Reduced Graphene Oxide/Organic Dye Composites for Bioelectroconversion of Saccharides: Application for Detection of Saccharides and α-Amylase Assessments.

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.

Capacitance-Based Biosensor for the Measurement of Total Loss of L-Amino Acids in Human Serum during Hemodialysis.

In this paper, we present a biosensor based on a gold nanoparticle (AuNP)-modified Pt electrode with an adjusted membrane containing cross-linked L-amino acid oxidase for the detection and quantification of total L-amino acids. The designed biosensor was tested and characterized using the capacitance-based principle, capacitance measurements after electrode polarization, disconnection from the circuit, and addition of the respective amount of the analyte. The method was implemented using the capacitive and catalytic properties of the Pt/AuNP electrode; nanostructures were able to store electric charge while at the same time catalyzing the oxidation of the redox reaction intermediate H2O2. In this way, the Pt/AuNP layer was charged after the addition of analytes, allowing for much more accurate measurements for samples with low amino acid concentrations. The combined biosensor electrode with the capacitance-based measurement method resulted in high sensitivity and a low limit of detection (LOD) for hydrogen peroxide (4.15 µC/µM and 0.86 µM, respectively) and high sensitivity, a low LOD, and a wide linear range for L-amino acids (0.73 µC/µM, 5.5 µM and 25-1500 µM, respectively). The designed biosensor was applied to measure the relative loss of amino acids in patients undergoing renal replacement therapy by analyzing amino acid levels in diluted serum samples before and after entering/leaving the hemodialysis apparatus. In general, the designed biosensor in conjunction with the proposed capacitance-based method was clinically tested and could also be applied for the detection of other analytes using analyte-specific oxidases.

Technical suitability and reliability of an in vivo and non-invasive biosensor-type glucose assessment as a potential biomarker for multiple stressors in fishes: an evaluation on Salmonids.

Regardless of the wide use of glucose measurements in stress evaluation, there are some inconsistencies in its acceptance as a stress marker. To meet the challenge and test the reliability/suitability of glucose measurement in practice, we simulated different environmental/anthropogenic exposure scenarios in this study. We aimed to provoke stress in fish followed by a 2-week stress recovery period and under the cumulative effect of leachate fish exposed to pathogenic oomycetes (Saprolegnia parasitica) to represent a possible infection in fish. We selected stream-resident and anadromous brown trout ecotypes (Salmo trutta) representing salmonids with different migratory behaviour strategies. Here, we analysed glucose content in fish-holding water, blood and gills to determine glucose suitability as a potential biomarker of fish response to environmental challenges. Additionally, swimming behavioural parameters and haematocrit were measured. The results indicated that the quantity of glucose released in the holding water of stressed fish increased considerably and remained substantially higher throughout the stress recovery period than the control level. Correspondingly, the circulating levels of glucose in blood and gills decreased over time in fish exposed to different stressors. A significant decrease in swimming activity of fish was observed during the first hours of leachate exposure and increased in fish exposed to S. parasitica compared to control. Our study is the first to ensure the validity and reliability of glucose response in evaluating physiological stress in fish under chemical and biological stimuli, indicating its sensitivity and response range of glucose measurement in fish-holding water.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

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.

Tethered Lipid Membranes as a Nanoscale Arrangement towards Non-Invasive Analysis of Acute Pancreatitis.

Extracellular heat shock proteins (HSPs) mediate immunological functions and are involved in pathologies such as infection, stress, and cancer. Here, we demonstrated the dependence of an amount of HSP70 and HSP90 in serum vs. severity of acute pancreatitis (AP) on a cohort of 49 patients. Tethered bilayer lipid membranes (tBLMs) have been developed to investigate HSPs' interactions with tBLMs that can be probed by electrochemical impedance spectroscopy (EIS). The results revealed that HSP70 and HSP90 interact via different mechanisms. HSP70 shows the damage of the membrane, while HSP90 increases the insulation properties of tBLM. These findings provide evidence that EIS offers a novel approach for the study of the changes in membrane integrity induced by HSPs proteins. Herein, we present an alternative electrochemical technique, without any immunoprobes, that allows for the monitoring of HSPs on nanoscaled tBLM arrangement in biologics samples such us human urine. This study demonstrates the great potential of tBLM to be used as a membrane based biosensor for novel, simple, and non-invasive label-free analytical system for the prediction of AP severity.

Acclimation effect on fish behavioural characteristics: determination of appropriate acclimation period for different species.

In the present study, the authors investigated the effect of acclimation duration (up to 4 h) on behavioural characteristics of taxonomically and functionally different fish species, i.e., the migratory rheophilic salmonids rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), and the non-migratory eurytopic European perch (Perca fluviatilis) and three-spined stickleback (Gasterosteus aculeatus). Specifically, the authors explored fish behavioural patterns based on specific endpoints (average, maximum and angular velocity) during the acclimation period, and determined the acclimation period suitable for the tested fish species. The performed behavioural data analysis showed that the minimum time needed to adjust fish activity to a more stable (baseline) level should be at least 2 h for O. mykiss and S. salar and 1 h for G. aculeatus. Nonetheless, P. fluviatilis behaviour did not show significant changes during the 4 h acclimation. The results of this study revealed that the effect of the acclimation duration on such rheophilic species as O. mykiss and S. salar was greater than that on the eurytopic species P. fluviatilis and G. aculeatus, indicating that acclimation period is important in managing fish stress before behavioural observations. For all species, the highest variability was found in the endpoint of maximum velocity, and the lowest in that of angular velocity. This study showed that before starting actual toxicity testing experiments, it is important to determine an appropriate, species-specific acclimation period.

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.

Thermally reduced graphene oxide: The study and use for reagentless amperometric D-fructose biosensors.

Aiming to create reagentless amperometric D-fructose biosensor, graphene based electrode materials have been synthesized by newly proposed thermal reduction of graphene oxide. The method allowed to separate and collect different fractions of thermally reduced graphene oxide (TRGO) with different physicochemical properties. The structural characteristics and surface morphologies of TRGO fractions were evaluated using SEM, XRD, TGA analysis, Raman spectroscopy and BET measurements. Three different fractions of TRGO were tested as electrode materials for D-fructose amperometric biosensors. The direct electron transfer (DET) from the active site of D-fructose dehydrogenase (FDH) to the electrode was achieved with all TRGO fractions. High values of the sensitivity (up to 14.5 µA mM(-1) cm(-2)) are of the same order as these for other D-fructose sensors based on the synergistic mediated processes. The relationships between the structure of TRGO fractions and the molecular processes determining the effect of DET in bioelectrocatalysis by FDH have been studied. Stability of the D-fructose biosensors was also assessed. The best results were achieved when immobilization of FDH was performed using a crosslinking with glutaraldehyde. For the best group, after a period of 5 days the sensitivity of the biosensor for D-fructose determination decreased by less than 20%.

Computational modeling of mediator oxidation by oxygen in an amperometric glucose biosensor.

In this paper, an amperometric glucose biosensor is modeled numerically. The model is based on non-stationary reaction-diffusion type equations. The model consists of four layers. An enzyme layer lies directly on a working electrode surface. The enzyme layer is attached to an electrode by a polyvinyl alcohol (PVA) coated terylene membrane. This membrane is modeled as a PVA layer and a terylene layer, which have different diffusivities. The fourth layer of the model is the diffusion layer, which is modeled using the Nernst approach. The system of partial differential equations is solved numerically using the finite difference technique. The operation of the biosensor was analyzed computationally with special emphasis on the biosensor response sensitivity to oxygen when the experiment was carried out in aerobic conditions. Particularly, numerical experiments show that the overall biosensor response sensitivity to oxygen is insignificant. The simulation results qualitatively explain and confirm the experimentally observed biosensor behavior.

Modelling carbon nanotubes-based mediatorless biosensor.

This paper presents a mathematical model of carbon nanotubes-based mediatorless biosensor. The developed model is based on nonlinear non-stationary reaction-diffusion equations. The model involves four layers (compartments): a layer of enzyme solution entrapped on a terylene membrane, a layer of the single walled carbon nanotubes deposited on a perforated membrane, and an outer diffusion layer. The biosensor response and sensitivity are investigated by changing the model parameters with a special emphasis on the mediatorless transfer of the electrons in the layer of the enzyme-loaded carbon nanotubes. The numerical simulation at transient and steady state conditions was carried out using the finite difference technique. The mathematical model and the numerical solution were validated by experimental data. The obtained agreement between the simulation results and the experimental data was admissible at different concentrations of the substrate.

Fine structure and related properties of the assembleable carbon nanotubes based electrode for new family of biosensors with chooseable selectivity.

Surfaces of constituent parts of biosensors based on single wall carbon nanotube layer were investigated and compare for properly functioning and faulty biosensors. Though the original technology is acceptable for changing of the selectivity, only glucose sensitive biosensors are investigated. Based on the results of the study, a correlation between the features of the nanoscale structures and parameters of amperiometric biosensors for assemblage of which an innovative approach is described. Original template of the electrodes has been prepared on a base of single wall carbon nanotube layer deposited on the supporting polycarbonate membrane. Original immobilisation of enzymes within special membrane allows functional modification of biosensors being accomplished by simple replacement of the enzymatic membrane. The original technology leads to a novel family of biosensors acceptable for detection of wide range of carbohydrates. The morphology and the local electric properties of the constituent parts of the biosensors are characterized by scanning probe microscopy. The sensitivity, selectivity and stability are described for typical types of the biosensors.

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.

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.

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.