| DNA biosensing has been developed in the past decades and the current state-of-the-art techniques have been employed to amplify DNA hybridization signals in different formats. The challenge nowadays in DNA biosensing lies in simplifying signal readouts and building field-friendly sensors that are amenable to point-of-need applications without compromising achieved sensing sensitivity.;This dissertation reports the development of a simple yet sensitive molecular amplification method, i.e. amplification-by-polymerization, on DNA detection. Chapter 1 described the research background of DNA biosensing from history, development, and significance.;Chapter 2 described the proof-of-concept experiments of RAFT polymerization-based DNA detection method. In particular, surface-initiated polymer growth was regulated by the immobilization of chain transfer agents on the Au surface where DNA hybridization occurred. A linear polymer growth was observed as a function of the reaction time, characteristic of "living" polymer reactions. Significant improvement in assay sensitivity was realized in comparison to the previously reported polymerization-based sensing method by enhancing polymer growth rate and reducing background noises caused by nonspecific adsorption. Direct visualization of fewer than 2,000 copies of a short oligonucleotide sequence was demonstrated in a detector-free fashion.;Chapter 3 describes the kinetics study of polymer growth atop DNA molecules via RAFT polymerization to optimize this DNA detection method. Growth kinetics of poly(monomethoxy-capped oligo(ethylene glycol) methacrylate) atop DNA molecules is investigated by monitoring the change of polymer film thickness as a function of reaction time. The reaction conditions, including the polymerization temperature, the initiator concentration, the CTA surface density, and the selection of monomers, were varied to examine their impacts on the grafting efficiency of DNA-polymer conjugates. Comparing to polymer growth atop small molecules, the experimental results suggest that DNA molecules significantly accelerate polymer growth, which is speculated as a result of the presence of highly charged DNA backbones and purine/pyrimidine moieties surrounding the reaction sites.;Chapter 4 describes the possible mechanism of DNA-accelerated RAFT polymerization on surface. A DNA templating theory was discussed to address the observed accelerated surface-initiated RAFT polymerization atop DNA molecules. This DNA templating effect was found to be tunable by adjusting ionic strength, pH value and polarity of the solution. At the same time, chemical structures of monomers and DNA templates, such as negatively-charged monomers and DNA with different sequence length and base, also have an important impact on DNA-templated polymerization.;Chapter 5 expands the application of this RAFT-based DNA biosensing to 3-D in-gel DNA detection. Hybridization of complementary DNA detection probes introduced chain transfer agents (CTA) into the gel via preconjugation to the probes. Surface-initiated polymer growth was prompted on the gel surface and the film growth on the surface where DNA hybridization occurred was monitored using infrared spectroscopy and atomic force microscopy. Visible change in the texture of the porous gel occurred after polymer growth, which offered an attractive detection alternative for in-gel DNA analysis.;Chapter 6 describes the use of a polymerization reaction as a signal amplification method to visualize sex-specific genomic DNA markers directly extracted from blood without PCR. Clear distinction between X and Y chromosomes was made both qualitatively by naked eye and quantitatively by ellipsometry. Assay optimization was conducted, including the selection of the proper blocking reagents, annealing temperature, and annealing time. The results confirm the potentials of the described sensing technique in eliminating PCR for complex genome DNA analysis, which advances one step closer to the development of a portable DNA sensing device for point-of-need applications. |
