Development of reusable electrochemical immunosensor for direct detection of biotin molecule / Khoo Mai Mai

Khoo, Mai Mai (2017) Development of reusable electrochemical immunosensor for direct detection of biotin molecule / Khoo Mai Mai. Masters thesis, University of Malaya.

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Abstract

Quantifying of small molecules such as biotin in complex matrices is very challenging in measurement sciences, despite the availability of several high technology instruments (e.g., chromatography and spectroscopy techniques) for small molecule detection. However, the use of such advanced instruments is limited by high costs, instrument portability, the necessity to pre-treat samples, and the need for skilled operators. Therefore, the focus of this study is on developing a reusable electrochemical immunosensor for the sensitive and specific detection of biotin, as the proposed immunosensor is capable of operating in turbid solutions, has potential for miniaturization or automation, and is easy to handle. Two electrochemical immunosensor versions were developed for biotin detection. The first version was fabricated with oligo(ethylene glycol), whereas the second version was fabricated with sulfanilic acid (Ph-SO3-) and (4-aminophenyl) trimethylammonium (Ph-NMe3+). The stepwise immunosensor fabrication of both immunosensor types was justified by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (FESEM). Biotin detection using immunosensors is based on the concept of a displacement assay. For repeated immunosensor use, electrode polarization at -800 mV was applied for 10 minutes to enhance the dissociation of the surface-bound antibody from the surface-bound biotin so the immunosensor surface could regenerate. After five regeneration cycles, the immunosensor surface exhibited little variation in electrochemical signal, with relative standard deviation (RSD) of 9.14%. The second immunosensor version was selected for further investigation, because it produced a significant signal in a low biotin concentration range. The 1,4-phenylenediamine (Ph-NH2)/Ph-SO3-/Ph-NMe3+ immunosensor fabricated at a molar ratio of 2:1.5:1.5 (Mix 3) was selected for further investigation, as it possessed a good anti-fouling property and good sensor sensitivity according to the decrement in charge transfer resistivity (RCT) following a displacement assay. The second immunosensor version indicated high reproducibility upon testing 10 immunosensors with RSD of 8.44%, high specificity towards biotin, and stability for 28 days at 4C. The developed immunosensor displayed high precision with RSD of 513% in intra-day (each concentration used 4 immunosensors) and inter-day (five consecutive days) precision studies based on the biotin detected at concentrations of 50, 75, and 100 μg mL1. According to the biotin concentrations stated on the packaging, the detection of biotin in infant formulas and supplements using immunosensors without sample preparation exhibited high recovery (>90%). The results obtained with the immunosensors from a real sample analysis were validated by high pressure liquid chromatography (HPLC). For the biotin concentration in serum, the linear relationship between detection signals obtained using the immunosensors and HPLC confirmed the correlation between the results obtained with both techniques. In conclusion, the Ph-NH2/Ph-SO3-/Ph-NMe3+-fabricated immunosensor developed for biotin detection was found to be reproducible, selective, resistive to non-specific protein adsorption, stable, accurate and precise for the direct detection of biotin in food, and clinical samples. Besides, cathodic polarization was considered a suitable approach for constructing a reusable immunosensor that is able to perform multiple measurements without detection ability loss.

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