Systematic evolution of ligands by exponential enrichment (SELEX) is a method to screen the nucleotide ligands from a large library of nucleotide sequences. Aptamers are the nucleotide ligands selected by SELEX method and can be easily and inexpensively produced. Chemical modification and integration into different analytical methods is also easy with aptamers.
Moreover, they have a strong affinity and a high specificity toward target molecules and thus are considered to be more advantageous as compared to antibodies. Aptamers are promising candidates in analytic, diagnostic and therapeutic applications. They can bind to a wide range of targets due to their three dimensional structure compatibility. Many researchers have used SELEX method to select the aptamers with high affinities. Recently, a microfluidic chip has been used for SELEX method, which was useful for the high-throughput applications enabling their selection with an increased speed and a reduced cost. Microfluidic chips are less expensive, easily reproducible and even disposable.
Capillary electrophoresis (CE) microfluidic chips are one of the efficient SELEX selection methods (CE-SELEX), which use electrophoresis to separate the nucleotides based on their mobility shifts. The bound nucleic acids could be collected, amplified and purified from the chip for multiple rounds of selection. CE-SELEX has been used to isolate aptamers for large targets like human immunoglobulin E and protein kinase C as well as for smaller targets like neuropeptides.
When compared to the traditional SELEX method, the CE-SELEX method has a high partitioning efficiency, which decreases the screening time. Therefore, the CE-SELEX method has been efficient for the selection of protein binding aptamers in low nanomolar range; however, it could be less effective for cell-surface targets. The other limitation for the CE-SELEX method is the requirement of a nanoliter volume of a random library. Moreover, separating unbound oligonucleotides is a crucial step for the aptamer selection in this process. Therefore, in order to get a fully-automated system, more integration with other microfluidic devices needs to be developed.
Sol-gel entrapment of biomolecules is another selection method that has been recently developed for the screening of aptamers. The aqueous environment in the nanoporous composites prepared by sol-gel process maintains the biological activity and stability of molecules. Researchers have developed many sol-gel microfluidic chips for the screening of high affinity aptamers. Sol-gel systems are used for targets such as recombinant yeast TATA binding proteins and yeast transcription factor IIB proteins.
For a higher elution efficiency, microheaters could be further incorporated in the microfluidic chip. The sol-gel microfluidic system enable the isolation of the aptamers that are specific to the target proteins. However, the microheater has not been used for an on-chip PCR process and the manufacturing of the sol-gel microfluidic system is relatively complex.
Furthermore, magnetic-chip-based microfluidic technologies have recently been developed for SELEX. However, such ferromagnetic structures are difficult to be fabricated, and a precise magnetic field is required to capture the magnetic beads. Magnetic-chip-based technology has been successfully used for the selection of aptamers against a light chain of recombinant botulinum neurotoxin type A and streptavidin. Another advantage of the magnetic-chip-based technology is its use in the screening of aptamers for target cells.
Magnetic-chip-based technologies cannot be integrated with micropumps, microvalves or on-chip PCR module. Recently, a microfluidic chip integrated with an incubation module and an on-chip nucleic acid amplification module has been reported for the rapid screening of aptamers. Components such as micropumps, microvalves, micromixers, and temperature control system could be integrated for a better efficiency of SELEX process. Such control systems could prevent the potential cross contaminations and reduce screening time. A magnetic-bead-based microfluidic technology integrated with micro-injector for the release of the PCR reagent, and a new array-type microheater for the maintenance of the temperature inside the PCR reaction has been recently reported.
In summary, the microfluidic technology has many advantages for aptamer screening over the conventional methods, including less sample/reagent consumption, lower power consumption, less contamination and automation. Microfluidic systems have been used for the selection of aptamers in cell-based assays, molecular diagnosis, immunoassays and biochemical assays. Microfluidic devices could provide accurate clinical analysis in less time and could be used for the development of point-of-care devices in the near future.
