Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity.

This study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold-nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active equivalent dehydrogenase via a single residue exchange.

My sweetheart has a basketball match: The relationship between love-hate and identification of individuals attending a euroleague match for recreational purposes.

The objective of this research was to examine the Love-Hate and Identification Relationship of Individuals Participating in Euroleague Match for Recreational Purposes. The study was conducted using a relational survey methodology. The study's population comprises persons who watching recreational purpose part in the Euroleague match held in Istanbul in 2023-2024 season, while the sample consists of 178 voluntary participants selected through convenience sampling. The participants completed the Fan Love-Hate Scale and Fan Identification Scale, in addition to being asked about their gender, marital status, age, educational status, and frequency of attending football matches per week. The data collected from the personal information form and scales was entered into the IBM SPSS 24.0 software package for analysis. Statistical analyses were conducted using the Independent Sample T test and One-way Anova methods. The LSD test was employed to ascertain the dissimilarity between the groups. The Pearson correlation analysis was utilized to ascertain the association between the variables of love-hate and identity. In summary, it is evident that demographic factors, including gender and age, significantly influence fan perceptions and sports identification. In contrast, there is no substantial correlation observed between attributes such as level of education achieved and the frequency of engaging in sports activities, and the aforementioned outcomes. The significant associations identified between the Fan Love-Hate Scale and the Sports Fan Identification Scale underscore the complex relationship between fans' emotional experiences and their connection to sports. Further investigations could be conducted to go deeper into the underlying causes that contribute to these relationships and inequalities, so resulting in a more thorough understanding of fan psychology.

Nature relatedness, flow experience, and environmental behaviors in nature-based leisure activities.

Introduction: Through nature-based leisure activities, spending time in nature offers opportunities to reduce stress, relax the mind, and enhance feelings of well-being. Being aware of the benefits provided by these activities increases the nature relatedness, and during the time spent in nature, it enables experiencing positive and satisfying moments by entering into a state of flow. The concepts of nature-relatedness and flow experience represent psychological experiences and characteristics that play an important role in enhancing psychological well-being and life quality. Methods: Based on structural equation models, the relationships among nature-relatedness, flow experience, and environmental behaviors were investigated. Data were collected from 379 individuals (212 male, 167 female) who regularly engage in nature-based leisure activities such as cycling, hiking, and fishing. The participants were predominantly male (55.9%) and aged 45 years and over (53.3%). Results: The nature-relatedness significantly influences flow experience (R 2 = 0.505, p < 0.01), environmental behavior (R 2 = 0.108, p < 0.01), environmental sensitivity (R 2 = 0.137, p < 0.01), and communication with nature (R 2 = 0.200, p < 0.01). Specifically, nature-relatedness directly enhanced environmental sensitivity (0.494 total effect), environmental behavior (0.604 total effect), and communication with nature (0.599 total effect) and did so both directly and indirectly through the mediation of flow experience. Discussion: A higher level of nature-relatedness can lead to a stronger flow experience, which in turn can increase positive. environmental behavior, environmental sensitivity, and communication with nature.

Mass transport controlled oxygen reduction at anthraquinone modified 3D-CNT electrodes with immobilized Trametes hirsuta laccase.

Carbon nanotubes covalently modified with anthraquinone were used as an electrode for the immobilization of Trametes hirsuta laccase. The adsorbed laccase is capable of oxygen reduction at a mass transport controlled rate (up to 3.5 mA cm(-2)) in the absence of a soluble mediator. The storage and operational stability of the electrode are excellent.

Activation/inhibition effects during the coelectrodeposition of PtAg nanoparticles: application for ORR in alkaline media.

PtAg bimetallic nanoparticles for oxygen reduction reaction (ORR) in alkaline media were prepared by pulse electrodeposition (PED). During PED the reduction of Ag(+) ions predominates, thus an increased Ag content in the co-deposit is accomplished. The mechanism for this anomalous co-deposition was elucidated by potential pulse experiments, which revealed that nuclei formation mainly occurs via the reduction of Pt(2+) ions. The growth of the particles is diffusion controlled leading to the formation of a Ag shell covering a PtAg alloyed region. However, the shell is not growing homogeneously on the PtAg alloy. Hence, regions of the PtAg alloy are exposed, which exhibit an enhanced ORR activity compared to a pure Ag surface.

Towards a high potential biocathode based on direct bioelectrochemistry between horseradish peroxidase and hierarchically structured carbon nanotubes.

Adsorption of horseradish peroxidase (HRP) on graphite rod electrodes sequentially modified with carbon microfibers (CMF) carrying carbon nanotubes in a hierarchically structured arrangement and finally pyrene hexanoic acid (PHA) for improving hydrophilicity of the electrode surface is the basis for the direct bioelectrocatalytic reduction of H(2)O(2) at potentials as high as about +600 mV. The high-potential direct bioelectrocatalytic reduction of H(2)O(2) is implying a direct bioelectrochemical communication between the Fe(IV)=O,P(+*) redox state known as compound I. The HRP loading was optimized leading to a current of 800 microA at a potential of 300 mV.

Local modulation of the redox state of p-nitrothiophenol self-assembled monolayers using the direct mode of scanning electrochemical microscopy.

Local reduction of the terminating nitro groups of a p-nitrothiophenol self-assembled monolayer (SAM) under formation of either hydroxylamine or amino groups is invoked using the direct mode of scanning electrochemical microscopy (SECM). By choosing the appropriate potential and a potential pulse sequence, the reduction of the SAM end groups to the desired oxidation state can be achieved, locally restricted to the area of the sample surface directly underneath the positioned SECM tip. Following the "writing" of redox microstructures within the SAM end groups, the local modification of the redox states is visualized ("reading") by using the feedback mode of SECM. The current at the Pt tip electrode is determined by the electron-transfer rate for reoxidation of the redox mediator at the sample surface. Thus, heterogeneities in the SAM surface, which are caused by local differences in the redox state of the end groups, are distinguishable due to the different electron-transfer rates governed by the redox state of the SAM end groups. To further unequivocally prove the successful local modification of the redox state of the SAM end groups during the writing process, the micropatterned surface is selectively modified with biotin at areas with reduced SAM end groups for further complementary binding of an avidin-enzyme conjugate. Selective post-functionalization with an avidin-alkaline phosphatase conjugate allows visualization of the microstructure using the generator-collector mode of SECM.

Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes.

Following previous electrochemical investigations of cellobiose dehydrogenase (CDH), the present investigation reports on the initial screening of the electrochemistry of three new CDHs, two from the white rot basidiomycetes Trametes villosa and Phanerochaete sordida and one from the soft rot ascomycete Myriococcum thermophilum, for their ability to directly exchange electrons with 10 different alkanethiol-modified Au electrodes. Direct electron transfer (DET) between the enzymes and some of the modified Au electrodes was shown, both, in the presence and in the absence of cellobiose. However, the length and the head functionality of the alkanethiols drastically influenced the efficiency of the DET reaction and also influenced the effect of pH on the biocatalytic/redox currents, suggesting the importance of structural/sequence differences between these CDH enzymes. In this respect, the white rot CDHs exhibit excellent biocatalytic and redox currents, whereas for the soft rot CDH the DET communication is much less efficient. Cyclic voltammograms indicate that the heme domain of the CDHs is the part of the enzymes that most readily exchanges electrons with the electrode. However, for P. sordida CDH on 11-mercaptoundecanol or dithiopropionic acid-modified Au electrodes, a second voltammetric wave was noticed suggesting that for some orientations of the enzyme, DET communication with the FAD cofactor can also be obtained.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission.

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium.

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.

Bioelectrochemical detection of L-lactate respiration using genetically modified Hansenula polymorpha yeast cells overexpressing flavocytochrome b2.

In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.

Scanning electrochemical microscopy (SECM) as a tool in biosensor research.

Scanning electrochemical microscopy (SECM) is discussed as a versatile tool to provide localized (electro)chemical information in the context of biosensor research. Advantages of localized electrochemical measurements will be discussed and a brief introduction to SECM and its operation modes will be given. Experimental challenges of the different detection modes of SECM and its applicability for different fields in biosensor research are discussed. Among these are the evaluation of immobilization techniques by probing the local distribution of biological activity, the visualization of diffusion profiles of reactants, cofactors, mediators, and products, and the elucidation of (local) kinetic parameters. The combination of SECM with other scanning-probe techniques allows to maximize the information on a given biosensing system. The potential of SECM as a tool in micro-fabrication aiming for the fabrication of microstructured biosensors will be shortly discussed.

Direct electron transfer--a favorite electron route for cellobiose dehydrogenase (CDH) from Trametes villosa. Comparison with CDH from Phanerochaete chrysosporium.

This paper presents some functional differences as well as similarities observed when comparing the newly discovered cellobiose dehydrogenase (CDH) from Trametes villosa (T.v.) with the well-characterized one from Phanerochaete chrysosporium (P.c.). The enzymes were physically adsorbed on spectrographic graphite electrodes placed in an amperometric flow through cell connected to a flow system. In the case of T.v.-CDH-modified graphite electrodes, a high direct electron transfer (DET) current was registered at the polarized electrode in the presence of the enzyme substrate reflecting a very efficient internal electron transfer (IET) process between the reduced FAD-cofactor and the oxidized heme-cofactor. In the case of P.c.-CDH-modified graphite electrodes, the DET process is not as efficient, and the current will greatly increase in the presence of a mediator (mediated electron transfer, MET). As a consequence, when comparing the two types of enzyme-modified electrodes an inverted DET/MET ratio for T.v.-CDH is shown, in comparison with P.c.-CDH. The rates of the catalytic reaction were estimated to be comparable for both enzymes, by measuring the combined DET + MET currents. The inverted DET/MET ratio for T.v.-CDH-modified electrodes might suggest that probably there is a better docking between the two domains of this enzyme and that the linker region of P.c.-CDH might have an active role in modulating the rate of the IET (by changing the interdomain distance), with respect to pH. Based on the new properties of T.v.-CDH emphasized in the present study, an analytical application of a third-generation biosensor for lactose was recently published.

Third-generation biosensor for lactose based on newly discovered cellobiose dehydrogenase.

The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm2) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL x min(-1)). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 microM, a sensitivity of 1100 microA x mM(-1) x cm(-2), a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 microM lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 microM lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%).

Biosensor based on cellobiose dehydrogenase for detection of catecholamines.

A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs Ag|AgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A.M(-1) x cm(-2) (approximately 1150 nA/microM) for noradrenaline, with a coverage of less than 2.5 microg of CDH adsorbed on the electrode surface (0.073 cm(2)). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones.