|Author:||Wong, Kwun Fung Jacky|
|Title:||Ultra-stable oligonucleotide-nanoparticle conjugates prepared by silica reinforcement method : properties and applications for colorimetric DNA detection|
DNA -- Analysis.
Hong Kong Polytechnic University -- Dissertations
|Department:||Interdisciplinary Division of Biomedical Engineering|
|Pages:||xxiv, 187 leaves : ill. (chiefly col.) ; 30 cm.|
|Abstract:||Gold nanoparticles have been widely utilized for molecular diagnosis, drug delivery, and nanostructuring. In particular, colorimetric methods of deoxyribonucleic acid (DNA) detection using oligonucleotide-gold nanoparticle conjugates has received considerable attention for point-of-care and on-site testing. Typically, these conjugates are prepared by chemisorption of monothiol-modified oligonucleotides onto gold nanoparticles' surface. Despite their high stability against salt-induced aggregation, they have limited chemical and thermal stabilities, as well as enzymatic amplification reaction compatibility, which prohibit them from real applications. This thesis describes a new silica reinforcement method designed to address these issues by coating the conjugate with a thin silica layer using (3-mercaptopropyl)trimethoxysilane. The silica-modified conjugate is simple-to-prepare and exhibits extremely high stability toward dithiothreitol (no noticeable aggregation after 24 h). In addition, the conjugate has high stability against gold core oxidative dissolution by sodium cyanide. These favorable chemical properties are attributed to the entrapment effect offered by the silica layer. Importantly, the silica layer is so thin that the unique sequence-specific hybridization-induced colorimetric detection of conjugate is preserved. With these properties, a closed-tube colorimetric isothermal amplification reaction (nicking endonuclease-assisted amplification) platform based on the silica-modified conjugate is demonstrated. Aiming at higher sensitivity, the incorporation of silica-modified conjugate into polymerase chain reaction is highly desirable. Compared to the conventional unmodified conjugate, the silica-modified conjugate has significantly higher thermal stability as well as lower non-specific adsorption of Taq DNA polymerase. With these, a closed-tube colorimetric polymerase chain reaction is demonstrated for the first time, the sensitivity of which is comparable with the standard gel electrophoresis technique and avoids carryover contamination due to enzymatic reaction incompatibility of unmodified conjugate (requirement of post-amplification addition of unmodified conjugates for detection). Besides oligonucleotide-gold nanoparticle conjugate, this silica reinforcement method is successfully applied to oligonucleotide-silver nanoparticle conjugate. In fact, it is potentially broadly applicable to other biomolecule/polymer-nanoparticle conjugates. To conclude, this silica coating method not only enables simple, sensitive, and contamination-free DNA analysis for point-of-care and on-site applications, but also holds promise for new medical diagnostic and therapeutic applications.|
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