| Abstract [eng] |
Nowadays third-generation biosensors, which use direct electron transfer (DET) between the active site of enzyme and electrode surface, are receiving a lot of attention. However, it is not effortless to develop effectively operating third-generation biosensors. As a result, carbon materials are used, which have many unique properties and can be modified with various functional groups. The purpose of this work was to synthesize samples of thermally reduced graphene oxide (TRGO), characterise and apply them in amperomeric third-generaton biosensors. Using modified Hummers‘ method graphite oxide (GO) was synthesized and thermally reduced thus obtaining three TRGO fractions (TRGO1, TRGO2, TRGO3). Investigation of materials revealed that after reduction, the structures partially regained electrical conductivity, lost most of their oxygen-containing functional groups and their interplanar spacings decreased. The highest electrical conductivity (2.31 S∙m-1 on a logarithmic scale), lowest amount of oxygen-containing functional groups and smallest interplanar distance (0.697 nm) revealed that TRGO3 was the most reduced. Carbon materials – graphite, GO and TRGO, were applied in biosensors, whose biological part consisted of urease and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). DET was implemented only using TRGO. Among the biosensors with PQQ-GDH and TRGO fractions, the biosensor using TRGO2 was the most effective with sensitivity 77.7±3.8 μA·mM-1·cm-2. TRGO2 had the largest surface area (708.0±0.3 m2·g-1), the highest number of defects and optimal amount of functional groups, which determined the highest sensitivity of this biosensor. All biosensors were more selective for monosaccharides due to their smaller molecular size and the optimal conditions required for the bioanalytical systems to operate effectively were 7.5 buffer solution pH, 0.2–0.3 V working electrode potential values and 10 mM buffer solution capacity. Comparing the performance of biosensors with urease, it has been found out that the sensitivity of biosensors with different TRGO2 fractions varied from 0,9±0,04 μA·mM-1·cm-2 to 1,3±0,03 μA·mM-1·cm-2. The sensitivity of biosensors using synthesized TRGO2 fractions under the same conditions is not identical, but it is sufficient and, together with the long linear part of the calibration curve (up to 25 mM), allows the constructed biosensors to be successfully used for determination of urea concentration. A third-generation GDH-TRGO biosensor was used to detect saccharides in real samples for future medical applications. The engineered GDH-TRGO biosensor was found to be suitable for the non-invasive detection of β-D-glucose and β-D-maltose when urine samples are diluted 10-fold while the working electrode potential is 0.1 V. |
