Ultrasensitive Detection of Hydrogen Peroxide Using Methylene Blue Grafted on Molecular Wires as Nanozyme with Catalase-like Activity.

In this study, we present the development of an electrochemical sensor designed for ultrasensitive detection of endogenous H2O2. This sensor relies on signal amplification achieved through nanozyme activity exhibited by methylene blue (MB) grafted onto a peptide support. The sensor exhibited excellent selectivity and sensitivity, with a limit of detection of 18 nM and a linear detection range of 20-200 nM. Thus, we have validated the concept of the MB-peptide system, serving as both an electroactive label and a catalyst for H2O2 decomposition under electrochemical conditions. The implemented signal amplification system enables the rapid detection of H2O2, with an overall assay time of 1-2 min, a significant improvement compared to amperometric detection using surface-immobilized enzymes.

Square-wave voltammetry of human blood serum.

A study on voltammetric analysis of blood serum diluted in a phosphate buffer is presented using advanced square-wave voltammetry at an edge plane pyrolytic graphite electrode. The results demonstrate that even in a complex medium like human blood serum, electrochemical characterization can be achieved through the use of advanced voltammetric techniques in conjunction with an appropriate commercially available electrode, such as the edge plane pyrolytic graphite electrode, which boosts superior electrocatalytic properties. Without undergoing any chemical treatment of the serum sample, the square-wave voltammetry technique reveals, for the first time, the electrode reactions of uric acid, bilirubin, and albumin in a single experiment, as represented by well-defined, separated, and intense voltammetric signals. All electrode processes are surface-confined, indicating that the edge plane sites of the electrode serve as an ideal platform for the competitive adsorption of electroactive species, despite the extensive chemical complexity of the serum samples. The speed and differential nature of square-wave voltammetry are crucial for obtaining an outstanding resolution of the voltammetric peaks, maintaining the quasi-reversible nature of the underlying electrode processes, while reducing the impact of follow-up chemical reactions that are coupled to the initial electron transfer for all three detected species, and minimizing fouling of the electrode surface.

Improved procedure for square-wave voltammetric sensing of fenhexamid residues on blueberries peel surface at the anodically pretreated boron-doped diamond electrode.

BACKGROUND: Fungicide fenhexamid (FH) has a high residual concentration on fruits and vegetables, thus, it is of high importance to monitor the level of FH residues on foodstuff samples. So far, the assay of FH residues in selected foodstuff samples has been conducted by electroanalytical methods on sp2 carbon-based electrodes that are well-known to be susceptible to severe fouling of the electrodes surfaces during electrochemical measurements. As an alternative, sp3 carbon-based electrode such as boron-doped diamond (BDD) can be used in the analysis of FH residues retained on the peel surface of foodstuff (blueberries) sample. RESULTS: In situ anodic pretreatment of the BDDE surface was found to be the most successful strategy to remediate the passivated BDDE surface by FH oxidation (by)products, and the best validation parameters, i.e., the widest linear range (3.0-100.0 µmol L-1), the highest sensitivity (0.0265 µA L µmol-1) and the lowest limit of detection (0.821 µmol L-1), were achieved on the anodically pretreated BDDE (APT-BDDE) in a Britton-Robinson buffer, pH 2.0, using square-wave voltammetry (SWV). The assay of FH residues retained on blueberries peel surface was performed on the APT-BDDE using SWV, and the obtained concentration of FH residues of 6.152 µmol L-1 (1.859 mg kg-1) was found to be below the maximum residue value fixed for blueberries by the European Union regulations (20 mg kg-1). SIGNIFICANCE AND NOVELTY: In this work, a protocol based on a very easy and fast foodstuff sample preparation procedure combined with the straightforward pretreatment approach of the BDDE surface was elaborated for the first time for the monitoring of the level of FH residues retained on the peel surface of blueberries samples. The presented reliable, cost-effective, and easy-to-use protocol could find its application as a rapid screening method for the control of food safety.

Electrode kinetics from a single experiment: multi-amplitude analysis in square-wave chronoamperometry.

The recently introduced technique of square-wave chronoamperometry (SWCA) is studied under conditions of progressively increasing height of potential pulses (square-wave amplitude) within a single experiment. In multi-amplitude square-wave chronoamperometry (MA-SWCA) a potential modulation consisting of square-wave forward and reverse potential pulses is imposed on a constant mid-potential; the amplitude of pulses increases progressively during the experiment. This allows the fast and reliable estimation of kinetic parameters at a constant pulse frequency in a single experiment, based on the resulting feature known as the amplitude-based quasireversible maximum. The proposed methodology is tested by simulating the responses of a simple quasireversible electrode reaction of a dissolved redox couple and a surface confined electrode reaction. Compared with conventional square-wave voltammetry (SWV) and SWCA, MA-SWCA shows advantages in estimation of the standard rate constant in terms of simplicity, speed and efficiency for both studied electrode mechanisms.

Bioactive Phenolic Compounds from Lingonberry (Vaccinium vitis-idaea L.): Extraction, Chemical Characterization, Fractionation and Cellular Antioxidant Activity.

Lingonberries contain high contents of bioactive compounds such as chlorogenic acids and anthocyanins. In addition to radical scavenging and antioxidant activities, these compounds can protect cells from DNA damage. For this reason, lingonberries might be well suited for nutraceuticals or natural biomedicines. To assess these applications, the present study characterized and identified the most effective extract, only consisting of anthocyanins, copigments or a mixture of both, obtained from a lingonberry juice concentrate. An extract was generated by using a XAD-7 column followed by fractionation into anthocyanins and copigments using adsorptive membrane chromatography. After identification of main polyphenols by HPLC-photodiode array-electrospray ionization-tandem mass spectrometry, free radical scavenging activity was analyzed by electron spin resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl radicals. Furthermore, cyclic voltammetry analyses and the Trolox equivalent antioxidant capacity (TEAC) assay were applied. Finally, the reactive oxygen species (ROS) reducing effects of the lingonberry extract and its fractions were evaluated in HepG2 cells. While the combination of anthocyanins and copigments possessed the highest antioxidant activities, all samples (XAD-7 extract, anthocyanin and copigment fraction) protected cells from oxidative stress. Thus, synergistic effects between phenolic compounds may be responsible for the high antioxidant potential of lingonberries, enabling their use as nutraceuticals.

Electrochemical Determination of Antioxidant Capacity of Traditional Homemade Fruit Vinegars Produced with Double Spontaneous Fermentation.

In the current study, the antioxidant activity of traditional homemade fruit vinegars (HMV) was estimated by measuring the rate of homogeneous redox reaction with 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS•+) using cyclic voltammetry. The antioxidant capacity of six HMV produced using traditional methods and the physicochemical characterization were measured in different vinegar production steps throughout a double spontaneous fermentation process, i.e., without any addition of yeasts or acetic acid bacteria. Their antioxidant capacity was compared with seven fruit commercial vinegars (ComV). Furthermore, the antioxidant capacity was independently measured with the TEAC (Trolox equivalent antioxidant capacity) assay, aiming at correlating with the electrochemical experimental data. Obtained results from both methods, the electrochemical and TEAC assays, interestingly indicated that all HMV have at least 10 times higher antioxidant activity than ComV. Furthermore, the large range of values for antioxidant capacity in samples of commercial vinegars from apples attested the importance of the raw material quality and technological procedures. The positive correlation between total phenolic content and antioxidant capacity measured by the two type of assays indicated that rose hip homemade vinegar (HMV5) has the highest antioxidant capacity. In contrast, the lowest levels of phenolic compounds and antioxidant capacity were found in apple and persimmon homemade vinegars (HMV1 and HMV6, respectively) which indicated that the type of fruit is crucial towards the production of high-quality vinegars. In this way, the use of traditional processes for the production of fruit vinegars proved to be very promising in terms of producing differentiated vinegars and, concomitantly, reaching high levels of health-promoting antioxidant capacities.

Electroreduction of Bi(III) Ions at a Cyclically Renewable Liquid Silver Amalgam Film Electrode in the Presence of Methionine.

The catalytic influence of methionine (Mt) on the electroreduction of Bi(III) ions on the novel, cyclically renewable liquid silver amalgam film electrode (R-AgLAFE) in a non-complexing electrolyte solution was examined. The presence of methionine leads to a multistep reaction mechanism, where the transfer of the first electron is the rate limiting step, which is the subject of catalytic augmentation. The catalytic activity of methionine is a consequence of its ability to remove water molecules from the bismuth ion coordination sphere, as well as to form active complexes on the electrode surface, facilitating the electron transfer process.

Differential Square-Wave Voltammetry.

A new voltammetric technique designed as a hybrid between differential pulse and square-wave voltammetry is proposed for the purpose of unifying the advantages of both techniques, i.e., the ability to provide mechanistic information, studying electrode kinetics of both sluggish and very fast electrode reactions, and the ability to suppress effectively residual background current. Voltammetric modulation of the hybrid technique consists of a staircase potential combined with square-wave potential modulation superimposed at the end of each potential step. By measuring the current at the end of each potential step and pulse, differential forward and backward voltammetric components can be composed, which is a unique ability of the hybrid technique. In addition, by analogy to square-wave voltammetry, a net differential component can be contracted with improved analytical performances compared to square-wave voltammetry. The proposed technique opens a new avenue for an advanced analysis of electrochemical processes and analytical application.

Electrochemical Quantification of Extracellular Local H2O2 Kinetics Originating from Single Cells.

AIMS: H2O2 is produced by all eukaryotic cells under physiological and pathological conditions. Due to its enormous relevance for cell signaling at low concentrations and antipathogenic function at high concentrations, precise quantification of extracellular local hydrogen peroxide concentrations ([H2O2]) originating from single cells is required. RESULTS: Using a scanning electrochemical microscope and bare platinum disk ultramicroelectrodes, we established sensitive long-term measurements of extracellular [H2O2] kinetics originating from single primary human monocytes (MCs) ex vivo. For the electrochemical techniques square wave voltammetry, cyclic and linear scan voltammetry, and chronoamperometry, detection limits for [H2O2] were determined to be 5, 50, and 500 nM, respectively. Following phorbol ester stimulation, local [H2O2] 5-8 µm above a single MC increased by 3.4 nM/s within the first 10 min before reaching a plateau. After extracellular addition of H2O2 to an unstimulated MC, the local [H2O2] decreased on average by 4.2 nM/s due to degradation processes of the cell. Using the scanning mode of the setup, we found that H2O2 is evenly distributed around the producing cell and can still be detected up to 30 µm away from the cell. The electrochemical single-cell measurements were validated in MC populations using electron spin resonance spectroscopy and the Amplex® UltraRed assay. Innovation and Conclusion: We demonstrate a highly sensitive, spatially, and temporally resolved electrochemical approach to monitor dynamics of production and degradation processes for H2O2 separately. Local extracellular [H2O2] kinetics originating from single cells is quantified in real time. Antioxid. Redox Signal. 29, 501-517.

New insights into the chemistry of Coenzyme Q-0: A voltammetric and spectroscopic study.

Coenzyme Q-0 (CoQ-0) is the only Coenzyme Q lacking an isoprenoid group on the quinoid ring, a feature important for its physico-chemical properties. Here, the redox behavior of CoQ-0 in buffered and non-buffered aqueous media was examined. In buffered aqueous media CoQ-0 redox chemistry can be described by a 2-electron-2-proton redox scheme, characteristic for all benzoquinones. In non-buffered media the number of electrons involved in the electrode reaction of CoQ-0 is still 2; however, the number of protons involved varies between 0 and 2. This results in two additional voltammetric signals, attributed to 2-electrons-1H(+) and 2-electrons-0H(+) redox processes, in which mono- and di-anionic compounds of CoQ-0 are formed. In addition, CoQ-0 exhibits a complex chemistry in strong alkaline environment. The reaction of CoQ-0 and OH(-) anions generates several hydroxyl derivatives as products. Their structures were identified with HPLC/MS. The prevailing radical reaction mechanism was analyzed by electron paramagnetic resonance spectroscopy. The hydroxyl derivatives of CoQ-0 have a strong antioxidative potential and form stable complexes with Ca(2+) ions. In summary, our results allow mechanistic insights into the redox properties of CoQ-0 and its hydroxylated derivatives and provide hints on possible applications.

New approach to electrode kinetic measurements in square-wave voltammetry: amplitude-based quasireversible maximum.

The influence of the potential pulse height of square-wave voltammetry (SWV) (i.e., the SW amplitude) is studied for a variety of quasireversible electrode mechanisms, including a simple solution-phase electrode reaction at a planar or spherical electrode, a solution phase electrode reaction coupled with a reversible follow-up chemical reaction, and a diffusionless surface confined electrode reaction. The electrode kinetics of all the electrode mechanisms depends critically on the SW amplitude, and the quasireversible kinetic region is a function of both frequency-related electrode kinetic parameters and the SW amplitude. Thus, a novel methodology for electrode kinetics measurements is proposed by altering the SW amplitude only, at a fixed frequency of the SW potential modulation, that is, at a constant scan rate of the voltammetric experiment. Electrode kinetic measurements at a constant SW frequency are of exceptional importance especially when complex electrode mechanisms are studied, which depend on several frequency-related kinetic parameters. The electrode kinetic measurements are based on a novel feature termed the "amplitude-based quasireversible maximum", manifested as a parabolic dependence of the amplitude-normalized net SW peak current versus the SW amplitude. The position of the amplitude-based quasireversible maximum depends on the standard rate constant of the electrode reaction, enabling estimation of this important kinetic parameter in a simple and fast procedure. The novel quasireversible maximum is attributed to all studied electrode mechanisms, implying that it is a general feature of most electrode mechanisms under conditions of SWV.

Hydroxylated derivatives of dimethoxy-1,4-benzoquinone as redox switchable earth-alkaline metal ligands and radical scavengers.

Benzoquinones (BQ) have important functions in many biological processes. In alkaline environments, BQs can be hydroxylated at quinoid ring proton positions. Very little is known about the chemical reaction leading to these structural transformations as well as about the properties of the obtained hydroxyl benzoquinones. We analyzed the behavior of the naturally occurring 2,6-dimethoxy-1,4-benzoquinone under alkaline conditions and show that upon substitution of methoxy-groups, poly-hydroxyl-derivatives (OHBQ) are formed. The emerging compounds with one or several hydroxyl-substituents on single or fused quinone-rings exist in oxidized or reduced states and are very stable under physiological conditions. In comparison with the parent BQs, OHBQs are stronger radical scavengers and redox switchable earth-alkaline metal ligands. Considering that hydroxylated quinones appear as biosynthetic intermediates or as products of enzymatic reactions, and that BQs present in food or administered as drugs can be hydroxylated by enzymatic pathways, highlights their potential importance in biological systems.

Development of a rapid and simple voltammetric method to determine total antioxidative capacity of edible oils.

In this work we report on a new, rapid and simple voltammetric method to determine the total antioxidant capacity (TAC) of the edible oils. The method explores the ABTS radical (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)) assay as a redox probe and it relays on measuring catalytic voltammetric currents. The electrocatalysis comprises redox regeneration of the electrochemically created ABTS(+) radical either by Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid) or by antioxidants present in studied oils. The detection limit of the method is determined to be 0.5 mg/L of Trolox equivalent, being a slightly lower than the corresponding UV-VIS spectrophotometric method. Applying the proposed voltammetric method the total antioxidant capacity of three types of commercially available cold-pressed edible oils are determined, and the results are found to be in a very good agreement with those obtained by UV-VIS spectrophotometry. The reported voltammetric method is cheap, rapid and simple, and it can be used as a sustainable alternative to the UV-VIS methods for the determination of total antioxidant capacitance of oils and other liquid lipophilic nutrients. Potent antioxidant capacity of studied oils was also confirmed by electron paramagnetic resonance spectroscopy of superoxide anion produced by macrophages.

Diagnostics of anodic stripping mechanisms under square-wave voltammetry conditions using bismuth film substrates.

A mechanistic study to provide diagnostics of anodic stripping electrode processes at bismuth-film electrodes is presented from both theoretical and experimental points of view. Theoretical models for three types of electrode mechanisms are developed under conditions of square-wave voltammetry, combining rigorous modeling based on integral equations and the step function method, resulting in derivation of a single numerical recurrent formula to predict the outcome of the voltammetric experiment. In the course of the deposition step, it has been assumed that a uniform film of the metal analyte is formed on the bismuth substrate, in situ deposited onto a glassy carbon electrode surface, without considering mass transfer within either the bismuth or the metal analyte film. Theoretical data are analyzed in terms of dimensionless critical parameters related with electrode kinetics, mass transfer, adsorption equilibria, and possible lateral interactions within the deposited metal particles. Theoretical analysis enables definition of simple criteria for differentiation and characterization of electrode processes. Comparing theoretical and experimental data, anodic stripping processes of zinc(II), cadmium(II), and lead(II) are successfully characterized, revealing significant differences in their reaction pathways. The proposed easy-to-perform diagnostic route is considered to be of a general use while the bismuth film exploited in this study served as a convenient nonmercury model substrate surface.

Calcium binding and transport by coenzyme Q.

Coenzyme Q10 (CoQ10) is one of the essential components of the mitochondrial electron-transport chain (ETC) with the primary function to transfer electrons along and protons across the inner mitochondrial membrane (IMM). The concomitant proton gradient across the IMM is essential for the process of oxidative phosphorylation and consequently ATP production. Cytochrome P450 (CYP450) monoxygenase enzymes are known to induce structural changes in a variety of compounds and are expressed in the IMM. However, it is unknown if CYP450 interacts with CoQ10 and how such an interaction would affect mitochondrial function. Using voltammetry, UV-vis spectrometry, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), fluorescence microscopy and high performance liquid chromatography-mass spectrometry (HPLC-MS), we show that both CoQ10 and its analogue CoQ1, when exposed to CYP450 or alkaline media, undergo structural changes through a complex reaction pathway and form quinone structures with distinct properties. Hereby, one or both methoxy groups at positions 2 and 3 on the quinone ring are replaced by hydroxyl groups in a time-dependent manner. In comparison with the native forms, the electrochemically reduced forms of the new hydroxylated CoQs have higher antioxidative potential and are also now able to bind and transport Ca(2+) across artificial biomimetic membranes. Our results open new perspectives on the physiological importance of CoQ10 and its analogues, not only as electron and proton transporters, but also as potential regulators of mitochondrial Ca(2+) and redox homeostasis.

Electrochemistry of coupled electron-ion transfer of a heme-like complex in an artificial organic membrane.

The electrochemistry of a heme-like complex 5,10,15,20-tetraphenylporphyriniron(III) chloride (Fe(III)-TPP-Cl) embedded in a lipophilic artificial membrane is studied by means of voltammetric techniques. The experimental system used comprises of a thin organic membrane hosting the redox probe, sandwiched between edge plane pyrolytic graphite electrode and an aqueous electrolyte solution. In the course of the voltammetric experiment the redox transformation of Fe(III)-TPP-Cl at the electrode|membrane interface is accompanied by concomitant ion transfer reactions across the membrane|water interface. The overall electrochemical process proceeds as a coupled electron-ion transfer reaction providing insight into energetic and kinetic aspects of the complex membrane charge-transfer reactions. The membrane electrochemistry of Fe(III)-TPP-Cl involves two quasireversible redox transformations typical for the Fe(III)/Fe(II) couple. Both redox processes are coupled with a chemical reactions proceeding inside the membrane as well as by complex ion transfer reactions across the membrane|water interface comprising chloride expulsion from the membrane and cation ingress from the aqueous phase.

Voltammetry based on fractional diffusion.

A cyclic voltammetric experiment governed by anomalous diffusion of an electroactive species is theoretically analyzed by means of fractional calculus. The diffusion mass transfer under semi-infinite conditions at a planar electrode is ascribed by a Fick's second law-like differential equation in which the time derivative of the concentration function is of a fractional order alpha, ranging from 0 to 1. Rigorous solutions relating the concentrations of electroactive species with the electric current are derived by means of the Wright function for the case of a simple reversible electrode reaction of two chemically stable redox-active species having identical diffusion coefficients. A general mathematical solution for a voltammetric experiment, relating the surface concentrations with the current and electrode potential, is presented in the form of an integral equation. On the basis of the latter solution, the cyclic voltammetric experiment is simulated under variety of conditions, in order to inspect the influence of the fractional parameter alpha and to reveal its physical significance. Aiming to explain peculiar features of cyclic voltammograms, concentration profiles of electroactive species, together with the Cottrell-like equation, are analyzed for various alpha values. It has been established that the shape of a cyclic voltammogram depends strongly on alpha, varying from a steady-state sigmoid shape when alpha --> 0 to a conventional peak-like shape for alpha --> 1. Whereas the midpeak potential is independent of alpha, the peak currents are proportional to upsilon(alpha/2), where upsilon is the sweep rate.

A new rapid and simple method to determine the kinetics of electrode reactions of biologically relevant compounds from the half-peak width of the square-wave voltammograms.

A new method is introduced to determine the kinetic parameters of electron transfer reactions of biologically important compounds, based on the measurements of the half-peak width (DeltaE(p/2)) of the square-wave voltammograms. A simple surface (diffusionless) redox reaction, and a simple electrode reaction occurring from dissolved state are considered as model systems. In the region of quasireversible electron transfer, the half-peak widths of theoretical square-wave voltammograms are linear functions of the logarithm of the dimensionless kinetic parameter ln(K) that characterizes the rate of the electron transfer reaction. The dimensionless kinetic parameter K is defined as K=k(s)(fD)(-0.5) for the redox reaction taking place from dissolved state, whereas for the surface redox reaction K is defined as K=k(s)/f (k(s) is the standard rate constant of electron transfer, f is the SW frequency, and D is the diffusion coefficient). A set of linear regression equations for the dependences DeltaE(p/2)vs. ln(K) are derived, which can be used for rapid and precise determination of the charge-transfer kinetic parameters. The estimated values for the standard rate constants of various biologically relevant redox systems using this approach are in very good agreement with the experimental values determined by other square-wave voltammetric methods. The square-wave voltammetric half-peak width method can be used as a simple and reliable alternative to other voltammetric methods developed for the kinetic characterization of electron transfer rates.

Protein-film voltammetry: a theoretical study of the temperature effect using square-wave voltammetry.

Square-wave voltammetry of surface redox reactions is considered as an adequate model for a protein-film voltammetric setup. Here we develop a theoretical approach to analyze the effects of temperature on square-wave voltammograms. The performed simulations address the surface redox reactions featuring slow, modest and fast electron transfer. The theoretical calculations show that the temperature affects the square-wave voltammetric responses in a complex way resulting in a variety of peak shapes. Temperature effects on the phenomena known as "quasireversible maximum" and "split SW peaks" are also analyzed. The simulated results can be used to analyze the redox mechanisms and kinetic parameters of electron transfer reactions in protein-film criovoltammetry and other surface-confined redox systems. Our analysis also shows how "abnormal" features present in some square-wave voltammetric studies can easily be misinterpreted by postulating "multiple species", "stable radicals", or additional processes. Finally we provide a simple algorithm to use the "quasireversible maximum" to determine the activation energy of electron transfer reactions by surface redox systems.

Probing the redox activity of T-lymphocytes deposited at electrode surfaces with voltammetric methods.

BACKGROUND: Reactive oxygen species and redox signaling play an important role in the regulation of many vital biological processes. However, they are also tightly connected with many pathological conditions. The detection and evaluation of these signaling events are very often accompanied with great difficulties. In this article, we describe the development of a novel electrochemically-based technique for monitoring the cellular redox state. METHODS AND RESULTS: T-cells were attached on the surface of a working electrode, which was modified with 2-palmitoylhydroquinone as a redox mediator. Using cyclic voltammetry, we were able to indirectly (via the redox mediator) monitor an electron transport from the cells towards the working electrode, which enabled us to evaluate the redox activity of the cells. CONCLUSIONS: This new technique is rather simple and sensitive and may be used in the future as a valid diagnostic procedure in various branches of biomedical science.