DNA hybridisation detection on chemically modified electrodes with bioelectrocatalytic amplification of signal
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Electrochemical detection of DNA hybridization is a viable alternative to optical and radio-labelled techniques. The benefits of using biosensors based on electrochemical systems are: high sensitivity, possible development of miniature systems interfaced to electronic devices as well as low cost of operation and small sample requirements. I detail in this thesis development of an electrochemical DNA hybridization assay, based on deposition of films consisting of single-strand DNA cross-linked with an osmium-based redox polymer on gold microelectrodes modified with self assembled monolayer of cysteamine. A signal, corresponding to hybridization between the immobilized probe ssDNA and a biotin-conjugated target DNA is amplified by addition of glucose oxidase-avidin conjugate and glucose substrate. The interaction between target DNA, glucose oxidase-avidin and osmium redox polymer layer generates a bioelectrocatalytic current in the presence of glucose. Catalytic currents corresponding to oxidation of glucose scale with complementary DNA concentration. A sensitivity improvement is gained when replacing gold macroelectrode (2 mm diameter) with microelectrode (40 microns in diameter). In a further study, modification of carbon electrodes via aryl diazonium electroreduction was investigated to provide a more robust sensing platform. The electrografted layer, consisting of aromatic amine, can be then used to anchor carboxymethylated dextran, allowing further attachment of single stranded DNA within the anchored film. Hybridisation of biotinylated complementary DNA sequence followed by reaction between biotin and glucose oxidase-avidin results in bioelectrocatalytic current in the presence of glucose and ferrocenemethanol in solution as electron transfer mediator. As was observed for the microelectrode sensor, the signals scale with the concentration of target DNA yielding a sigmoidal curve, when plotting current versus logarithm of concentration. The more robust sensing platform allows for use of blocking agents with this assay. Presence of milk powder and detergent (sodium dodecyl sulphate) in hybridisation solution improved the specificity of the sensor. DNA sensing requires analysis of an enormous number of samples. A high through-put approach using the sensing platform developed can be achieved using disposable electrodes. The same approach to sensing platform development was thus applied to screen-printed carbon electrodes. Such screen-printed sensors, as in the previous cases, allowed distinguishing between complementary and non-complementary sequences of target DNA. The standard deviation of the signals was, however, higher for screen-printed electrodes than that observed for graphite disc electrodes. Future work should focus on improving the precision of the assay. This would involve seeking well-defined and reproducible electrodes and also investigating the use of reagents which have minimum effect on background currents of carbon electrodes.