High sensitivity DNA detection using gold nanoparticles and conducting polymers
Spain, Elaine (2011) High sensitivity DNA detection using gold nanoparticles and conducting polymers. PhD thesis, Dublin City University.
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The detection and quantitation of specific nucleic acid (NA) sequences continues to grow in importance driven by issues ranging from personalized medicine to companion diagnostics such as antibiotic selection for infectious diseases. In order to enhance the sensitivity of electrochemical detection of DNA, novel conducting polymer- metal nanoparticle composites have been created. An electrode modified with nanostructured gold (AuNP-elec) has been used to increase the surface of the electrode and therefore the amount of single strand DNA (ss-DNA) immobilised. This modification results in more capture strands being available to bind target strands thus improving the detection limit of the ssDNA biosensor. This sensor follows a three step procedure involving the immobilisation of a capture strand, its hybridisation with the target followed by complementary strand binding to a horse radish peroxidase, HRP, labelled oligo. By carefully selecting the immobilisation buffers as well as optimising the hybridisation times, a significantly improved current response was obtained. Electrochemical detection of both methods was carried out through a suitable substrate (H2O2) for the enzyme labelled duplex.
Polyaniline (PANI) has been synthesized by electrochemical, chemical and vapour oxidation methods. These PANI films have then been modified with electrochemically deposited chemically grown gold nanoparticles to give a nanocomposite material and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold
electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a horseradish peroxidase (HRP)-labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150 pM to 1 μM and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.
PEDOT films have been created by solution-casting an oxidant on the target substrate and then drying it followed by exposure to the monomer vapour. The dried substrate containing the oxidant is then placed over the liquid surface of the conjugated monomers in a closed chamber. The conjugated monomer liquid can easily volatilize and the substrate is fully exposed to the conjugated monomer vapour phase. The PEDOT films were also modified with AuNPs and compared to the electrochemical and chemical synthesis of PEDOT materials. Semi-log plots of the pathogen DNA concentration (150 pM to 1 μM) vs. faradaic current were determined. The PEDOT biosensor after AuNP deposition showed a linear response over a wide dynamic range from 1 nM to 1 μM with a detection limit of 3.8 fM. The implication of these findings and the possibility of extending such high sensitivity detection techniques for detecting other DNA molecules are discussed.
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