Atom Transfer Radical Polymerization (ATRP)in Amplification-by-Polymerization for DNA Sensing

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Title: Atom Transfer Radical Polymerization (ATRP)in Amplification-by-Polymerization for DNA Sensing
Author: Lou, Xinhui
Advisors: Bruce Novak, Committee Member
Lin He, Committee Chair
Daniel L. Feldheim, Committee Member
Morteza G. Khaledi, Committee Member
Abstract: DNA sensing has attracted a lot of attention due to its importance in biological and medical fields. Numerous methods have been developed to amplify DNA hybridization signals to meet different needs. The purpose of this research was to develop a simple yet efficient method to detect sequence specific DNA in a home testing kit format according to the concept of amplification-by-polymerization. This dissertation reports the development of an atom transfer radical polymerization (ATRP)-based DNA detection method. The background knowledge on current DNA sensing methods and the applications of polymers in DNA sensing technologies are described in Chapter 1. Chapter 2 describes the proof-of-concept experiments of this DNA detection method. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on gold surface where specific DNA sequences located. These initiators subsequently triggered the growth of polymer at the end of DNA molecules. Only the perfectly matched targets were distinctively observed by the naked eye due to the formation of polymer that altered the substrate opacity. The demonstrated capability to detect DNA with direct visualization laid the groundwork for the future development of detector-free testing kits in DNA sensing. Chapter 3 describes the two strategies to further improve the sensitivity of this detection method: by formation of branched polymer through repetitive ATRP and by minimization of the background noise through optimization of the passivation layer in DNA monolayers. Chapter 4 describes the kinetics of DNA⁄polymer formation using different catalyst systems. The effects of the composition and concentration of the catalysts used during DNA-accelerated ATRP reaction were evaluated. The results showed a strong correlation between polymer formation and the reaction conditions. The results also showed that the presence of DNA molecules significantly fastened the growth rates of both PHEMA and POEGMA in ATRP. This accelerating effect was suspected as a combined result of the highly charged DNA backbones and the unique chemical structure of DNA molecules. Chapter 5 describes the two applications of this ATRP-based detector-free DNA detection method: in single nucleotide polymorphism (SNP) detection and in human gender determination. Chapter 6 describes a colorimetric ATRP-based DNA detection method based on the increased stability of polymer coated gold nanoparticles (GNPs). In this method, hybridizations, ligation and ATRP were conducted on ssDNA labeled GNPs. GNPs remained red color or aggregated after ATRP at the presence or absence of complementary target DNA in the hybridization step, respectively. The reported method provides a generic approach for biomolecular hybrid formation on a solid surface and could open up new possibilities in the applications of DNA detection and gene delivery. Chapter 7 describes a direct comparison between CNBr chemical ligation and T4 ligation. Much higher ligation efficiency and specificity of CNBr ligation was found compared to T4 ligation, which renders the potential applications of CNBr ligation in DNA sensing to replace enzymatic ligation.
Date: 2007-08-07
Degree: PhD
Discipline: Chemistry

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