Development of Electrochemical Biosensor for Ageing Biomarkers

Abstract

The ageing population is now a serious problem around the world resulting in healthy ageing is the global goal to address this issue. To effectively control the incidence of ageing diseases caused by the high-level concentration of reactive oxygen species in the human body, the electrochemical biosensor is one of the most common ways for detection. However, the lack of easy operation, high selectivity, and quick response is still the challenge of the quantitative measurement for superoxide anion (O2 - ∙) in the blood environment. This study is aimed to develop an O2 - ∙ biosensor that could fit in a hand-held device to provide quick and accurate results. The generation methods of O2 - ∙ were first discussed due to their characteristics of poor stability. Compared with the chemical generation, the biological method showed a 10 % higher activity of enzymes and good stability of generated O2 - ∙ for at least 5.5 hours. This study was focused on two biosensors, the superoxide dismutase (SOD)-based biosensor due to the high selectivity for O2 - ∙, and a coupled SOD- horseradish peroxidase (HRP) biosensor for the high accuracy of the O2 - ∙ quantitative measurements. The immobilization of the enzyme SOD and HRP were achieved by layer-by-layer adsorption and cross-linking technique respectively. The calibration curves for these two biosensors were generated and the effect of interferences was discussed subsequently. Results showed both biosensors had good linearity between the detection range of 0 to 0.5 mM O2 - ∙, with the sensitivity and limit of detection for the SOD-based biosensor as 67.13 µA/(mM·cm2 ) and 5.6 μM and for coupled SOD-HRP biosensor as 23.8 µA/(mM·cm2 ) and 6 μM respectively. Only glucose showed a positive bias on SOD-based biosensor but no effect on coupled SOD-HRP biosensor. The O2 - ∙ detection in blood samples was achieved by both two biosensors and the bias from glucose was considered in the calibration for the SOD-based biosensor. The blood sample was diluted at different multiples to demonstrate the accuracy of the results. Compared with the chemiluminescence method, the biosensors showed the advantages of a quick response in 100 seconds, high selectivity for O2 - ∙ and successful O2 - ∙ quantitative measurement of around 0.2 mM in volunteer blood samples.