The Synergy of Thermally Reduced Graphene Oxide in Amperometric Urea Biosensor: Application for Medical Technologies.

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.

Application of oxygen-independent biosensor for testing yeast fermentation capacity.

The pyrroloquinoline quinone (PQQ)-dependent soluble glucose dehydrogenase based carbon paste electrodes were investigated and applied for glucose monitoring in the oxygen deficient media. Reagentless biosensors possessing a wide linear range (up to 5 mM glucose with a detection limit of 0.12 mM) were designed. The oxygen-insensitive response of the biosensor creates the opportunity to use it as a flow-through device for continuous monitoring of glucose in media during the wine yeast fermentation process. The analysis of glucose assimilation rate by yeast strains using the developed biosensor correlated well (R2=0.9938) with convenient yeast testing methods.

The influence of external factors on the operational stability of the biosensor response.

The behaviour of the electrochemical glucose biosensor based on the glucose oxidase was examined in the diffusion and the kinetic modes of the action. The sensitivity and linearity of the biosensor can be monitored changing the permeability of the outer membrane of the biosensor. The mathematical model based on the enzymatic conversion of the substrate and the diffusion of the substrate was created. The influence of the fluctuations of the membrane thickness, the diffusion coefficients and pH were modelled and their impact was evaluated at different modes of an action of the biosensor. Taking into account that limited acceptable fluctuations of the biosensor response should not exceed 5%, we calculated how K(M(app.)) and V(max) can move to satisfy this requirement. In a deep diffusive mode (thick highly acetylated membrane), the fluctuations of K(M(app.)) up to 400% do not influence significantly the biosensor response. In the diffusion mode of action of the biosensor, the limit of the V(max) fluctuations is on the level of 34%. The increase of the thickness of the membrane 5 times, increases the limit of fluctuations only to 19%. The reduction of the permeability of the membrane 4 times increases the level of limited fluctuations about 10-16%. The novelty of this work is binding into one system the fluctuations of pH and diffusion parameters and demonstrating the interdependence of them as an integrated factor of the reliability of the biosensor response.

Amperometric immunosensor for diagnosis of BLV infection.

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody-antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N',N'-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.

Modified graphitized carbon black as transducing material for reagentless H2O2 and enzyme sensors.

Direct electron transfer between redox enzymes and electrodes is the basis for the third generation biosensors. We established direct electron transfer between quinohemoprotein alcohol dehydrogenase (PQQ-ADH) and modified carbon black (CBs) electrodes. Furthermore, for the first time, this phenomenon was observed for pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH). Reagentless enzyme biosensors suitable for the determination of ethanol, glucose and sensors for hydrogen peroxide were designed using CB electrodes and screen-printing technique. Aiming to create an optimal transducing material for biosensors, a set of CB batches was synthesized using the matrix of Plackett-Burman experimental design. Depending on the obtained surface functional groups as well as the nano-scale carbon structures in CBs batches, the maximal direct electron transfer current of glucose and ethanol biosensors can vary from 20 to 300 nA and from 30 to 6300 nA for glucose and ethanol, respectively. Using modified CB electrodes, an electrocatalytic oxidation of H(2)O(2) takes place at more negative potentials (0.1-0.4V versus Ag/AgCl). Moreover, H(2)O(2) oxidation efficiency depends on the amount and morphology of fine fraction in the modified CBs.

Wiring of PQQ-dehydrogenases.

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.

Reagentless biosensors based on co-entrapment of a soluble redox polymer and an enzyme within an electrochemically deposited polymer film.

A novel biosensor architecture, which is based on the combination of a manual and a non-manual deposition technique for sensor components on the electrode surface is reported. A water-soluble Os-poly(vinyl-imidazole) redox hydrogel is deposited on a graphite electrode by drop-coating (i.e. manually) followed by the electrochemically-induced deposition of an enzyme-containing non-conducting polymer film. The local polymer deposition is initiated by electrochemical generation of H(3)O(+) exclusively at the electrode surface causing a pH-shift to be established in the diffusion zone around the electrode (i.e. non-manually). This pH-shift leads to the protonation of a dissolved polyanionic polymer which in consequence changes significantly its solubility and is hence precipitating on the electrode surface. In the presence of a suitable enzyme, such as quinohemoprotein alcohol dehydrogenase (QH-ADH), the polymer precipitation leads to an entrapment of the redox enzyme within the polymer film. Simultaneously, the water-soluble Os-poly(vinyl-imidazole) redox hydrogel, which is slowly dissolving from the electrode surface after addition of the electrolyte, is co-entrapped within the precipitating polymer layer. This provides the pre-requisite for an efficient electron-transfer pathway from the redox enzyme via the polymer-bound redox centres to the electrode surface. The sensor preparation protocol has been optimised aiming on a high mediator concentration in the polymer film and an effective electron transfer.

Bioelectrochemical application of some PQQ-dependent enzymes.

This paper focuses on the use of PQQ-dependent enzymes (PQQ enzymes) in amperometrical biosensors and gives emphasis on their innovative designs and applications. The study covers some aspects in the evolution of biosensors based on PQQ enzymes. Main attention is focused on the electrochemical properties of PQQ enzymes as very promising materials for the formation of electrochemical biosensors. Immobilization approaches and redox mediators recently used in PQQ enzymes based biosensors are reviewed. The acceptance of polypyrrole as a very promising immobilization matrix for some PQQ enzymes is discussed.