Carbon fiber based DNA biosensor design

This thesis focuses on a carbon fiber microelectrode based DNA biosensor design. Initially, electrochemical detection techniques were investigated for DNA hybridization. Methylene blue (MB) as a hybridization indicator was prefered in this study. Firstly, an electrochemical DNA biosensor was developed based on the recognition of target DNA by hybridization detection with immobilized capture synthesized 15 mer single-stranded deoxyribonucleic acid (ssDNA) capture probe on a chemically modified carbon fiber microelectrode (CFME). The capture probe was covalently attached to amine-labeled ssDNA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosulfosuccinimide (NHS) modified CFME. Square wave voltammetry (SWV) was used to monitor the hybridization reaction on the capture probe electrode. The covalently immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on CFME surface by using methylene blue (MB) as a hybridization indicator. The decrease of the peak current of MB, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The peak current of MB was found to increase in the following order: hybrid-modified CFME, single-base mismatch hybrid-modified CFME, noncomplementary modified CFME. The results showed that the developed biosensor had a good calibration range between 0.5 and 10 ppm, with a detection limit of 0.72 ppm. Secondly, the study reports a systematic and detailed investigation on the interactions between methylene blue (MB) and DNA immobilized onto carbon fiber microelectrode (CFME). In this study, designed CFME based DNA biosensor was successfully carried out considering three different hybridization mechanisms to obtain more specific and selective results. The consistency of the results was tested with the various probe configurations (unlabeled and amino-labeled). The voltammetric signals of MB were measured single stranded DNA (ssDNA)-modified CFME and double stranded DNA (dsDNA)-modified CFME by means of square wave voltammetry (SWV). The electrochemical parameters for MB on binding to DNA onto single CFME in the solution and at the electrode surface were described. This study shows that before or after hybridization, MB accumulation is related with probe configuration. Thirdly, the research also investigated that the dilution agent effect onto interactions between methylene blue (MB) and DNA using carbon fiber microelectrode (CFME) based DNA biosensor. In this study, designed CFME based DNA biosensor was successfully carried out considering the effect of probe dilution agent (e.g. 3-mercaptopropionic acid) against proposed hybridization mechanism types. This study shows that probe dilution agent is a significant factor in determining the type of DNA hybridization mechanisms. Overall, it can be concluded that CFME could be used as an effective electrode for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects. It provides very useful information for developing carbon fiber based sequence-specific electrochemical DNA sensors. In this study, designed CFME based DNA biosensor was used successfully in the detection of specific DNA sequences.