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Amperometric response of different modified electrodes to O 2 •−-DMSO. Curve (a) is the same base line for (b) to (e) but in pure DMSO. (b) Bare Au, (c) SOD/Cys/Au, (d) Cu +2 /Cys/Au, (e) Cys/Au electrodes. Each amperometric step indicates the response of modified electrode after addition of O 2 •−-DMSO solution (to 3 mL of PBS while stirring at 500 rpm). Amperometric tests were carried out at 0.25 V vs Ag/AgCl. Inset shows ∆I vs. pure DMSO (a) and O 2 •−-DMSO (b to d) volume.
Source publication
Cysteine self assembled monolayer-modified gold (Cys/Au) electrode is used to immobilize superoxide dismutase (SOD) and establish a direct electron transfer between enzyme and electrode surface. However, due to the redox activity of copper ion on Cys monolayer, there would be an ambiguity in electrochemical studies of immobilized SOD on Cys/Au elec...
Contexts in source publication
Context 1
... these results indicates that the source of the inherent electrochemical activity of Cys/Au electrode, is trace contaminant of Cu 2+ in buffer solution. (Fig. 4d). Also, SOD is a remarkably superefficient enzyme with a reaction rate approaching the diffusion limit [46]. Therefore, it makes sense that when SOD is absorbed on Cys SAM the O ...
Citations
... To determine whether the presence of Cu + -bound to CRIP2 conferred redox capabilities to the protein, we performed cyclic voltammetry assays, as previously described (138). Briefly, to enhance electron transfer, the electrode surface was modified using gold alkanethiols selfassembled monolayers (SAMs; (139)(140)(141)(142)), as these favor protein stability and prevent enzyme denaturation over the electrode surface (143,144). SAMs also possess low reliance on the electrode surface and superior electron transfer rates (139). ...
... SAMs also possess low reliance on the electrode surface and superior electron transfer rates (139). In addition, alkanethiols, such as L-Cys, exhibit a robust interaction with the gold surface and possess terminal groups with high affinity for the enzymatic structure, facilitating the electron transfer process, and contribute to the stable immobilization of an enzyme on the electrode surface without affecting catalytic activity (139,141,142). To investigate parameters related to the electron transfer processes of hsCRIP2 metallated with Cu + , the protein was mounted over an electrode modified with SAM and in the presence and absence of L-Cys. ...
Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.
... Most of these compounds, being developed so far, were based on manganese, cupric and ferric complexes with chelators such as porphyrins [7], macrocyclics [8,9], schiff-bases [10], imidazoles [11] and amino acids derivatives [12]. In our recently published report we realized that the cupric ions which coordinated to cysteine and immobilized on gold electrode represents specificity to O 2 •− [13]. So, it was concluded that copper cysteine complex could act as a SOD mimic enzyme and might serve as a good O 2 ...
... An interesting challenge in nanocatalysts is to gain metal nanoparticles capped with metal complexes which could mimic metalloenzymes [23]. In this sense, by capping gold nanoparticles on copper-cysteine (Cu-Cys) complex [13], a novel nanocomposite (gold/copper-cysteine, GNPs/Cu-Cys) was developed which shows SOD mimetic activity. Then, the SOD mimetic activity of the nanocomposite against O 2 ...
A nanocomposite consisting of gold nanoparticles and the copper(II) complex of cysteine (GNP/Cu-Cys) is shown to represent a useful mimic for the enzyme superoxide dismutase (SOD). The relative activities of plain GNPs, Cu metal, Cys, Cu-Cys and GNP/Cu-Cys were determined and compared to those of native SOD. The value for half-maximal inhibitory concentration of the nanocomposite is 0.3 μg∙mL‾1 which is 3 times higher than that of the native enzyme. The GNPs/Cu-Cys nanocomposite was immobilized on a carbon paste electrode and used as a biomimetic sensor for the detection of superoxide anions. The resulting sensor has a linear range over the concentration range from 3.1 to 326 μM, a detection limit of 2.8 μM (at an S/N of 3), and a sensitivity of 0.018 μA∙μM.cm−2. The selectivity for superoxide anions over potential interferents such as hydrogen peroxide, uric acid and citric acid is excellent.
Graphical abstract
The electrochemical signal for superoxide anion is produced by reducing O2•− at a carbon paste electrode modified with gold nanoparticles and the cysteine-copper complex.
... Thus, both reactions could be used for superoxide conversion on a gold electrode, resulting in either a positive or negative concentration dependent current response on superoxide. Cyclic voltammetric studies on cysteine coupled CuZnSOD electrodes revealed two prominent redox peaks with formal potentials of around 56 mV [49] Interestingly, by cyclic voltammetric and amperometric studies, Rafiee-Pour [104] found even higher signals from Cu 2+ ions, as if using CuZnSOD enzyme instead. The copper ions could be present as impurities in the measurement solution, which is highly supposable for complex cell media. ...
... Nevertheless, the results show the applicability for the usage of SOD linked electrodes in superoxide determination. Applied redox potentials vs. Ag/AgCl for the amperometric detection of superoxide were in the range of -200 mV for superoxide reduction [98] and 250 to 300 mV for its oxidation [104,98]. ...
Reactive oxygen species were found to contribute to diverse physiological and pathophysiological phenomena, like immune reactions, the progress of ageing and the development of cancer diseases. The measurement of reactive oxygen species from biological samples is not yet accomplished in a reliable way. A detection of extremely short lived and low concentrated species requires specific demands on the used sensor setup and principle.
The possibility for electrochemical in vitro sensing of superoxide radicals was investigated within this work. Conventional approaches were adapted to micromachined sensor setup and a novel sensing approach was suggested for stable and reliable superoxide measurement.
Sensor calibration was a main challenge, since the analyte shows instability and has to be produced in situ. Comprehensive studies on the artificial production of superoxide radicals were conducted by spectrophotometry and electron paramagnetic resonance
spectroscopic investigations. Herewith, a reliable and controllable superoxide production could be assured, which was essential for sensor development. A novel sensor principle, based on the direct electrochemical oxidation on gold electrodes, is suggested for the
first time for the measurement of superoxide release from model cells. Electrochemical sensor setup was designed and realised by micromachining technologies. Thin-film gold microelectrodes, coated with semi-permeable membrane, were found to fulfill best the
requirements for a stable and sensitive detection of superoxide release. The concept of fully integrated sensor chips into conventional tissue culture flasks provides an easy-tohandle and expandable sensing platform. The close vicinity of the sensor electrodes to the cultivated cells enables a sensitive measurement of extracellular short-lived species with their intrinsic short free diffusion length. Strategies for the enhancement of sensor performance include a proper choice of operation potential window for the electrochemical conversion of the analyte. Sensor selectivity and stability was improved by the introduction of suitable cell compatible protection layers, based on simple coating procedures. A cell cultivation directly on-chip could be demonstrated with human breast cancer and mouse fibroblast cell lines. The cultivation and measurement of model cells in a sensor equipped conventional containment, like culture tissue flasks or in a standard well format showed indisputable advantages over conventional approaches, like fluorescence or EPR based techniques. A direct cultivation and electrochemical measurement in the same setup dramatically decrease the effort for cell preparation and minimise intrinsic stress reaction from extensive chemical or mechanical treatment. The proposed sensor setup represents a kind of ideal sensor for the in vitro measurement of superoxide, since the influence of the setup to the cells as well as to the standardised cell culturing routines is minimized. The proposed detection method, without the need for enzymatic recognition elements, provides a much more stable and reliable approach, which easily exceeds the state of the art in superoxide sensing.
In the present work, SOD mimetic nanozyme (NACu-Cys) consisting of Cu-Cys complex and nano-albumin (NA) were synthesized. After characterizing the nanozyme, its superoxide dismutase (SOD) behavior was evaluated by inhibition of the pyrogallol autoxidation method. The results revealed that NACu-Cys exhibited SOD mimetic activity with a half inhibition concentration (IC50) value of 7.0 × 10-3 µM and a turnover number (kcat) of 5.4 × 107 s-1. In the next step, this nanozyme was applied as a protective agent against oxidative stress induced by sperm cryopreservation. Increasing the motility, raising the viability and reducing the apoptosis occurred as a result of NACu-Cys additions to human sperm freezing medium. Comparison between the natural SOD and SOD mimic behavior of NACu-Cys revealed that this nanoparticle has the ability to be used as oxidative stress decrescent during cryopreservation process.
It has been suggested that electron transfer (ET) processes occurring in complex environments capable of glass transitions, specifically in biomolecules, under certain conditions may experience the medium’s nonlinear response and nonergodic kinetic patterns. The interiors of self-assembled organic films (SAMs) deposited on solid conducting platforms (electrodes) are known to undergo glassy dynamics as well, hence they may also exhibit the abovementioned ‘irregularities’. We took advantage of Cu2+ ions as redox-active probes trapped in the Au-deposited −COOH-terminated SAMs, either L-cysteine, or 3-mercaptopropionic acid diluted by the inert 2-mercaptoethanol, to systematically study the impact of glassy dynamics on ET using the fast-scan voltammetry technique and its temperature and high-pressure extensions. We found that respective kinetic data can be rationalized within the extended Marcus theory, taking into account the frictionally controlled (adiabatic) mechanism for short-range ET, and complications due to the medium’s nonlinear response and broken ergodicity. This combination shows up in essential deviations from the conventional energy gap (overpotential) dependence and in essentially nonlinear temperature (Arrhenius) and high-pressure patterns, respectively. Biomimetic aspects for these systems are also discussed in the context of recently published results for interfacial ET involving self-assembled blue copper protein (azurin) placed in contact with a glassy environment.
Lead as one part of heavy metals group pose a detrimental risk to human health and the environment due to its toxicity, even exposure to minuscule quantities can be life threatening. Levels of lead are not stable in the environment and vary according to industrial production, urbanization, climate changes and many other factors. It is not surprising that rapid, sensitive and simple analytical determination of metal ions in environment is very important. Electrochemical techniques are one of the best methods for detecting metals due to their low cost, high sensitivity and portability. The aim of this study was to compare various microwave digestion procedures for lead(II) ions isolation in vultures samples of vultures available in very small quantities (5-20 mg) followed by differential pulse voltammetry as a detection method. We found that the combination of (65%, w/w) HNO 3 + (30%, w/w) H 2 O 2 is advantageous for the resulting electrochemical response. To determine the effect of mineralization on the height of the lead(II) ions peak, eight different mineralization programs were tested. It clearly follows from the results obtained that 200 W power for 30 min. is the most optimal for mineralisation Int. J. Electrochem. Sci., Vol. 6, 2011 5981 of vultures' samples. In the following part of the study, the influence of four different locations of samples in the rotor of used microwave system was investigated. Under the optimized conditions, samples of blood and eggs from vultures poisoned by lead(II) ions were prepared and electrochemically analysed. Three types of working electrodes as hanging mercury drop electrode, carbon paste electrode and screen printed electrode made of multi-walled nanotubes (MWNTs) were employed for this purpose. All three electrodes were sensitive enough to be used for analysis of real samples. Based on the results obtained it clearly follows that the level of lead(II) ions in intoxicated vultures ranged from 1 to 5 µg/ml of blood. In addition, it was found that the lead toxicosis was diagnosed in all vultures properly. Besides lead, cadmium was determined electrochemically and Ca, Mg, Fe, Zn and Cu spectrometrically. Besides the content of lead(II) and cadmium(II) ions, we determined metallothionein as a protein connected with protection of vertebrates against metal ions using differential pulse Brdicka reaction. The level of MT was determined as 1.82 ± 0.31 µg/mg of proteins in the intoxicated vultures compared with the control group 0.71 ± 0.19 µg/mg of proteins, which means more than a 250% increase of MT levels.
