Clinical diagnosis and monitoring can greatly benefit from the latest advances in analytical chemistry, most notably the development of separation techniques. The interdisciplinary collaboration of physicians and chemists can result in original and new insights into several diagnostic methods. The increase in innovations and developments in proteomics and genomic research, and rising R&D cost are propelling the adoption of electrophoresis. Top-down proteomics or the analysis of intact proteins could contribute significantly to systems biology understanding and the characterization of drug targets in the drug discovery process. One obstacle to higher efficiency top-down proteomics studies is effective sample separation, which has motivated recent research into capillary electrophoresis (CE) methods.
Two-dimensional gel electrophoresis (2DGE) has been profound in its ability to resolve up to thousands of sample proteins by separating them by molecular weight (MW) and isoelectric point. Separated protein spots could later be excised from the gel slab and mass analyzed. While robust, 2DGE has lagged and taken a complementary position to LC-MS owing to its lack of complexity and sensitivity, that is demanded in research. Gel electrophoresis does not provide adequate reproducibility for protein quantification, which is becoming increasingly important. The separation technology has limited dynamic range and imposes significant sample handling requirements with little amenability to high-throughput automation.
The growing adoption of other technologies with better efficiency and results such as mass spectrometry, microarrays, and chromatography and time consuming analysis are restraining the growth of the electrophoresis technologies market. Much as robust bottom-up proteomics came about through the successful interface of nano-scale LC and electrospray, the future of top-down proteomics may hinge on the demonstration and standardization of CE-electrospray ionization ESI-MS systems. Hybrid technologies like chromatography-mass spectrometry have provided superior dynamic range and reproducibility while operating in-line with mass spectrometry.
Trends in capillary electrophoresis. Capillary electrophoresis which is widely deployed as an orthogonal or complementary form of separation and fractionation had limited prospects for widespread adoption in proteomics research market. However, this has changed with the recent shift in analytical priorities from high-throughput protein identification to protein characterization. Intact protein analysis is becoming increasingly important to proteomics research for the identification of drug targets and elucidation of cell system pathways. CE is a fast separation technique that can handle very small sample volumes and is potentially suitable for analysis of clinical samples, obtained either invasively (blood, cerebrospinal fluid, and sputum) or non-invasively (urine, saliva, sweat, and exhaled breath condensate). CE is also easily miniaturized and can be potentially used in mobile laboratories and point-of-care diagnostics.
Several factors have made CE, particularly capillary zone electrophoresis, a superior option to liquid chromatography for use in top-down proteomics. CE better handles large proteins and polypeptides as compared to reverse-phase LC which can take a longer time and produce broad spectrum peaks. Faster separation and greater sensitivity with resolution of analytes in the low nanogram range can be accomplished with CZE over HPLC. CZE utilizes a loading capacity that is one to three orders of magnitude lower than that of HPLC and delivers a higher efficiency in separation.
The potential advantages of CE in top-down proteomics applications has not translated to an upsurge in adoption, due to the current limitations in common practice and proteomics MS platforms. The efficiency and speed of CZE separation outstrip the spectra acquisition rate of the current MS system. Small sample loading capacity can also be a hindrance by limiting the detectable dynamic range and MS instruments sensitivity.
Portable capillary electrophoresis. The main advantage of CE has been its portability for in situ measurements, which improves the assessment for the best sampling sites without any effort and problems with sample transport and storage, and the ability to deal with very tiny sample volumes (< 20 µL), in addition to fast (2 -10 minutes) detection time and a detection limit from 0.1 to 1 µM.
The portable capillary electrophoresis (P-CE) has a battery system which gives it electrical autonomy. It is a capillary electrophoresis assisted by external pressure. Electrical consumption is not required to generate the pressure, which increases the instrument autonomy. The development of P-CE has just started as this technology is yet to gain impetus in adoption and use. The P-CE design of this invention is based on the miniaturization and subsequent charging battery of electronic and non-electronic elements of a conventional capillary electrophoresis (not microchip). Data acquisition is performed with a high-resolution data recorder which transfers the signal to the computer via an USB port.
LOC technology in electrophoresis. Another major trend in the market is the increased popularity of lab-on-a-chip technology in electrophoresis. This technology combined with electrophoresis is used for screening proteins and other molecules. It helps overcome the limitations of SDS-PAGE and other separation techniques that depend on conventional capillary electrophoresis. Current technologies offer lab-on-a-chip that have already demonstrated several advantages over conventional CE systems, including minimal reagent and sample consumption, higher efficiency, speed and throughput, portability, integration, and increased parallelism and automation. The key principle behind developing and manufacturing these microchips is using well-established fabrication techniques. Microchip electrophoresis (MCE) was one of the earliest applications of the micro-total analysis system (µ-TAS) concept, whose aim is to reduce analysis time and reagent and sample consumption while increasing throughput and portability by miniaturizing analytical laboratory procedures onto a microfluidic chip. MCE-based instruments have had some commercial success and have found applications in many disciplines.
MCE is likely to remain a focus in the field of electrophoresis in an endeavor to create sensitive, universal systems for a range of applications. Key areas that are being addressed over the past 2 years are in efforts to deal with matrix effects, and to minimize those, as well as to make simpler, more robust methods, particularly for implementation in portable devices where the aim is to reduce and minimize the need for complex manual handling. This aspect will receive more attention as the push toward complete sample-in/answer-out system continues. Importantly, there is no approach that is universal'selection of the right approach for the right application remains a challenge and is perhaps the hardest part of the process.