2024 Theses Doctoral
Nucleic Acid Probes for Microfluidic Measurement of Intracellular Biomarkers toward Biodosimetry Applications
Nucleic acid probes against intracellular targets can find essential applications in biodosimetry. This thesis describes the development of multiple nucleic acid probes, including aptamers and molecular beacons, against radiation-responsive intracellular molecular targets via microfluidic technology. Aptamers are single-stranded oligonucleotide molecules that bind with high affinity and specificity to a wide range of target molecules. The method of systematic evolution of ligands by exponential enrichment (SELEX) plays an essential role in the isolation of aptamers from a randomized oligonucleotide library. To date, significant modifications and improvements of the SELEX process have been achieved, engendering various forms of implementation from conventional SELEX to microfluidics-based full-chip SELEX. While full-chip SELEX is generally considered advantageous over conventional SELEX, there has not yet been a conclusive comparison between the methods. Herein, we present a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection, and fully integrated microfluidic affinity selection and PCR amplification. Using immunoglobulin E (IgE) as a model target molecule, we compare these strategies in terms of the time and cost for each step of the SELEX process, including affinity selection, amplification, and oligonucleotide conditioning. Target-binding oligonucleotides in the enriched pools are sequenced and compared to assess the relative efficacy of the SELEX strategies. We show that the microfluidic strategies are more time- and cost-efficient than conventional SELEX.
Aptamers are an alternative category of affinity probes that are much smaller in size, making them ideal probes for intracellular targets. However, few aptamers are developed against intracellular targets, and the few intracellular-targeting aptamers are mainly used as intramers engineered to be expressed inside the target cells, which are unsuitable for intracellular biodosimetry applications. Herein, we use a radiation biomarker BAX as a target, and present an intracellular aptamer developed via microfluidic technologies. The isolated BAX aptamer would allow for in situ labeling of intracellular BAX protein, and we have preliminarily demonstrated the dose-dependent labeling in ex vivo human blood samples. This method could enable the development of aptamers for a panel of intracellular proteins towards radiation biodosimetry applications.
Aptamer development involves a screening process following the sequencing of the enriched pool. This process would usually be performed with affinity determination methods, which are often time-consuming and may hinder the development of aptamers. Herein, we reported a graphene-based nanosensor designed for the aptamer screening process. Screening of enriched pool against IgE protein was performed on this sensor. By comparative validation, this sensor showed the capacity to identify the strong binders in the enriched aptamer candidate pools and can be used to expedite aptamer development.
Molecular beacons are single-stranded oligonucleotides that adopt a stem-loop structure for in situ hybridization. They can be designed to target radiation-responsive mRNAs, which is a class of biomarkers that are attractive for biodosimetry. We design and use molecular beacons as probes for the measurement of radiation-induced changes of intracellular mRNA in a microfluidic device for the determination of radiation dosage. Our experiments, in which fixed TK6 cells labeled with a molecular beacon specific to BAX mRNA exhibited dose-dependent fluorescence in a manner consistent with RT-qPCR analysis, demonstrate the potential utility of this approach in point-of-care biodosimetry. Molecular beacons against FDXR mRNA have also been developed preliminarily.
In summary, this thesis presents the development of multiple molecular probes for intracellular targets, aiming to be applied towards biodosimetry applications. Opportunities for future research are discussed at the end of the thesis, including enhancements in microfluidic measurements of intracellular biomarkers, the development of nucleic acid probes for multiplexed measurements, and the creation of integrated microfluidic devices for point-of-care intracellular biodosimetry.
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This item is currently under embargo. It will be available starting 2025-07-18.
More About This Work
- Academic Units
- Mechanical Engineering
- Thesis Advisors
- Lin, Qiao
- Degree
- Ph.D., Columbia University
- Published Here
- August 7, 2024