A Functionalised Aptamer Electrochemical Biosensor Platform

The ability to utilise new knowledge of biomarkers from genomic and proteomic data will have a great impact on molecular diagnosis. Biomarker detection could be achieved by utilising a capture molecule that associates specifically with the target biomarker. The work described in this thesis focuses on a platform comprising a lysozyme binding aptamer and an amperometric electrode (an electrochemical aptasensor). To couple the binding reaction to a change in current, the aptamer is modified with a redox group, ferrocene. Two types of signalling aptamer were constructed, one comprised the aptamer self-assembled on gold and hybridised to a short complementary oligonucleotide carrying a ferrocene group. The second incorporated the binding sequence into a molecular beacon, one end of which self-assembled onto the electrode, the other end carried the ferrocene group. Both of these showed a lysozyme dependent change in current on a gold electrode. Further characterisation of the first aptasensor suggested that the nucleic acid formed a multilayer structure on the electrode surface and that lysozyme binding induced conformational change moved ferrocene close to the surface, increasing the current. In contrast, the second aptamer usually showed a decrease in current in the presence of lysozyme suggesting that the binding resulted in the ferrocene moving away from the surface. In order to evaluate the possible use of these aptasensors for continuous in vivo measurement, needle shaped microelectrodes arrays were produced and the beacon aptamer immobilised on the surface. These electrodes had high impedance which resulted in low sensitivity, however lysozyme binding could still be detected using electrochemical impedance spectroscopy with ferrocyanide in solution. These microspike arrays could also be used for glucose sensing following modification with glucose oxidase