Analysis Of The Latest Applications Of Microfluidic Chips In The Field Of Proteomics
Various molecules such as DNA, RNA and chemicals have often been analyzed with numerous MF systems for both general purpose and disease diagnostics. MF have also been regarded as a general theme of fabrication for a significant number of devices used to diagnose disease including pathogen detection. Microfluidic chip-based methods have definitively played an important part in high-throughput genomic studies.
A benchmark in genome sequencing, the Capillary array electrophoresis (CAE) is a sequencing method where multiple capillaries are used in parallel for high-throughput sequencing of target DNAs. Directly derived from the genome; the proteome is responsible for the regulation of both gene expression and cellular metabolism. Therefore, in order to understand biology at the systems-level, the quantitative investigation of the proteome is paramount. In this research, we discuss the latest applications of microfluidic chips in the field of proteomics with an aspect of preconcentration, on-chip separation, single-cell proteomics and mass spectrometry coupling. In proteomic studies, the utmost basic requirement is the preparation of proteins, as when working with naturally low-concentration proteins, preconcentration is required to ensure that the desired proteins are in the detectable range. Preconcentration of proteins on microfluidic chips using various means have been reported. For example, Song et al. has reported the use of laser-patterned nano-porous membrane at a cross-channel junction on a microfluidic chip. When voltage is applied to charged proteins, the linear preelectrophoretic concentration can be realized at the membrane surface with an increase in the concentration of 2-4 times. By utilizing phase-changing sacrificial layers, Kelley and co-workers were able to show that protein concentration is possible through miniaturized electric field gradient focusing on a microfluidic chip. Recently, Wang et al. has reported a microfluidic sample preconcentration device derived from the electro-kinetic trapping method. Utilizing a flat nano-fluidic filter formed within a microchannel, acting as n ion-selective membrane for electro-kinetic trapping of charged molecules. Hence, electro-kinetic trapping of proteins can be held at the nano-fluidic filter region for several hours at a concentration factor as high as 106-108. Kim et al. reported a PDMS microfluidic chip useful in the enrichment of proteins; a thin-walled PDMS section of around ~20µm was fashioned between two micro-channels. Subject to an electric field, preconcentration of negatively charged proteins could easily be achieved on the anode side of the section with a 103-106-fold increase.
Ultimately, complex sample matrixes and ion suppression in the MS ion source means that the use of mass spectrometry in proteomics is still fraught with technical difficulties. A vital step for proteomics research is the purification of target proteins from complex biological samples which requires a sequence of procedures as desalting, matrix removal, target enrichment, and separation. The advancement of microfluidic-based technologies for proteomic applications through the streamlining of relevant analytical processes into a platform has been instrumental in the acceleration of protein and post-translational modification identification. Ji et al. developed a droplet-based proteolysis reaction for application in online tryptic digestion of proteins separated by HPLC, with the resulting peptides directly identified via ESI-MS/MS. To overcome these issues, significant efforts were made towards the development of enrichment technologies for phosphopeptides to augment their detection, for example, titanium dioxide (TiO2)-based affinity enrichment. To this end, other advancements include hydrophilic interaction chromatography, and immobilized metal affinity chromatography. For the global screening of phosphoproteome in primary human leukocytes, the TiO2-based phosphochip-Q-TOF was developed by Heck’s group. Despite numerous attempts at studies into membrane proteins due to their importance in drug targeting, they have proven to be difficult to solubilize and are known to be liable to aggregation which makes them difficult to analyze. Although detergents have been employed to help solubilize them, they cause substantial interference in downstream analysis as is the case with MS. Although many conventional approaches have been employed to segregate proteins from interfering detergents, such as dialysis, hydrophobic absorption or ion exchange chromatography. Recent improvement in this quandary is the microfluidic electrocapture method, which has been developed for preconcentration and separation of analytes from interferents for detection via ESI-MS. Single-cell proteomics allows us to gain an invaluable glimpse into the protein expression of proteins in individual cells within a heterogeneous cellular population. To achieve this, different approaches have been suggested to realize single-cell proteomic studies. For example, Ewing and fellow researchers demonstrated the mapping of both cellular and subcellular contents from single cells through time-of-flight secondary ion mass spectrometry. Recently, researchers have turned to microfluidic systems as attractive platforms for single-cell proteomics due to their potential for automation and compatibility with cells in regard to their size. Another reseanoterch group headed by Ramsey demonstrated a microfluidic device that integrates cell handing, lysis and electrophoretic separation as well as fluorescent detection. Loaded cells were transported via hydrodynamics into reservoirs to a region where they could be focused and quickly lysed using.
The most familiar protein modification of the human proteome is glycosylation; which is closely associated with many crucial biological functions such as cell development, adhesion and differentiation, immune response and host-pathogen interaction. Furthermore, glycans have been noted for their significant impact on the properties of proteins in regards to folding, immunogenicity, solubility as well as secretion, and thermal stability, etc. Many physiological and cellular processes are influenced by networks of protein to protein interaction. Diseases such as Parkinson’s and Alzheimer’s are attributed to the aggregation of abnormal proteins. Based on this, the disruption of protein-protein interaction is a viable therapeutic strategy for these diseases. A typical approach used for protein-protein interaction analysis is the Yeast two-hybrid, pull-down MS. Latest developments in surface plasmon resonance (SPR) and MS provides the option for better integration that could be directed to the identification and characterization of protein-protein interactions. As an example, the incorporation of the SPR technique was applied to detect proteins interacting with particular peptides or immobilized proteins on a gold sensor chip. The MS/MS method of setup greatly increases the odds for protein identification through not just peptide mass data, but also through amino-acid sequencing. In the area of proteomics, Mass spectrometry (MS) based techniques have been widely utilized as evidenced by peptide mapping, post-translational modification, and protein-protein interactions. Thanks to rapid advancements and breakthroughs in microfabrication technology, issues related to microfluidic chips such as rapidity, throughput, and cost effectiveness have been addressed when combined with MS analysis. Wheeler et al. used electro-wetting-on-dielectric-based technique for digital microfluidics to demonstrate inline sample purification for proteomic analysis when using MALDI-MS. Dodge et al. used the PDMS microfluidic device which combined a number of techniques, such as online protein electrophoretic separation, selection and protein digestion for analysis with MALDI MS. Despite the recognition, the potential for MF in diagnosis has been slow to materialize. Currently, thousands of research publications have been made, however, outcomes resulting in successful devices are significantly less than expected. Although there are commercially available LoC products for DNA analysis, protein crystallization and simple chemical reactions, there is a demand for a so called “killer application” in the context of clinical diagnostics as PoC diagnostics have not quite lived up to expectations. A possible explanation may be due to the complexity of the systems involved, although many diagnostically relevant complex biochemical processes have been demonstrated on-chip. Another possible explanation may be due to the initial resistance in the adaptation of new technology. Market diction states that new technologies are user-driven not technology-driven. Case in point, traditional methods are preferred by users due to habits with new displacing technologies requiring ease of operation, training, and simplicity. Majority of PoC diagnostics tools employed at hospitals and laboratories are simply not approachable by the common folk. Therefore, efforts should be made to improve on user-friendly diagnostic concepts such as the use of symbols to indicate the detection of antigens, antibodies, viruses, or any other biological targets for analysis. Analysis of in-the-field samples, such as blood and saliva does not follow the trend of purified samples typically processed n general laboratories and is therefore more challenging and complicated to process in an MF device. Therefore ideally, new devices need to be designed for use in the virtual environments rather than controlled laboratory conditions. A possible reason for the impedance to realization of academic research to practical application (devices) may be due to a lack of funding. The manufacture of MF devices can be a costly endeavor with expensive instruments beyond the reach of certain laboratories. As these MF platforms were designed with research in mind, they do not adequately address mass production of practical devices. The vast majority of manufacturing methods available on LoC devices involve micromachining on to glass or silicon, or soft lithography on PDMS. All quite costly.
In order to overcome resistance to adaptation from consumers of current products, MF technology must substantially outperform, or be more cost-effective than products already in present use. The increased importance given to global health has led to a surge in demand for viable, cost-effective, high through output and integrated PoC devices which is only likely to become increasingly higher in the years to come. As an emergent technology in commercial diagnostics, insofar as bringing the technology from the laboratory to the field is concerned, MF holds tremendous potential. Taking into consideration the inherent advantages the technology has to offer, that MF devices will succeed conventional techniques is difficult to argue. Continued development of MF for applications such as manufacturing methods and flexible platform technologies that can easily be customized for individualized diagnostic tests will be the catalyst for success. A successful commercial adaptation will also lead to an excursion into other areas beyond the realm of biological domain.
