Development of enzymatic and aptamer-based electrochemical biosensors using carbon supported mesoporous silica and gold electrodes for the determination of glucose and insulin

Diabetes is a chronic and metabolic disease that causes disorders in regulating blood sugar. Blood sugar regulation occurs with the hormone insulin, secreted by the pancreas. It enables glucose in the blood to be taken into the cells and converted into energy. In patients with Type 1 diabetes, the pancreas cannot secrete enough insulin, as in Type 2 diabetes, the cells become insensitive to insulin. In this case, external insulin may be required to keep blood sugar at normal levels. For this purpose, electrochemical and aptamer-based biosensors can be used in insulin therapy. This study aimed to develop two new biosensors for the determination of glucose and insulin. In the part of the biosensor developed to determine glucose, the suitability of silica-derived mesoporous materials for enzyme immobilization and its performance in electrochemical bioelectronic systems were investigated. For this purpose, conductivity has been gained by adding graphene oxide (GO), a thin, flexible and conductive layer, to silicasourced mesoporous materials. MCM-41, a regular porous and high surface area mesoporous material, was used as the silica-derived mesoporous material. GO/MCM-41 solution consisting of a mixture of GO and MCM-41 (1:1) by volume was prepared. Electrochemical characterizations were made by coating this solution on printed circuit carbon electrodes. Linear scanning voltammetry and alternating voltammetry methods were used in characterization studies. As a result of electrochemical characterization studies, the GO/MCM-41 solution, which showed good performance, was selected, and its performance in biosensor application was examined. GO/MCM-41/Fc modified electrode was obtained by coating 75 mM ferrocene on GO/MCM-41 coated on SPE. Ferrocene is a redox-active compound and provides an electrochemical signal. Then, the glucose oxidase (GOx) enzyme was arrested on the GO/MCM-41/Fc modified electrode. GOx is an enzyme that catalyzes the oxidation of glucose. During the immobilization of the enzyme, a polymer film was formed using chitosan. The optimal enzyme arrest time was determined for GO/MCM-41/Fc/Chit. The determined enzyme arrest time is four days at +4 °C; for glucose determination of the prepared biosensor, linear operating range, detection limit, selectivity, reproducibility, shelf life and real sample analysis were performed. The linear operating range of the biosensor for glucose determination was found to be 1 - 10 mM. The limit of detection (LOD) was calculated as 0.516 mM. The reproducibility of the biosensor was evaluated by the relative standard error of its response to 5 mM glucose concentration at ten different times. The relative standard error was found to be 5.84%. The shelf life of the biosensor was evaluated with the relative standard mistake of the 1st-day response of the shelf life responses on the 3rd, 7th and 27th days, respectively. The standard relative error was 6.44%, 9.82% and 5.86%. The selectivity of the biosensor was evaluated by the effect of substances such as uric acid, ascorbic acid and insulin that could potentially interfere with the response. It was concluded that these items did not significantly affect the response. For real sample analysis, the amount of glucose in sour cherry, strawberry and apricot jams was calculated from the calibration curve. The calculated glucose was partially compatible with the values given by KOSKA. An aptamer-based electrochemical biosensor system was designed in the other part of the study. In this system, gold (Au) is coated on the printed circuit carbon electrodes (SPE) by electroplating and insulin-recognizing aptamer is attached to the Au surface. The binding conditions and surface occupancy of the aptamer were characterized. Then, the interaction of aptamer with insulin was investigated by signal on-off measurement method. The incubation times of aptamer, mercaptohexanol and insulin were then determined as 180 min, 60 min and 25 min by response surface method using the Design-Expert program, respectively. The response of the aptamer for different insulin concentrations was determined using the electrochemical square-wave voltammetry method and the calibration curve was obtained by taking the differences according to the aptasensor background response. The linear working range of the developed aptasensor in aqueous solution was determined as 25 - 150 pM and the detection limit value was 18.45 pM (at 95% confidence interval). Finally, interfering substances were identified (streptavidin, glucose, thrombin, and uric acid) and aptasensor was validated. No significant interfering effect was observed for the aptasensor. The lowest relative standard deviation and accuracy of the aptasensor response were found to be 9.5% and 6.4%. The stability loss of the aptasensor within 10 days was found to be 8%.