Abstract
Deoxyribonucleic acids (DNAs) are generally labeled as hereditary material that can carry genetic information for living cells. In recent years, DNAs, as a natural material, have been an attractive candidate in biomedical science and engineering research studies because of their excellent biocompatibility and low cytotoxicity. DNAs can be a structural engineering material for constructing sequence-specific nano-objects with predictable conformation and highly programmable reaction circuits. This allows them to serve as effective detectors for pathogens and diseases, and play a role in diagnostic imaging and gene therapy studies. Moreover, DNAs can be modified with various functional groups to conjugate with other materials, generate signal reporters and execute multi-functional events, such as linking, imaging, and targeting. In this thesis, we introduce three applications using functionalized DNAs with different remarkable properties - visual detection of lead ions through nanoparticle-amplified magnetophoresis, fluorescence quantification of intracellular sodium ions, and in vitro cellular miRNA imaging and gene silencing.1.Visual detection of lead ions based on nanoparticle-amplified magnetophoresis and Mie scattering
We built a visual lead ion detector based on GR-5 DNAzyme with nanoparticle-amplified magnetophoresis and Mie scattering. GR-5 DNAzyme is a specifically designed DNA that folds into a complex tertiary structure and is cleavable by lead ions. In the presence of lead ions, released fragments from cleaved GR-5 DNAzymes can connect magnetic microparticles (MMPs) and gold nanoparticles (AuNPs) through DNA hybridization. Then, the connected AuNPs are released to connect a second pair of MMPs and polystyrene microparticles (PMPs). Due to the high surface-to-volume ratio, hundreds of oligonucleotides on released AuNPs are available to form MMPs-AuNPs-PMPs to achieve an amplification effect. Using magnetophoresis, solution turbidity changes from milky to clear because of the reduction of Mie scattering when fewer free PMPs are suspended in the solution. Our results show a limit of detection at 0.15 nM with a linear range from 0 to 0.7 nM, and the selectivity is at least 50000 to 1. This detection method can also be applied to detect lead ions in solutions with different pH values or tap water. This method can be used in low-resource settings and provide the same sensitivity as detection methods using fluorescence labels or electrochemistry.
2.Intracellular sodium detection based on mRNA activation
We developed a bioactive control DNA device to measure intracellular sodium ion concentration in specific cell types without the impact of nanodevice susceptibility towards the environmental background. This device can detect the intracellular sodium ion concentration or changes in membrane potential for the study of the role of metal ions in cell growth pathways or as indicators of physiological and pathological conditions. With the aid of blocking strands, the cleavage property of DNAzyme is inhibited outside the specific cell types, which minimizes false-positive signals caused by sodium in the environment, such as cell culture growth medium and buffer for DNA hybridization. The trigger mechanism does not require any external motivations such as light irradiation or specific modification. The DNAzyme is automatically activated by the release of blocking strands through strand displacement with the specific mRNA inside the selected cell type. This platform offers broad and promising applications for the onsite detection of intracellular metal ions in specific cell type.
3.Y-shaped DNA complex with dual function for miR-21 detection and cancer therapy
We developed a dual system with a sensing and silencing effect based on a Y-shaped DNA nanodevice. Here, the Y-shaped structure is built with 3 single DNA strands functioning as a linker unit, a signal unit, and a therapy unit respectively. The nanodevice aims to activate conformational change in response to a single trigger, identify the cell phenotype by detecting cancer cells' biomarkers, and execute dual- functional events (miRNA sensing and gene silencing) with signal reporting. Inside cancer cells, the device could detect the target biomarker miR-21 and activate gene therapy to cause expression reduction of cell death antagonists, Bcl-2. In contrast, the side effect on normal cells can be minimized by the bioactive control of the 'close-gate' nanodevice. This method might have potential applications in the sensing of biomarkers and gene therapy as a new intelligent theranostic system.
In summary, owing to the functionalization versatility of DNA, we believe that DNA can act as a mediator and be applied into different biosensors, both in vitro and in vivo. Moreover, combined with the technological advancement in cellular analysis, we expect the proposed methods to be helpful in real-time in vivo studies and the development of commercial products in the future.
| Date of Award | 27 Oct 2022 |
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| Original language | English |
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| Supervisor | Ting Hsuan CHEN (Supervisor) |