Thermally reduced graphene oxide (TRGO) is a graphene-based nanomaterial that has been identified as promising for the development of amperometric biosensors. Urease, in combination with TRGO, allowed us to create a mediator-free amperometric biosensor with the intention of precise detection of urea in clinical trials. Beyond simplicity of the technology, the biosensor exhibited high sensitivity (2.3 ± 0.1 µA cm⁻² mM⁻¹), great operational and storage stabilities (up to seven months), and appropriate reproducibility (relative standard deviation (RSD) about 2%). The analytical recovery of the TRGO-based biosensor in urine of 101 ÷ 104% with RSD of 1.2 ÷ 1.7% and in blood of 92.7 ÷ 96.4%, RSD of 1.0 ÷ 2.5%, confirmed that the biosensor is acceptable and reliable. These properties allowed us to apply the biosensor in the monitoring of urea levels in samples of urine, blood, and spent dialysate collected during hemodialysis. Accuracy of the biosensor was validated by good correlation (R = 0.9898 and R = 0.9982) for dialysate and blood, utilizing approved methods. The advantages of the proposed biosensing technology could benefit the development of point-of-care and non-invasive medical instruments.

Investigations of L-glutamate release in living organisms can help to identify novel L-glutamate-related pathophysiological pathways, since abnormal transmission of L-glutamate can cause many neurological diseases. For the first time, a nitrogen-modified graphene oxide (GO) sample (RGO) is prepared through a simple and facile one-pot hydrothermal reduction of GO in the presence of 20 wt.% of the dye malachite green and is used for amperometric biosensing. The biosensor demonstrates adequate stability and is easy to prepare and calibrate. The biosensor detects the current generated during the electrooxidation of hydrogen peroxide released in the L-glutamate that is converted to the alpha-ketoglutarate catalyzed by L-glutamate oxidase. The biosensor consists of a semipermeable membrane, with L-glutamate oxidase (EC 1.4.3.11) immobilized in albumin and RGO and the working Pt electrode. First, the basic version of the L-glutamate biosensor is examined in PBS to investigate its sensitivity, reliability, and stability. To demonstrate the applicability of the L-glutamate biosensor in the analysis of complex real samples, quantification of L-glutamate in bovine brain extract is performed and the accuracy of the biosensor is confirmed by alternative methods. The enhanced version of the L-glutamate biosensor is applied for L-glutamate release investigations in a newly developed strain of rats (DAT-knockout, DAT-KO).

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM⁻¹cm⁻² was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

In this study, PQQ-dependent glucose dehydrogenase (PQQ-GDH) was immobilized onto reduced graphene oxide (rGO) modified with organic dyes from three different classes (acridine, arylmethane, and diazo); namely, neutral red (NR), malachite green (MG), and congo red (CR) formed three types of biosensors. All three rGO/organic dye composites were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. The impact of three rGO/organic dye modifications employed in bioelectrocatalytic systems on changes in enzyme activity and substrate selectivity was investigated. The highest sensitivity of 39 µA/cm² was obtained for 1 mM of glucose when a rGO_MG/PQQ-GDH biosensor was used. A significant improvement in the electrochemical response of biosensors was attributed to the higher amount of pyrrolic nitrogen groups on the surface of the rGO/organic dye composites. Modifications of rGO by NR and MG not only improved the surfaces for efficient direct electron transfer (DET) but also influenced the enzyme selectivity through proper binding and orientation of the enzyme. The accuracy of the biosensor’s action was confirmed by the spectrophotometric analysis. Perspectives for using the proposed bioelectrocatalytic systems operating on DET principles for total or single monosaccharide and/or disaccharide determination/bioconversion systems or for diagnoses have been presented through examples of bioconversion of D-glucose, D-xylose, and maltose.