Nucleic-acid recognition interfaces: How the greater ability of RNA duplexes to bend towards the surface influences electrochemical sensor performance

The influence of RNA versus DNA on the performance of electrochemical biosensors where redox-labelled nucleic acid duplexes bend towards the electrode surface has been assessed. Faster electron transfer was observed for duplexes containing RNA, suggesting duplexes with RNA are more flexible. These data are of particular importance for microRNA biosensors

Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation

Electrochemical microRNA biosenosrs

To develop point of care devices for cancer by detecting blood-based biomarkers, there are several challenges that the current research aims to overcome including higher sensitivity, greater selectivity, and rapid assaying time. To achieve these goals, the results of three research chapters are presented in this PhD dissertation. The first two chapters aim to establish a detection method for microRNA while also seeks to advance fundamental insight into the impact of surface chemistry on the performance of nucleic acids biosensors. In the first chapter, the effect of interfacial design on the mismatch discrimination capability from methylene blue, as a non-covalently bound redox label, at low-density DNA films was studied. A decrease in the ability of the double helix to access the electrode surface was found to result in an improvement in the single mismatch discrimination. Although selective discrimination of the target microRNA from single base pair mismatch was achieved, the detection scheme required multi incubation and rinsing steps, which is not desirable. To address this issue, the applied sensing method in the second chapter involves the use of methylene blue covalently attached to probe nucleic acid, whose distance from the electrode surface is altered as a result of the conformational changes of probe upon hybridization. Using this method, the impact of surface chemistry of nucleic acids duplexes (the choice of probe and target being DNA or RNA) on their electrochemical properties has been investigated. It was found that DNA/RNA has a unique and highly bent conformation, compared to the well-studied DNA/DNA duplex. The optimal conditions for the construction of microRNA detection have been identified using this system. To achieve ultralow concentration detection, the third chapter reports on combining the developed detection method for microRNA in the second chapter, with the concept of using gold coated magnetic particles as dispersible electrodes. This is to address the limited mass transfer rates at the nanoscale. Using this combination, an ultrasensitive and rapid detection of microRNA, with a lowest detected concentration of 1 aM and a response time of 30 min, was achieved

Dual Signaling DNA Electrochemistry: An Approach To Understand DNA Interfaces

Electrochemical DNA biosensors composed of a redox marker modified nucleic acid probe tethered to a solid electrode is a common experimental construct for detecting DNA and RNA targets, proteins, inorganic ions, and even small molecules. This class of biosensors generally relies on the binding-induced conformational changes in the distance of the redox marker relative to the electrode surface such that the charge transfer is altered. The conventional design is to attach the redox species to the distal end of a surface-bound nucleic acid strand. Here we show the impact of the position of the redox marker, whether on the distal or proximal end of the DNA monolayer, on the DNA interface electrochemistry. Somewhat unexpectedly, greater currents were obtained when the redox molecules were located on the distal end of the surface-bound DNA monolayer, notionally furthest away from the electrode, compared with currents when the redox species were located on the proximal end, close to the electrode. Our results suggest that a limitation in ion accessibility is the reason why smaller currents were obtained for the redox markers located at the bottom of the DNA monolayer. This understanding shows that to allow the quantification of the amount of redox labeled target DNA strand that hybridizes to probe DNA immobilized on the electrode surface, the redox species must be on the distal end of the surface-bound duplex

Nucleic acid hybridization on an electrically reconfigurable network of gold-coated magnetic nanoparticles enables microRNA detection in blood

There is intense interest in quantifying the levels of microRNA because of its importance as a blood-borne biomarker. The challenge has been to develop methods that can monitor microRNA expression both over broad concentration ranges and in ultralow amounts directly in a patient’s blood. Here, we show that, through electric-field-induced reconfiguration of a network of gold-coated magnetic nanoparticles modified by probe DNA (DNA–Au@MNPs), it is possible to create a highly sensitive sensor for direct analysis of nucleic acids in samples as complex as whole blood. The sensor is the first to be able to detect concentrations of microRNA from 10 aM to 1 nM in unprocessed blood samples. It can distinguish small variations in microRNA concentrations in blood samples of mice with growing tumours. The ultrasensitive and direct detection of microRNA using an electrically reconfigurable DNA–Au@MNPs network makes the reported device a promising tool for cancer diagnostics