between these two species is based only on the difference in their molecular weights (MWs). It is worth noting that all non-target proteins are not investigated in this immunoassay. Only the reporter molecules and their complex are under examination.

Since the reporter molecules are labeled with fluorescent dye, a single-point laser can be used to induce the fluorescence. This laser-induced fluorescence (LIF) is monitored by a detector that will detect the presence of the unbound reporter and its complex, and then relate the intensity of the detected fluorescence to the concentration of both species. This fluorescence detection system is placed across the separation channel near the buffer waste reservoir. The concepts of detection and quantification are described next.

Fluorescent detection, electropherogram, and gel-like plots [3]

Fluorescence Detection and Results

The single-point laser-induced fluorescence (LIF) is a mean used to detect and quantify the reporter molecules and their complex. When the molecules across the laser beam, the attached dyes emit fluorescence whose intensity is measured by the detector. The detector then generates the electropherogram corresponding to the measured intensity. The area under the electropherogram peak can be related to the concentration of the target protein.

The unbound reporter can be distinguished from the complex based on the fact that the unbound reporter molecule has lower molecular weight (MW) than its complex. Therefore, the unbound reporter molecules travel electrokinetically faster in the separation gel and are detected first. The corresponding intensity gives the first peak in the electropherogram.

In case of a single pair of ligand and receptor, there will normally be two peaks in the electropherogram. Usually the first peak belongs to the receptor or reporter of smaller MW, followed by the second peak corresponding to the ligand-receptor complex of larger MW. This information can also be represented by gel-like plots, which are computer-generated. In the gel-like plots, the topmost band represents the unbound reporter molecules that arrive first. The brightness of the band conveys the information about the degree of fluorescence intensity detected. The width of the band corresponds to the width of the peak, which conveys the information about the migrating time of the analyte.

It is worth mentioning that the probability of binding between both receptor and ligand is a significant factor to the sensitivity and accuracy of the immunoassay. Therefore, the enrichment process plays an important role in raising this probability. To enhance this enrichment process, either preprocessing the sample (off-chip) or increasing the period of the electrophoretic sample loading is proven to be useful.

#### III. Clinical Applications

One of clinical applications reported was the use of this microfluidic gel electrophoretic device in assisting oral diagnosis.[1] [3] It was used for early diagnosis of the periodontal disease and to monitor the disease state and its development from human saliva. Periodontal disease or periodontitis is a putative oral disease that destroys collagen and causes a major tissue damage, connective tissue attachment loss, and bone loss. Found in the periodontitis patient saliva were matrix metalloproteinase-8 (MMP-8), interleukin-1 beta (IL-1B), and C-telopeptide pyridinoline cross-links (ICTP). These proteins were identified as disease biomarkers and became the target proteins for detection and monitoring this oral disease.[1] Herr et al. demonstrated the detection and quantification of MMP-8 using this microfluidic gel electrophoretic device.

In their study, the monoclonal antibody for MMP-8 was used as a reporter, which had the specificity for binding only to the MMP-8 in the saliva. The use of this monoclonal antibody (${\displaystyle a}$MMP-8) had an advantage in that it eliminated the need for a matched pair antigen-antibody. In the reporter mixture, the bovine serum albumin (BSA) protein standard was also added as a reference. Both ${\displaystyle a}$MMP-8* and BSA* were fluorescently labeled for the detection purpose. Note that the asterisk is used to denote the fluorescently labeled analyte. According to their study, 1 nM of ${\displaystyle a}$MMP-8* and 1 nM of BSA* were used in the mixture.

In the microfluidic device, ${\displaystyle a}$MMP-8* bound only to MMP-8 biomarker forming a complex of similar charge-to-mass ratio. The unbounded antibody and its complex were separated from each other by the separation gel based on the difference in their sizes and detected individually as described in the Principle of Operation for microfluidic gel electrophoresis. In this immunoassay application, the BSA* with the highest mobility was detected first, followed by the remaining ${\displaystyle a}$MMP-8* and MMP-8 complex, respectively. The corresponding peaks were shown in the electropherogram and gel-like plots. The peak area or the width of the band in gel-like plot was used to calculate the concentration of MMP-8.

To be able to quantify the concentration of the endogenous MMP-8 in the saliva sample collected from the patients, a calibration curve was required to be generated first. The calibration curve was obtained from the analyses of known MMP-8 concentrations. A series of experiments were performed by adding known concentrations of the recombinant MMP-8 in the diluted saliva from healthy patients and then running the microfluidic gel electrophoresis. From the electropherogram, the peak areas of the MMP-8 complex were normalized with the peak area of BSA* and then plotted against the corresponding concentrations. A nonlinear least-squares fitting method using a four-parameter logistic model [3] was used to obtained the calibration curve. Based on this calibration curve, the concentration of the endogenous MMP-8 complex was predicted from its normalized peak area. It was reported that the average concentrations of the MMP-8 in the healthy and disease subjects were 64.6 +/- 16.4 ng/mL and 623.8 +/- 204.0 ng/mL, respectively.[3] It is worth noting that the concentration of MMP-8 in the patients classified as periodontally diseased exhibited the dynamic activity of the disease.

The validity of the results obtained from the microfluidic gel electrophoresis was verified by comparing with those obtained from the conventional enzyme-linked immunosorbent assay (ELISA). As reported by Herr et al., the results obtained from the microfluidic device were highly correlated with those from ELISA with r2 = 0.979, where r is Pearson product-moment correlation coefficient. This microfluidic-based immunoassay, however, had several advantages over the conventional counterpart in that it bypassed the time-consuming reaction and washing steps required by ELISA, was more automated, required only single antibody making it applicable for wider range of applications, and needed much less amount of saliva sample per analysis. In addition, it did not require surface immobilization of the antibody.

In addition to measuring the concentration of MMP-8 in the periodontitis patients, a clinical examination was also performed to correlate the analytical data with physiological symptoms. It was found that MMP-8 was highly correlated with bone and tissue loss, but having no correlation with bleeding upon probing. With highly sensitive detection of MMP-8 biomarker, this microfluidic-based immunoassay could provide early diagnosis that would improve the clinical treatment of this disease. Furthermore, with the use of MMP-8 inhibitor to reduce collagen degradation, it was promising that this microfluidic gel electrophoretic device could be used to monitor and track the progress of MMP-8 inhibitor therapy as well.

### Microfluidic Capillary Electrophoresis

In this section, another type of microfluidic electrophoresis will be described. Unlike microfluidic gel electrophoresis, microfluidic capillary electrophoresis operates on the principle of electrokinetic of bulk flow. The difference in charge-to-mass ratio of analytes is the fundamental of electrophoretic separation in capillary microfluidic channel. Without the need of sieving medium, the microfluidic capillary electrophoresis becomes more favorable than its gel counterpart in terms of the ease in fabrication and reusability. The detail fabrication process and principle of operation including clinical applications of this device are described as follows. Note that the fabrication process and principle of operation explained below are based on the published work by Backofen et al. 2002.[10]

#### I. Fabrication Process

The fabrication of microfluidic structure using PDMS is easier and much less expensive than using glass. The process begins with creating a master for molding the PDMS, which can be obtained by patterning a photoresist. After developing, a mixture of PDMS and cross-linker is poured over the patterned photoresist and then cured. The PDMS is peeled out and cut to create an access to the reservoir. In the final step, this patterned PDMS is attached permanently to a glass base and ready to use. The detail step-by-step procedure is described below.

Diagram describing a fabrication process to create a master for PDMS molding using negative photoresist and standard photolithography [10]

Creating a Master for PDMS Molding

Materials:[10]

2. 4-inch silicon wafer
3. SU-8 50 negative photoresist
4. Propylene glycol methyl ether acetate developer

Beginning with a preparation process, a photomask is created first. The photomask used is usually a contact photomask that can be made of glass or simply a transparency with the designed microfluidic structural layout printed on it. The photomask is used to transfer this microfluidic pattern on to a photoresist, which is spin-coated onto a silicon wafer. It is worth noting that the choice of photoresist to be used affects the design of photomask. For example, if using the negative photoresist, all the microfluidic patterns need to be designed using clear field so that the UV light can go through and polymerize the contact areas of the negative photoresist. The unexposed areas will be washed away in a developer. On the other hand, the designed patterns are the dark field of photomask for the positive photoresist.

Before coating the photoresist, the silicon wafer is needed to be cleaned first. A standard RCA cleaning process is applicable. Then, the negative photoresist is spin-coated on the silicon wafer and pre-baked. Exposing under the UV light, the coated negative photoresist is patterned with the designed microfluidic layout from the photomask. The exposed photoresist is then post-baked and developed. As a result, only the exposed areas of the negative photoresist remain as shown in the diagram.

Diagram showing the PDMS molding process using the developed, patterned photoresist [10]

Molding PDMS to Fabricate Microfluidic Structure

Materials:[10]

1. PDMS oligomer

After the master for PDMS molding is ready, a mixture of PDMS and cross-linker is prepared. A 10:1 ratio of the polymer and cross-linker is applicable.[10] The degassed mixture is then poured onto the wafer to cover the entire photoresist master. Finally, the molded PDMS is cured in the oven at 70 °C for 1 hour.[10] This process step is summarized in the diagram. In addition, a cross-sectional view of the PDMS molded on the wafer is also provided.

After curing, the PDMS is peeled out of the wafer. The PDMS with the transferred microfluidic structure is further processed by creating an opening for each circular reservoir region. This opening will be used to load sample or reagent and a place to apply vacuum and pressure, which is regulated by a syringe.

Diagram showing the complete microfluidic capillary electrophoretic device [10]

Finalize Microfluidic Capillary Electrophoretic Device

Materials:

1. Glass plate
2. Needle probe

A glass plate with coated patterned electrodes is used to enclose the microfluidic channels and reservoirs, and at the same time, provides electrical connections to the fluid in reservoirs. The electrodes can be fabricated on glass substrate by means of evaporation of chrome and platinum. Chrome is used as an adhesive layer between glass substrate and platinum electrodes. The patterning of the metals can be achieved by a regular photolithography, which requires another photomask (a transparency), to form the electrodes on glass. It is worth noting that a combination of patterned electrodes and needle probe is used in the final microfluidic capillary electrophoretic system as shown in the diagram. The platinum needle probe is used to supply high electric potential for electrophoretic purpose and simultaneously as an electrochemical sensor.

The PDMS is permanently attached to the glass plate by oxidation using plasma. A covalent bonding between oxygen and silicon atoms (O-Si-O) provides a permanent seal between both materials. The complete microfluidic capillary electrophoretic device is shown in the diagram.

#### II. Principle of Operation

In general, the operation of microfluidic capillary electrophoretic device can be described by three fundamental steps – loading sample/reagents, forming sample plug, and electrophoretic separation. The loading process described here involves manual loading using syringes to regulate the flow. After all sample and regents are loaded, a sample plug is formed. This step needs to be designed carefully since the concentration of the analytes relies on this step. In the last step, the analytes are separated electrophoretically. Unlike the size-based separation in the (microfluidic) gel electrophoresis, the separation by (microfluidic) capillary electrophoresis is based on the differences in charge-to-mass ratios of the analytes.

It is worth noting that the microfluidic capillary electrophoretic system being discussed here is used as an analytical platform where all the sample preprocessing steps are performed off-chip. The following description focuses mainly on the analysis of negatively charged analyte in the sample. Note that the principle of operation described below is based in part on the published work by Backofen et al. 2002.[10]

All microfluidic channels filled manually and equilibrated by electrophoresis [10]

The system setup begins with loading the microfluidic reservoirs with a buffer and preprocessed sample solution such that all the reservoirs except the sample reservoir contain the buffer. Continuing with filling the microfluidic channels with buffer, syringes are connected to the top openings of the reservoirs and used to regulate the flow. Vacuum and pressure are applied until the channels are filled with the buffer.

The next process is to equilibrate the sample and buffer by means of electrophoresis. To do so, the high voltage sources are connected to the system as shown in the diagram. One possible configuration is the use of multiple supply units such as two units of U1 = 0.5 kV and a single unit of U2 = 1.2 kV. This process allows the buffer and sample to localize in the channels as shown in the diagram.

Sample solution flowing through the small connecting channel and entering the channels leading to the buffer and buffer waste reservoirs [10]

Forming Sample Plug

For the sample to be analyzed, a predefined and controllable portion of the sample needs to be injected into the separation channel. This predefined and controllable portion of the sample is referred to as a sample plug. According to the microfluidic structure being discussed, the sample plug is formed by a hydrodynamic flow of the sample solution due to the difference in the solution levels in the reservoirs. This hydrodynamic flow only occurs when all the high voltage sources are switched off.

As depicted by the diagram, the portion of sample solution flows through the small connecting channel into the channels leading to the buffer and buffer waste reservoirs. This sample portion can be divided into two ports indicated by blue and yellow dashed outlines as shown in the inset. The blue outline is the sample plug whose volume can be specified by design. The yellow dashed outline indicates the portion of the sample that will flow back toward the sample waste reservoir when switching the high voltage sources back on.

Sample plug electrokinetically moving along the separation channel toward the buffer waste reservoir [10]

Electrophoretic Separation

When switching on all the high voltage sources, the negatively charged analyte molecules and buffer ions start to flow again by electrophoresis. At the small connecting channel, the portion of sample solution begins to split into two parts and then move away in the opposite directions. As depicted in the inset, the part of sample solution indicated by blue-dashed outline, or the sample plug, electrokinetically moves along the separation channel toward the buffer waste reservoir. On the other hand, the part of sample solution outlined by the yellow dashed line electrokinetically move away toward the sample waste reservoir. The small connecting channel is then filled by the buffer as occurred originally.

In the separation channel, the negatively charge analytes in the sample plug are separated due to the differences in their charge-to-mass ratios. Each analyte is detected by the electrochemical sensor placed at the end buffer waste reservoir. For the electrochemical sensor used in this microfluidic capillary electrophoretic system, the detection of each analyte is based on a reduction in voltage drop across the needle probe and reference electrode. These voltage drops can be plotted in the electropherogram and can be used to study the analyte components such as proteins or peptides in the sample solution.

#### III. Clinical Applications

The detection and quantification of biomarkers in patients with skin lesions reported by Guzman et al. 2008 [5] is one of the clinical applications for the microfluidic capillary electrophoresis. Based on the same fundamental concept described previously, a more sophisticated design of microfluidic capillary electrophoresis so-called the immunoaffinity capillary electrophoresis (IACE) was used in their study. IACE is a lab-on-a-chip that utilizes the microfluidic technology to incorporate the affinity-based purification, enrichment, and electrophoretic separation processes in one single microchip.

In the clinical study of biomarkers for this inflammatory disease, IACE was used to analyze the micro-dissected samples from the patients with different stages of skin damage. Twelve different antibodies were used to capture twelve corresponding target proteins/peptides being considered as biomarkers. This affinity-binding also assisted the isolation of target analytes from non-target analytes in microfluidic environment, thus improving the subsequent enrichment process and reducing background noise during the analysis. As reported, the integrated enrichment feature increased the sensitivity and enhanced the low detection limit of IACE. The concentration as low as a few nanogram per milliliter could be detected and quantified. With this highly sensitive capability, it was also reported that IACE could be used to monitor the progressive patterns of the disease. The results obtained by IACE were proven consistent with the gold standard like the traditional histopathology.

### Notes

1. Herr AE, Hatch AV, Giannobile WV, Throckmorton DJ, Tran HM, Brennan JS, Singh AK. "Integrated microfluidic platform for oral diagnostics" Ann NY Acad Sci. Author manuscript available in PMC (2008).
2. Xu X, Li L, Weber SG. "Electrochemical and optical detectors for capillary and chip separations" Trends Analyt Chem. Author manuscript available in PMC (2008).
3. Herr AE, Hatch AV, Throckmorton DJ, Tran HM, Brennan JS, Giannobile WV, Singh AK. "Microfluidic immunoassays as rapid saliva-based clinical diagnostics" Proc of Nat Acad Sci. 104(13):5268-5273 (2007).
4. Barry R, Ivanov D. "Microfluidics in biotechnology" J Nanobiotechnology 2:2 (2004).
5. a b c d Guzman NA, Blanc T, Phillips TM. "Immunoaffinity capillary electrophoresis as a powerful strategy for the quantification of low-abundance biomarkers, drugs, and metabolites in biological matrices" Electrophoresis. Author manuscript available in PMC (2009).
6. Gong M, Wehmeyer KR, Limbach PA, Arias F, Heineman WR. "On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis" Anal Chem. Author manuscript available in PMC (2008).
7. Gong M, Wehmeyer KR, Stalcup AM, Limbach PA, Heineman WR. "Study of injection bias in a simple hydrodynamic injection in microchip capillary electrophoresis" Electrophoresis. Author manuscript available in PMC (2008).
8. Hsieh J-F, Chen S-T. "Comparative studies on the analysis of glycoproteins and lipopolysaccharides by the gel-based microchip and SDS-PAGE" Biomicrofluidics 1 (2007).
9. a b c Wako Chemicals ( http://www.wakousa.com/specialty/specialty_focus.html )
10. Backofen U, Matysik F-M, Lunte CE. "A chip-based electrophoresis system with electrochemical detection and hydrodynamic injection" Anal Chem. Author manuscript available in PMC (2008).