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New Technologies for Electrophoresis

Nov 26, 2023Nov 26, 2023

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at [email protected].

Conceived during the late 1980s, the "lab-on-a-chip" idea sought to create the equivalent of an entire analytical laboratory on matchbook-sized slabs of silicon, glass, and plastic. Most of these devices were based on capillary electrophoresis, an analytical mode that remains dominant in microfluidic analytical devices.

The dream of fabricating anything resembling an entire laboratory on a matchbook-sized substrate was never realized, but commercial instrumentation has emerged that performs straightforward electrophoresis on chips. These mostly serve the life sciences, including medical diagnostics. A market research report estimates a robust seven percent yearly growth rate for such systems.

PerkinElmer offers two microfluidic electrophoresis platforms—the LabChip® GX Touch™ nucleic acid analyzer for DNA and RNA, and the LabChip® GXII Touch™ protein characterization system for proteins and glycans. Both systems provide high-throughput platforms for electrophoresis-based biomolecule separation, sizing, and quantitation. PerkinElmer developed special microfabrication methods that embed low-micron-sized channels into thin glass and quartz microchips. The microchips interface with the instrument via plastic caddies to support easy sample loading and engagement with the instrumentation.

"The primary advantages of microfluidic-based vs gel-based biomolecule analysis are the throughput, resolution, ease of use, sensitivity, and versatility," says James Atwood PhD, general manager of automation and microfluidics at PerkinElmer. "Traditional gel electrophoresis is time consuming, labor-intensive, prone to variability, and consumes large quantities of precious samples."

LabChip microfluidic-based assays consume just 150 nanoliters of sample per separation and are compatible with nucleic acid and protein samples in the low pg/μL and ng/mL concentration ranges, respectively. Throughput of up to 384 samples per cycle for LabChip far exceeds that of gel-based systems and biomolecules, such as N-linked glycans, which are not amendable to electrophoresis gel-based separation but can be separated and detected in microfluidic systems. Another important advantage of microfluidic-based biomolecule separation is separation and quantitation of native proteins, based on charge alone, to detect charge variants. Gel-based electrophoresis cannot resolve protein charge variants.

In typical gel electrophoresis-based mass spectrometry workflows, the compounds are first separated in the gel then the bands corresponding to the biomolecules of interest are excised. In proteomic workflows, proteins are enzymatically degraded, and peptide fragments are extracted from the gel prior to LC-MS/MS. While this approach has a number of advantages, including being an orthogonal separation mode, it is extremely tedious.

Several microfluidic-based systems have been developed that enable the direct coupling of a microfluidic chip to an HPLC system and mass spectrometer. In these microchromatography systems, the microfluidic channels are packed with a stationary phase that is specific to the desired separation mode. This enables the automated capture and/or separation of biomolecules directly on-chip with elution, ionization, and detection in the mass spectrometer. In contrast to gel-based approaches, the microchromatography systems are automation-friendly and can be tailored to target specific biomolecules of interest based on modifying the packing material.

The chips used with the Agilent 2100 Bioanalyzer system consist of a plastic caddy with 16 wells used for reagent and sample application. Each chip is labeled with identifiers for assay type, lot number, and the assay-specific chip setup. The glass chip incorporates etched microchannels and is glued onto the backside of the caddy. "The production process of the glass chip and separation channel is similar to that of semiconductor devices," says Eva Graf, product manager for bioanalyzers at Agilent Technologies (Santa Clara, CA). Agilent uses special protective masks for protein and nucleic acid chips, which mirror the channel structure. When the glass chips are exposed to the etching agent, only the channels are etched into the glass. The remaining chip surface is protected by the mask.

The microchannel system connects all wells of the caddy with the injection cross and separation channel. Before applying samples, the microchannels are filled with a mix of separative gel and fluorescent dye. During the chip run, a high voltage is applied in multiple steps according to an assay-specific script reflecting the layout of the microchannels in the glass chip. "The script coordinates pre-draw, electrokinetic injection and, of course, electrophoretic separation of the samples," Graf explains. "Simultaneous electrophoretic separation and pre-draw of the subsequent sample reduces the analysis time to minutes per sample." Separated analytes are then detected within the separation channel by laser-induced fluorescence and the signal is translated into gel-like images and electropherograms.

Electrophoresis-on-a-chip allows highly resolving separation requiring only minimal sample volume, which makes the technology attractive for quality control of precious samples before any downstream application.

For Agilent, top electrophoresis-chip applications include library quality control in next-generation sequencing workflows. According to Graf, accurate calculation of size distribution and sample concentration before sequencing is crucial to achieving optimal cluster density, and assessing the quantity and quality of experimental starting material is essential for research success. "When using RNA as starting material for gene expression analysis using NGS, microarrays, or qPCR, RNase degradation is a common reason for failed experiments. The chip-based RNA assays make it easy to visually detect even small degradation effects, and the RNA integrity number allows objective sample qualification."

By contrast, traditional gel electrophoresis only allows estimation of sample integrity, which is prone to user-dependent interpretation. "High sensitivity and a large linear dynamic range of the DNA assays make it an optimal tool for analyzing fragments amplified by PCR or digested by restriction enzymes, and enables easy evaluation of cleavage efficiency of synthesized gRNA in genome editing," Graf says.

On the protein analysis side, EPCs provide a rapid, reliable way to replace analytical SDS-PAGE methods. Typical applications include assessment of size, purity, and concentration of proteins during processes like expression of recombinant proteins, protein purification, stability studies, or general antibody analysis.

Lab managers evaluate instrumentation based on its capabilities, not its buzz factor. Purchasers of chip-based systems should therefore make sure that systems under consideration provide the depth and breadth of assays they expect to run.

Graf recommends systems that offer assays "appropriate for your sample type, especially with regards to sample concentration, sample size, sample type (DNA, RNA, protein, etc.)," always with an eye on sample throughput. "Also, consider the vendor's services for support, warranty, repair, and installation, and the system's ease of use, all of which can help reduce lab downtime to a minimum."

Sample volumes have become a major selling point with all analytical instruments. Smaller volumes consume less reagent and less sample, and allow researchers to do more with rare or scarce samples. Agilent's 2100 Bioanalyzer, for example, requires just one microliter of sample for RNA and DNA assays.

Regulatory compliance is an issue for pharmaceutical R&D labs, with 21 CFR part 11 being the applicable regulation covering electronic records. Compliance also plays into standard operations for diagnostics labs and any facility with ongoing interaction with the patent or legal system, for example, forensics.

Cost is always a consideration, but for instrumentation that labs use every day, cost of ownership must be considered with regard to energy and reagent consumption. Service plans tend to be comprehensive, with generally rapid response, but very busy labs should also consider the economic impact of downtime.

Related Infographic: Polyacrylamide Gel Electrophoresis