Bionanotechnology approaches to the amplification-free detection of nucleic acid

Thomson, David Andrew Coley (2012). Bionanotechnology approaches to the amplification-free detection of nucleic acid PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Thomson, David Andrew Coley
Thesis Title Bionanotechnology approaches to the amplification-free detection of nucleic acid
Formatted title
Bionanotechnology Approaches to the Amplification-Free Detection of Nucleic Acid
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Matthew Cooper
Trent Munro
Krassen Dimitrov
Total pages 148
Total colour pages 33
Total black and white pages 115
Language eng
Subjects 030302 Nanochemistry and Supramolecular Chemistry
090303 Biomedical Instrumentation
060199 Biochemistry and Cell Biology not elsewhere classified
Formatted abstract
The detection of viruses in complex biological samples is important in the management of infectious disease caused by human viral pathogens. Amplification-free quantitative detection of viral nucleic acid enables simplified diagnostic devices with characteristics appropriate for point-of-care and resource-limited settings. A new nanoparticle assay was developed for detection of a Herpes simplex Virus (HSV) DNA sequence within complex, minimally processed clinical samples. In a sequence-specific manner, viral nucleic acids link magnetic nanoparticles to fluorescent nanoparticle reporter labels. Following washing steps, the number of fluorescent nanoparticles was quantified, leading to a correlated read-out of target molecule concentration.

The assay was first established using commercially available conjugation methods to develop reagents specific for HSV. This format provided a limit of detection in the low picomolar range, with non-specific interactions defining a key aspect of the assay performance. Undesired nonspecific interactions lead to an investigation of particle coating chemistries to reduce non-specific binding. These techniques were based on the living radical polymerisation (LRP) of a copolymer of azide-modified and hydroxyl-terminated oligoethylene glycol methacrylate (OEGMA) polymer brushes from the surface of magnetic nanoparticles. Use of polyethylene oxide based polymers lead to improvements in assay sensitivity by reducing background noise. The azide substituents within the copolymer were conjugated to alkyne-substituted HSV specific capture probes via an orthogonal and efficient copper-catalyzed azide alkyne cycloaddition (CuAAC). LRP and CuAAC were used to synthesise nanoparticle reagents that enabled amplification-free detection of viral DNA in minimally processed clinical samples.

Two microfluidic devices were developed for fluorescent nanoparticle readout, including a static chamber and a continuous-flow magnetophoresis device. The magnetophoresis experimental system provided a method for  continuous-flow magnetic particle extraction using an opposed magnet configuration and a passive gravity-actuated system for sample and buffer delivery. Reporter and magnetic nanoparticle complexes were read out using confocal fluorescence microscopy. In a comparison study, the static-chamber device gave improved performance, as determined by a signal-to-noise comparison at 50 pM concentration of DNA. After a final round of buffer component optimisation, including the addition of a critical reagent (proteinase K) and a number of surfactants, final performance of the assay was assessed with the static-chamber readout method. The assay provided equivalent performance of 500 fM (25 attomole) of DNA in buffer samples and neat serum. The study demonstrated the utility of the assay format, surface modifications techniques, buffer components and microfluidic device for DNA assays conducted in minimally processed clinical samples.
Keyword Amplification-Free

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Created: Wed, 19 Jun 2013, 22:07:58 EST by David Thomson on behalf of Scholarly Communication and Digitisation Service