1
Miniaturized Capillary Electrophoresis for the Separation and Identification of Biomolecules

Suresh K. Kailasa1 Vaibhavkumar N. Mehta2, and Jigneshkumar V. Rohit3

1Department of Chemistry, Sardar Vallabhbhai National Institute of Technology, Surat-395007, Gujarat, India

2ASPEE Shakilam Biotechnology Institute, Navsari Agricultural University, Surat-395007, Gujarat, India

3Department of Chemistry, National Institute of Technology Srinagar, Hazratbal Kashmir 190006, Kashmir, India

1.1 Introduction

Microchip capillary electrophoresis (MCE) is one of the efficient bioanalytical tools for rapid separation and detection of bioactive molecules with high separation resolution [1-3]. It has proven to be a prominent tool for identification of nucleic acids and proteins in food and clinical microbiology [2]. Separation of biomolecules is a key platform for quantitative and qualitative analysis of target biomolecules in biological matrices. For the first time, Manz's group integrated a simple analytical procedure on a small glass chip for the separation and detection of target chemical species [4], collectively referred to as "lab-on-a-chip" or micro total analysis systems (µTAS). In this concept, MCE is included due to the separation mechanism in a microchip with very short channels. As a result, target molecules from the mixture are effectively separated by using high electric field strengths. MCE has been widely applied as a rapid separation tool in various fields of science, i.e. proteomics, genomics, biomarkers, and forensics [5-9]. These reviews reported that MCE has shown better performance for the separation of target analytes compared to traditional capillary electrophoresis. The MCE has successfully separated >30 000 proteins from a single cell [10].

In this chapter, we summarize the recent developments of MCE for separation and identification of nucleic acids and proteins from clinical and food samples. We briefly describe the history and role of MCE in clinical and food microbial research. A section is devoted to applications of MCE for separation and identification of nucleic acids, proteins, and biomarkers from clinical and food samples. The analytical features of MCE for rapid separation and detection of biomolecules are tabulated, which provides significant information to scientists to know potential advancements of MCE in molecular biology.

1.2 Brief Summary of MCE

Generally, MCE consists of four core parts: microfluidic chip, electric field, separation, and detector. The electric field is applied for sample concentration and separation. Figure 1.1 displays a T-shaped microfluidic chip. The microfluidic chip contains few reservoirs such as sample and buffer reservoirs. These reservoirs should be filled with a background solution, and sieving gels and pipetting and syringe pumps are used for fluidic control. Once the microfluidic chip is set up with these parts, a high electric field is applied to the reservoirs (sample) to separate the target analytes. The detector is placed at the end of the separation channel, which results in registering the zones for separation and transmitting the data for signal processing unit, which generates an electropherogram. In this section, an overview of fabrication of microfluidic chips, sample preparation (on-microfluidic chip), separation, and analyte detection is given.

1.2.1 Fabrication of Microfluidic Chips

So far, several methods have been adopted for the fabrication of microfluidic chips, such as reactive ion etching, wet etching, photolithography, conventional machining, hot embossing, injection molding, soft lithography, in situ construction, laser ablation, and plasma etching [11, 12]. Silicon or glass is used as raw materials for the fabrication of microfluidic chips. Microfluidic electrophoresis chips consist of two reservoirs (sample and buffer) connected to the separation channel. A wide variety of materials including ceramics, glass, and polymers (poly(methyl methacrylate), cyclic olefin copolymers, polycarbonate, polystyrene, and fluorescent poly(p-xylylene) polymer (Parylene-C) have been used for preparation of microfluidic chips. Paper and fabric-based disposal chips have also received attention in MCE [7]. Electroosmotic flow (EOF) is generated in microfluidic chips as the reservoirs are filled with a background solution or electrolyte. Since EOF significantly obstructs separation, microfluidic chips are coated with various chemicals and hydrogels to suppress EOF.

Figure 1.1 Separation and identification of amplified-PCR products of T-cell lymphoma. 50-bp DNA ladder (i); mixture of TCR? and Cµ (ii); positive control Cµ(130-bp) (iii); TCR? (90-bp) (iv); and negative control (v). The dotted linesrepresent the applied electric field. Figure reprinted from Ref. [25] with permission.

1.2.2 Designing Microfluidic Channels

Crossed-channel and T-shaped microfluidic chips are widely used in MCE, and the microchip channel is connected perpendicularly to other channels (sample and buffer). Microfluidic chips are also prepared with different designs. For example, a microfluidic chip (Agilent BioanalyzerT chip) is prepared with 16 reservoirs, of which 12 are for sample reservoirs and four for references and reagents. Accordingly, MCE has been successfully applied for the analysis of various bioactive molecules with high precision and accuracy. Microfluidic chip channels with a width of 10-100 µm and a depth of 15-40 µm are considered the best design for separation of analytes. Also, separation channel area is designed to be 165 mm and 8 × 8 mm2 and the number of channels of microchips is increased to 12-384 for separation of nucleic acids and multiple genotyping (384) molecules with reduced time and increased accuracy [3]. Furthermore, microchips are designed with 8, 12, 16, 48, and 384 parallel channels for rapid and efficient separation of a wide variety of analytes.

The electric field (voltage application) and hydrodynamic pressure are applied for sample injection in MCE. On-chip peristaltic pump is used for hydrodynamic injection [13]. Inkjet and array techniques are droplet injection systems, which provide high throughput and the sample injection volume ranges from nano- to picoliters [14]. As microfluidic chips are compact in size, electrokinetic injection is the preferred method of sample injection, where the injection volume of the sample is strongly dependent on the applied voltage and injection time [15]. Further, hydrodynamic injection system requires a pump or pipette, which limits its use in MCE. It is usually carried out by variations in pressure, vacuum, reservoir (sample waste), and fluid levels of sample.

Electrokinetic injection system contains several injection modes, i.e. floating, gated, dynamic, and pinched. In the pinched injection mode, a voltage is applied at various channels including sample, buffer waste, and buffer reservoirs, and, as a result, the sample is injected into the channel junctions, and further enters the separation channels. Although the pinched injection mode is well illustrated and low volume of analyte plugs, it decreases the sensitivity due to sample plug. In the floating injection mode, which is similar to the pinched design, the potential is not required in the buffer and buffer waste reservoirs, increasing the sample load due to diffusion of the sample into the separation channel. Voltage can be applied at the sample and buffer reservoirs, and two waste reservoirs are grounded in this mode. The sample is injected into the separation channel by switching off the voltage at the reservoir (buffer), loading several amounts of the sample, which could help to improve the sensitivity of MCE. Then, the voltage is again switched on at the buffer reservoir. In dynamic injection mode, electroosmosis is required to inject the sample into separation channels, which can also improve the sample load.

1.2.3 Electrophoretic Separation

Target analytes are effectively separated in separation channels via electrophoretic separation by applying an electric field. Electrophoretic migration of analytes occurs due to the electric field and flow of liquid, collectively known as EOF. Various electrophoretic separations including electrokinetic chromatography, gel electrophoresis, and zone electrophoresis have been described in MCE [7]. Dielectrophoresis (DEP), gel electrophoresis, and zone electrophoresis modes are generally used in MCE. The DEP allows the separation of particles and cells by applying irregular electric fields (nonuniform) [7]. PC-3 human prostate cancer cells and polystyrene microbeads were separated by ionic liquid electrodes [16], and target analytes and particles were successfully separated via the on-chip procedure from human plasma [17]. The charged analytes migrate using the background solution (BGS) and are separated through electrophoretic mobility of target analytes, which leads to detection of analytes on the basis of descending mobility. As a result, small organic molecules including metabolites and drugs are effectively separated by the zone electrophoresis mode.

Electrophoretic mobility of analytes takes place when an electric field is applied along a sieving matrix. The analytes...