Affinity molecules can serve as precision tools for selective recognition and measurement of specific biomolecules in the fields of therapeutic drug monitoring and quality control in recombinant protein production. In therapeutic drug monitoring, affinity molecules can enable the accurate quantification of drug concentrations within physiological fluids, enhancing both the safety and efficacy of clinical treatments. In the realm of recombinant protein production, these molecules can allow precise isolation and measurement of desired recombinant proteins from complex mixtures by selectively targeting specific protein tags or domains, ensuring the consistency and purity of protein products. Currently, antibodies are most commonly used affinity reagents in these fields but are limited by production complexity, batch variability, high cost, and low stability. Aptamers, known as ‘chemical antibodies’ but composed of nucleotides, are considered potential next-generation affinity reagents. Aptamers are obtained via a synthetic process, termed SELEX, of iterative affinity selection and polymerase chain reaction (PCR) amplification of target-binding members from a randomized oligonucleotide library. This process is traditionally labor and resource-intensive and time-consuming. In this thesis, microfluidic technology is employed to enable time-efficient and cost-effective generation of aptamers for monoclonal antibodies and recombinant proteins toward applications in therapeutic drug monitoring and quality control of recombinant protein production.
This thesis starts with a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection (called “chip-selection SELEX”), and fully integrated microfluidic affinity selection and PCR amplification (termed “full-chip SELEX”). The comparison results indicate that chip-selection SELEX offers the lowest cost and highest efficiency in aptamer isolation. We then use chip-selection SELEX to streamline the process of isolating anti-idiotype aptamers targeting human monoclonal antibodies against spike protein of SARS-CoV-2 virus. The process is completed within only 5 rounds of SELEX within two days, which represented a significant improvement when compared to conventional methods whose completion generally requires more than 10 SELEX rounds in up to a month. These anti-idiotype aptamers are combined with a graphene-based affinity nanosensor to enable rapid antibody concentration measurements to inform therapeutic decisions in a timely manner. In addition, a microfluidic dual-aptamer sandwich assay with highly efficient isolation of aptamers is developed to enable rapid and cost-effective detection of tag-fused recombinant proteins. This approach addresses both the limitations of current dual-aptamer assays and commonly encountered difficulties in the lack of aptamers available for such assays, by first using chip-selection SELEX to generate aptamers and then employing these aptamers to implement a microfluidic dual-aptamer assay for quality control during recombinant protein production.
Despite the high efficiency in aptamer isolation using chip-selection SELEX, the full-chip SELEX platform is still desired for minimal manual operation and reagent consumption. The current full-chip SELEX platform has low isolation efficiency and could not offer information of affinity selection process. Herein, by introducing asymmetric PCR into the full-chip SELEX process, we improve the efficiency in aptamer isolation and can successfully monitor the selection progress. This real-time monitoring capability allows us to identify the optimal point to terminate the SELEX process, preventing the potential loss of aptamer candidates and reducing the overall consumption of time and reagents. In addition, introducing solution phase-based asymmetric PCR addresses a notable technical challenge of on-chip PCR bead replenishment, toward complete automation of the full-chip SELEX platform. Furthermore, a holder equipped with connection pins is designed to enable the reversible connection between gold electrodes and electrical wires. This design promotes the reusability of gold electrode-deposited glass substrates, resulting in a substantial reduction in chip fabrication costs.
In addition to the SELEX protocol development effort, we also present efficient and cost-effective microfluidic approaches for post-SELEX aptamer characterization, including aptamer identification and kinetic aptamer-target binding measurements. To mitigate the expensive and time-consuming nature of aptamer identification from SELEX-generated target-binding sequence pools, we present an approach that is based on a cost-effective and efficient procedure to generate modified single-stranded DNA copies of the aptamer candidates and then assess the affinity of the resulting modified ssDNA strands to target molecules. The approach is applied to identify aptamers from 12 candidates with consistent results, but at a cost three times lower than that of established methods. We also present a microfluidic fluorescence assay, which exploits a synergistic combination of microfluidic technology and fluorescence microscopy, to realize cost-effective and multiplexed measurement of kinetics of aptamer-target analyte binding without requiring special-purpose equipment.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/wz6g-ft93 |
| Date | January 2024 |
| Creators | Wen, Kechun |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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