High sensitivity nucleic acid detection using metal nanowires and nanotubes
McCooey, Andrea (2015) High sensitivity nucleic acid detection using metal nanowires and nanotubes. PhD thesis, Dublin City University.
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DNA and RNA can be classed as biomarkers of disease, i.e. proteins, antibodies, nucleic acids and cells that are found in abnormal amounts in body fluids or tissue when disease is present. As the number of deaths attributed to diseases such as cancer increase year on year, a strong emphasis is now being placed on the development of point of care devices which test for levels of these biomarkers in blood, serum, saliva, urine or other sample matrices.
An electrochemical biosensor has been developed which aims to amplify the signal associated with a single nucleic acid binding event, rather than amplifying the target nucleic acid, as per PCR or NASBA. This sensor involves a three step procedure, consisting of the immobilisation of a capture strand of single strand DNA to a gold electrode, it’s hybridisation with a complementary target, followed by target hybridisation to a complementary probe strand which is labelled with a nanowire.
Nanowires have a number of unique physical and electronic properties that are different from those of spherical nanoparticles, which makes them ideal for use in an electrochemical biosensor. The advantage of nanowires and nanotubes is due to their increased surface area to volume ratio, which changes or enhances the properties of the material they are made of, but also nanotubes and rods can be further functionalised with nanoparticles or biomolecules such as proteins, enzymes or nucleic acids, which can create multi-functional structures.
A template-based electrodeposition process has been employed in order to deposit ordered arrays of uniformly-sized metal nanowires. The electrochemical and spectroscopic properties of these nanowires were investigated with respect to electrodeposition time. Hollow nanotubes were also electrochemically deposited within the walls of polycarbonate membranes. Nanotubes offer the advantage of increased surface area along the inside wall as well as the outside wall, which can enhance the signal associated with the binding of target DNA. The inside walls of nanotubes can also be functionalised with nanoparticles or biomolecules which can add a multi-functional dimension to these structures. The electrochemical and spectroscopic properties of these nanotubes were also investigated and compared to those of the solid nanowires.
Gold nanowires were electrodeposited using the pores within a polycarbonate membrane as a template. Gold nanostructures are well known to catalyse the reduction of hydrogen peroxide. The catalytic activity of gold nanowires and nanotubes bound through DNA hybridization was assessed by monitoring the difference in current associated with the reduction of hydrogen peroxide in a solution of 0.01M H2SO4 before and after peroxide addition. Single stranded capture DNA was bound to the nanowires and an underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen Staphylococcus Aureus, (S. aureus), which causes mastitis. The modified electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. Semi-log plots of the pathogen DNA concentration vs. change in faradaic current were linear from 1 fM to 10 μM. The hollow nanowires also produced a response that was linear from 1 fM to 10μM, but with a dramatically decreased sensitivity when compared with the solid nanowires (10.3 μA dec-1 vs. 15.5 μA dec-1).
Gold-Copper core:shell nanowires have been electrodeposited within the pores of a track etched polycarbonate membrane filter. The sacrificial “stripping” of the outer metal shell of the nanowire has been identified as an alternative detection strategy for nucleic acid detection, which is important for application in a multiplexed assay. Use of this method could allow for several types of target DNA to be detected in a single assay just by manipulating the properties of different metals. Core: shell nanowires functionalised with probe strand DNA that is complementary to that of the pathogen S. Aureus were immobilised onto an electrode surface via a DNA sandwich assay. The charge associated with the reductive desorption of the core-shell nanowires were linearly dependant on the log of the target DNA concentration from 1nM to 100 μM.
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