Recent innovations in stain-free electrophoresis greatly reduce impact on protein characterization workflow allowing laboratory professionals to now run samples in less than 30 minutes, thereby drastically cutting costs and time required to determine sample purity.
The development of electrophoresis as a separation analysis technique has created a high-throughput purification of macromolecules based on their charge and size, thus generating purified molecules. This has led to the increasing use of electrophoresis by pharmaceutical and biotechnology firms, research organizations, hospitals, diagnostic centers, and academic institutions. Different types of electrophoresis system are now available depending upon their specific application in routine clinical laboratories. Cellulose acetate electrophoresis is becoming an important technology in clinical diagnosis and is used for both quantitative and qualitative analysis. Known for rapid and efficient separation of small samples, this makes electrophoresis suitable for examination of both small and macromolecular biomarkers in complex samples during protein analysis.
The rising prevalence and awareness of cancer have resulted in gaining of importance for the early diagnosis of cancer. Capillary electrophoresis (CE) is continuously evolving since its first introduction in the 1980s. The development of new CE separation procedures, the coupling of these systems to more sensitive and versatile detection systems, and the advances in miniaturization technology have allowed the application of CE to the resolution of new and complex analytical problems, overcoming the traditional disadvantages associated with this method. Also, recent innovations in stain-free electrophoresis greatly reduce impact on protein characterization workflow that is important in protein therapeutics. With new technology, laboratory professionals can now run samples in less than 30 minutes.
The global electrophoresis market is expected to reach USD 2.8 billion by 2022 from an estimated USD 2.15 billion in 2017, reflecting a CAGR of 5.4 percent, according to MarketsandMarkets. Rising incidence of cancer, infectious diseases, and genetic disorders; increase in funding for research on genomic, proteomic, and electrophoresis techniques; growing number of industry–academia research collaborations; growing use of CE with mass spectroscopy; and increase in the number of clinical, forensic, and research laboratories are the major factors driving the growth of the electrophoresis market.
The gel documentation systems segment is expected to register the highest CAGR owing to increasing demand for gel electrophoresis in proteomics research and personalized medicines. The reagent segment accounts for the largest share of the global market that is primarily attributed to the increasing demand for 2D electrophoresis (2DE) for protein separation for various applications such as biomarker discovery and protein mapping. Over the years, genomic and proteomic technologies have gained significant importance in fields of clinical diagnosis and drug discovery and development.
North America accounts for the largest share of the electrophoresis market, followed by Europe and Asia-Pacific. The patent expiration of several blockbuster drugs of major pharmaceutical companies is, in turn, increasing investments for new drug discovery; a key market driver in North America. On the other hand, the Asia-Pacific region is expected to witness the highest CAGR owing to the growing proteomics and genomics research and increasing research funding, rising adoption of advanced electrophoresis techniques, increasing investments by pharmaceutical and biotechnology companies, and growing research collaborations and funding.
Owing to the high versatility, major advancements have been made with regard to the instrumental setups over the years. The focus of the industry remains introduction of the best technologies that offer high-throughput and improved sensitivity. New strategies have been proposed to develop high-sensitive, portable, and miniaturized devices.
CE–MS. The last decade has witnessed a revival in technological developments for CE–MS, while at the same time moving away from being a purely academic tool to one increasingly being adopted by industrial scientists. Until a few years ago there was only one commercial supplier offering a technology solution, meaning little progression. But more recently, new players have entered the market with new technologies. This will get better and better, making CE–MS more sensitive and more robust. Players are making impressive strides toward miniaturizing the technology, with the development of microfluidic CE–MS. Miniaturization will enable a lot of high-throughput work, without compromising the level of information they offer.
Accelerating gel electrophoresis through stain-free technology. Although SDS-PAGE can satisfy requirements for both sample purity and yield, the process can take a full day. When time and costs are critical concerns, researchers often cannot afford to spend a full day verifying protein quality after each step of a purification workflow that includes multiple columns. Technology has attempted to reduce the time required for electrophoresis by increasing the speed of gel runs and decreasing the time required for staining. But the greatest time decrease occurs when staining is completely removed from a procedure. Stain-free technology reduces run times to ≤30 minutes, thereby drastically cutting costs and time required to determine sample purity. The technology uses ultraviolet (UV) irradiation to target aromatic amino acids in native proteins. Stain-free technology also provides increased dynamic range, increased sensitivity, and a lower limit of detection compared with standard staining methods. Quality is not sacrificed for speed.
2D electrophoresis. Association of this technology with MS, in combination with computer-assisted software for image evaluation, has enabled 2DE analysis in comprehensive qualitative and quantitative examination of proteomes as well as in separation and selection of proteins. Currently, 2DE technology has gained special attention for its enormous ability to study differentially expressed proteins between different groups for comparative proteomic analysis to screen biomarkers and identification of drug targets for therapeutic management. Good sample preparation for solubilization of proteins, followed by protein extraction, is critical for efficient and reproducible 2DE. Conventional approaches for protein solubilization and modification do not reliably provide the best samples for the technology. Now the commercially available kits for protein extraction and other purposes of 2DE are considerably simple to use and have improved the sensitivity and reproducibility of the 2DE technology, yet they have limited use.
High-speed CE with LIF detection. Currently, the miniaturization of high-speed CE systems has become one of the major development directions of the technology, which can provide various portable instruments for point-of-care testing, in-situ analysis, and extraterrestrial exploration by means of their small size, high-resolution separation, and fast analysis time. So far, most of miniaturized high-speed CE systems are developed on the basis of microchip-based CE technique with advantages of automated picoliter-scale sample injection and separation and high system integration. In the early stage of the development of miniaturized CE instruments, electrochemical detectors were adopted frequently due to their simple structure and small size. Therefore, although it still presents great challenges in achieving miniaturization of laser-induced fluorescence (LIF) detectors due to their complex optical structure, currently LIF detection has become the major detection technique used in miniaturized CE instruments.
Microchip capillary electrophoresis. MCE has come up as a better alternative for monitoring and characterizing classical and novel formats of antibody-based bio-therapeutics as it offers a high-throughput approach to speed up sample throughput with a resolution superior to conventional CE. The vendors have come up with products which enable automated analysis of 96–384 samples in one run. MCE–SDS assays offer several advantages compared to the conventional CE–SDS in terms of faster time and a better resolution.
Software development for microfluidics-based CE systems. Relying on the experience gained from building the previous systems, this new approach for building electrophoretic separation systems was developed, based on a commercial breadboard system for miniaturized microfluidic parts, offering high design flexibility and small size as in lab-on-chip systems, yet using standard silica capillaries and obtaining results comparable to commercial CE instruments. However, this presents a challenge for system builders who want to efficiently build and use purpose-made instruments for conducting scientific experiments that can now be tackled by the development of the software package instrumenting. The package allows system builders to build a useful graphical user interface (GUI) for their experimental setups, allowing automation of multiple components controlled by separate microcontrollers. A code could be reused between projects using the same hardware units.
A Bright Future
Overall, electrophoresis has become an established analytical tool in the clinical laboratory, forensic laboratory, and biopharmaceutical companies. In these areas, it is expected that developments in CE will particularly focus on further improving the throughput that is, using narrower and shorter capillaries in array format and going from seconds instead of minutes for separation times. CE–MS, over the past few decades, has remained out of the limelight. However, there is now revived momentum to exploit some of the distinctive advantages it can offer. These include scientists hoping to discover robust clinical biomarkers, which will propel the healthcare industry into a new era of personalized medicine. However some critical hurdles still need to be tackled before it is ready for actual clinical studies. Recent work in the area indicates that electrophoresis is heading in the right direction.
An increasing number of protein therapeutics in the industry necessitates the development of faster, more cost-effective, and generally more effective workflows. Greater competition in this area means that biomanufacturers need to decrease their time to market. The development of new technologies can help meet the demand for new products. Stain-free CE technology drastically reduces run times while increasing sensitivity, dynamic range, and the lower limit of detection. When coupled with advances to automated chromatography systems, protein purification workflows can be completed faster and at lower cost than ever before at both small and process scales. Low cost and sensitive approach for DNA sequencing, the major role of electrophoresis in drug discovery, rising use of electrophoresis techniques as a result of growing importance of antibody research in the development of biotherapeutic are some of the other factors that will drive the use of electrophoresis further in research applications. With the existing innovative technologies and the forthcoming upgradations in these instruments, the market is expected to flourish by leaps and bounds.
Advances in Electrophoresis: Relevance to Proteomics and Genomics
Electrophoresis has been a principal driving force behind advancements in biological research covering metabolomics, proteomics, and genomics. The global market is expected to reach USD 2.3 billion in 2020 with gel electrophoresis (GE) accounting for 61 percent and capillary electrophoresis (CE) contributing to the rest of the share in the market. Advent of informatics in terms of sophisticated signal analysis algorithms improved the biological of outcomes of 2D GE. 2D PAGE with high-resolution capacity to separate post-translational protein entities is the need of the hour, which has driven the development of 2D fluorescence difference GE (2D DIGE) capable of providing higher accuracy of quantitative data with minimal occurrence of false positives. By tagging the protein samples with fluorescent dyes, subtle variations in protein abundances between the two samples can be sharply detected by this technology.
Another time saving and environment friendly innovation in GE technologies is stain-free technology, wherein tri-halo compounds incorporated in the SDS-PAGE gel modify the tryptophan residues in the protein samples following UV irradiation thus emitting a fluorescence signal. This prevents staining and destaining steps thus making the proteins to be visualized directly. A major breakthrough in comparative proteomic analysis is 3D GE that can accommodate 1536 samples directly from a microwell or up to 36 immobilized pH gradient strips pre-separated by isoelectric focusing can be loaded. Sample migration and separation occurs in the third spatial dimension perpendicular to the loading surface. Labelled proteins are detected by laser induced fluorescence.
Advances in the different CE techniques (non-aqueous CE, microemulsion electrokinetic chromatography, capillary isotachophoresis, capillary electrochromatography, and immunoaffinity CE) and detection techniques (mass spectrometry, light-emitting diode, fluorescence, chemiluminescence, and contactless conductivity) with on-line sample pretreatment enabled its utilization over a wide range of applications encompassing genome sequencing, pharmaceutical analysis, and other biological applications. The most important milestone achieved by this technology was the successful completion of the Human Genome Project.
Dr Shaik Mohammad Naushad
Head – Biochemical Genetics and Pharmacogenomics,
Sandor Lifesciences Pvt. Ltd.
Growing Applications of Electrophoresis
Gel electrophoresis is a most common standard laboratory technique used for separation of macromolecules like DNA, RNA, or proteins. It is based on the differential migration features of charged molecules in an electric field. The mobility of a charged molecule through an electric field depends on several factors like net charge on the molecule, field strength, ionic strength, size, shape of the molecule, and properties of the matrix through which the molecule migrates (e.g., pore size, viscosity). Matrix of gels acts as a molecular sieve through which molecules move on application of electric currents. The results can be analyzed quantitatively by visualizing the gel using UV light and a gel imaging device.
The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. This technique has enormous application in forensics, molecular biology, genetics, microbiology, and biochemistry fields. In the molecular biology field it is used for determining quality of DNA/RNA, analysis of PCR products, mutation detection, southern and northern blotting, sequencing etc. It is also used as a preparative technique prior to use of other methods such as RFLP, PCR, cloning, DNA sequencing, or blotting.
The technique of electrophoresis has been known since 1930 but it still proves to be an essential aid in diagnosis and therapeutic follow-up of patients with plasma cell disorders. It is routinely used in clinical laboratories for screening protein abnormalities in various biological fluids. The technique is used for electrophoresis of serum, urine, CSF proteins, enzymes (ALP, LDH, and CK), lipoproteins, and hemoglobin. Changes in the relative concentration of protein fractions in serum protein electrophoresis (SPE) allow easy recognition of pathological disorders associated with nephrotic syndrome, inflammatory reaction, and hepatic diseases.
Dr S Tasleem Raza
Professor – Department of Biochemistry,
Era’s Lucknow Medical College