Dr. Victor DICULESCU

Background: Hepatocellular carcinoma is associated with high mortality and increasing incidence. Sorafenib, a cornerstone of therapy for advanced hepatocellular carcinoma, presents certain disadvantages, including low bioavailability and poor water solubility. This work describes a new strategy for sorafenib-targeted delivery aimed at improving treatment efficiency and reducing side effects. Methods: Magnetic nanoparticles coated with azelaic acid were modified with aptamer molecules that specifically recognize human liver cancer cell line HepG2, ensuring specificity for the tumor tissue. The nanoparticles were further loaded with sorafenib. The obtained drug delivery system was extensively characterized using UV-Vis spectrophotometry, transmission electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electrochemical impedance spectroscopy. Results: The drug delivery system demonstrated a higher release of sorafenib at acidic pH compared to pH 7.4. The cell internalization of the bare and aptamer-modified magnetic nanoparticles was assessed in HepG2 and human normal foreskin fibroblasts BJ cell lines, demonstrating that the aptamer significantly enhances internalization in tumor cells, while having no impact on healthy cells. Conclusions: The sorafenib-modified nanoparticles exhibited excellent cytocompatibility with BJ cells across all tested concentrations, while showing cytotoxicity towards HepG2 cells at higher concentrations, confirming the selectivity of the system.

Paper-based devices hold great promise in biosensing, but the choice of electrode materials influences performance. Here, we report a paper-based electrochemical sensor developed for nucleic acid quantification, in a sandwich-type architecture integrating 3-electrode systems on metallized electrospun polymeric fibers. A 3D-printed hydrophobic barrier on the chromatographic paper defines injection and testing zones. Fluid diffusion through paper and concentration gradients are considered in the design. Electrochemical characterization is performed using 40 mu L of methylene blue solution, which interacts with double-stranded nucleic acids, reducing its redox activity. This interaction mechanism within the paper substrate is confirmed by spectroscopy. The sensor achieves detection of nucleic acids in 3 min with 2 mu L of solution. Real sample analysis is performed for the quantification of PCR-amplified genes with a limit of detection of 1.38 ng mu L-1. The device serves as a promising point-of-care diagnostics tool for the direct quantification of amplified genetic material.

This article describes a new synthesis of nanoscaled Y2O3 that is bioinspired. It has been confirmed that Callistemon viminalis flower extract works well as a chelator when used to bioengineer high-shape anisotropy nanorods of single-phase Y2O3 . X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, fourier transform infrared spectroscopy and photoluminescence spectroscopy were used to analyse the structural, morphological, surface and optical features. The photoluminescent spectra of the bio-engineered nanorods show blue emissions. As the annealing temperature was increased from 300 to 500 degrees C, the blue colour purity values of the synthesized Y2O3 nanorods were 58.1, 80.7 and 77.0% at 300, 400 and 500 degrees C respectively. The chromaticity coordinates (0.2020, 0.1931), (0.1660, 0.1082) and (0.1714, 0.1226) from the photoluminescence spectra of the biosynthesized Y2O3 nanorods were used to determine these values. The CIE y-component coordinate values of the bioengineered blue-emitting nanophosphors suggest their potential for applications in display technology and white light-emitting diodes.

This study describes the development of a pyruvate kinase (PyK)-biosensor for the evaluation of PyK activity, as a diagnostic tool for early cancer screening and detection of kinase inhibitors used in cancer treatment, with the evaluation of the inhibition mechanism. The biosensor was constructed by co-immobilizing the enzymes PyK and pyruvate oxidase (PyOx) on Au film electrodes by crosslinking with glutaraldehyde (GA) and evaluated electrochemically by cyclic voltammetry (CV) and fixed potential amperometry (CA). First, the experimental conditions were optimized in terms of applied potential, enzyme ratio PyK:PyOx and enzyme substrate concentration: phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP). The biosensor sensitivity towards PEP detection was 2.11 +/- 0.08 mu A mM- 1 cm- 2, with very high reproducibility and repeatability, which made it suitable for inhibition studies of PyK inhibitor. The inhibition mechanism of shikonin was determined in relation to both PEP and ADP, with the calculation of IC50 values and binding constants (Ki). Detection of shikonin was possible at very low concentrations in the linear range of 0.1-4.0 pM. The electrochemical results were validated by UV-Vis spectrophotometry. The developed biosensor is a valuable tool for drug screening by enabling enzyme catalytic function examination with applicability to identify inhibitors, estimate their affinity, inhibition mechanism linked to their molecular mechanisms of action and evaluate selectivity, of great interest in both pharmaceutical and medical domains.

This study investigates the development of electrochemical genosensors using gold-coated electrospun polymeric fibers electrodes, Au/PMMA/PET and immobilized phosphorothioated oligonucleotides. Scanning electron microscopy (SEM) with energy-dispersive X-rays spectroscopy (EDS) revealed a uniform distribution of oligonucleotides on the fibers, contrary to planar gold electrodes Au/Ti/SiO2/Si, where network-like films were observed. X-ray photoelectron spectroscopy (XPS) confirmed the successful immobilization of the phosphorothioated oligonucleotides via strong covalent gold-sulfur bonds, while surface plasmon resonance (SPR) indicated superior binding affinity, with significantly lower equilibrium dissociation constants, when compared to unmodified probes. The detection of BCR/ABL fusion gene of chronic myeloid leukemia using differential pulse voltammetry and methylene blue as electroactive indicator, showed that the Au/PMMA/PET electrodes achieved a sensitivity of 379 +/- 12 mu A cm(-)(2) pM(-)(1) and a limit of detection of similar to 5.00 +/- 0.01 fM, outperforming the Au/Ti/SiO2/Si planar electrodes. Reduced non-specific adsorption was observed on the Au/PMMA/PET electrodes and attributed to the inherent charges introduced during the electrospinning process, which created localized electrostatic fields that repelled weakly adsorbing molecules. These findings demonstrate the potential of Au/PMMA/PET electrodes as a robust platform for further development of high-performance clinical diagnostic devices.

Overexpression of pyruvate kinase (PyK) is linked to many kinds of malignant tumors, representing therefore one of the most promising therapeutic targets for cancer treatment. Inhibition of PyK slows down tumor growth or causes tumor cell death, minimizing cancer cell proliferation, and understanding inhibitor mechanism of action can significantly improve cancer therapy. The present work describes the use of an amperometric bienzymatic biosensor, based on PyK and pyruvate oxidase (PyOx), in enzyme inhibition studies of four kinase inhibitors, CPG77675, Nilotinib, Ruxolitinib, Cerdulatinib. Their inhibition mechanism is studied and discussed in detail, with a thorough evaluation of their enzyme-inhibitor complex binding constants (Ki) and the inhibitor concentration required for 50% inhibition (IC50), employing standard inhibition procedure graphical methods. The biosensor is successfully applied for the quantification of the inhibitors by fixed potential amperometry, with excellent detection limit values in the pM range. It is the first detection method reported for the anticancer drugs CPG77675 and Cerdulatinib. The electrochemical assay based on the biosensor brings several advantages over the available assay kits for high-throughput screening (HTS) of kinase inhibitors, namely: low cost, easy operability and robustness demonstrated by biosensor high reproducibility and both operational and storage stability, offering an opportunity to discover new inhibitors and optimize their therapeutic index.

In this paper, graphene was obtained on a copper substrate using the CVD method, and then it was transferred to various substrates such as glass and SiO2/Si patterned with metallic interdigitated electrodes. The graphene thus obtained was characterized using Raman spectroscopy, scanning electron microscopy (SEM), current-voltage measurements, and electrochemical methods, in order to be used for sensing applications.

This work describes the development of a flexible uric acid (UA) biosensor based on palladium-coated submicrometer electrospun poly(methylmethacrylate) (PMMA) fibers metalized with gold and attached to polyethylene terephthalate substrate (Pd/Au/PMMA/PET). The morphological characterization conducted by scanning electron microscopy revealed nanoscale Pd dendritic structures. Electrochemical investigations in the absence and in the presence of redox probes demonstrated that these Pd nanostructures are responsible for a six-fold increase in the electroactive area and enhanced electron transfer kinetics when compared to the gold-coated electrospun fibers. The UA biosensor obtained by immobilizing the uricase enzyme (UrOx) onto the Pd/Au/PMMA/PET electrode surface, allowed UA detection with a sensitivity of 431 mu A cm(-2) mM(-1) and a limit of detection of 12 mu M. Investigation of the redox reactions of hydrogen peroxide (a product of the enzymatic oxidation of UA by UrOx) at the Pd/Au/PMMA/PET electrode demonstrated that the working principle of the biosensor is based on the reduction of PdO produced at the electrode surface during the spontaneous reduction of hydrogen peroxide on Pd. This allows a biosensor operating potential of -0.05 V (vs Ag/AgCl) with high selectivity. The UrOx/Pd/Au/PMMA/PET biosensor was applied for UA detection in body fluids (sweat, urine, and blood serum) with recovery values between 98 and 105%, which were validated by high-performance liquid chromatography analysis. The stability of the device was evaluated over a period of 3 months, retaining 78% of the initial sensitivity, and reproducibility with RSD = 4.9% was achieved. The analytical performance of the biosensor under harsh mechanical deformations and at physiological temperatures demonstrated the potential applications of the device to wearable sensing platforms.

Kynurenic acid (KA) is an active metabolite of tryptophan with notable biological effects, such as antioxidant, neuroprotective, and anti-inflammatory properties. It often undergoes changes of the concentration in biological fluids in chronic diseases. Thus, detecting KA is of great importance for diagnosing inflammatory and neurodegenerative conditions, monitoring disease progression, and assessing responses to pharmacological treatment. This study aimed to design a tailored, flexible platform for sensitive and direct electrochemical detection of KA in biological fluids. Carbon-based electrodes were custom-printed in the lab using specialized inks and flexible substrates. The working electrodes were further functionalized with graphene oxide and subsequently electrochemically reduced to increase the sensitivity toward the analyte. An optimized differential pulse voltammetry protocol was developed for KA detection. The elaborated platform was firstly characterized and then evaluated regarding the analytical performances. It showed a good limit of detection (3 nM and demonstrated the capability to detect KA across a broad concentration range (0.01-500 mu M). Finally, the elaborated flexible platform, was succesfully applied for KA determination in serum and saliva samples, in comparison with an optimized HPLC-UV method. The developed platform is the first example of in-lab printed flexible platform reported in literature so far for KA detection. It is also the first study reported in the literature of detection of KA in raw saliva collected from 10 subjects. The sensitivity towards the target analyte, coupled with the adaptability and portability, showcases the potential of this platform for thus illustrating great potential for further development of wearable sensors and biomedical applications.

Flexible porous materials have gained a high interest due to their impact on the development of electrochemical point-ofcare devices for monitoring the state of health of individuals. Among the porous materials, paper and textiles are most commonly used due to their innate capillary action on fluids. In this article, attention is paid to the retention of analytes in paper and textile porous substrates, and possible procedures to overcome this effect are discussed. The patterning of hydrophilic and hydrophobic regions for sample flow manipulation, and the folding properties of the flexible substrates for 3D architectures capable of transfer of analytes, are considered in relation to current electrode materials and detection methodologies.

Bortezomib is an inhibitor of proteasomes and an anti-cancer drug. Although bortezomib is considered a safe drug, as confirmed by cytotoxicity assays, recent reports highlighted the possibility of interaction between bortezomib and cellular components, with detrimental long-term effects. The evaluation of the interaction between bortezomib and dsDNA was investigated in bulk solution and using a dsDNA electrochemical biosensor. The binding of bortezomib to dsDNA involved its electroactive centers and led to small morphological modifications in the dsDNA double helix, which were electrochemically identified through changes in the guanine and adenine residue oxidation peaks and confirmed by electrophoretic and spectrophotometric measurements. The redox product of bortezomib amino group oxidation was electrochemically generated in situ on the surface of the dsDNA electrochemical biosensor. The redox product of bortezomib was shown to interact primarily with guanine residues, preventing their oxidation and leading to the formation of bortezomib-guanine adducts, which was confirmed by control experiments with polyhomonucleotides electrochemical biosensors and mass spectrometry. An interaction mechanism between dsDNA and bortezomib is proposed, and the formation of the bortezomib redox product-guanine adduct explained.

Proteins are vital components of living cells and the loss of their native functions has been associated with a wide variety of medical conditions. From this point of view, investigation of the protein microenvironment is crucial to support the development of therapeutic approaches capable of ensuring cellular functions. Therefore, analytical assays for the detection, quantification, and characterization of proteins, drugs, and protein-drug complexes play an essential role in fundamental research and clinical applications. Electrochemistry arises as an alternative methodology for fast assessment of proteins and drugs and is attractive due to the adaptability to miniaturization and scalability of electroanalytical devices, which then can be further employed as strategies towards personalized medical care. Thus, this review summarizes electrochemical investigations in the past 10 years on protein-based analytical devices and biosensors. A general overview of electrochemical assays that integrate proteins with nanostructured materials and conductive polymers is presented. Applications of electrochemical assays and biosensors were divided into four categories. First, those designed for drug screening strategies that focus on targeting specific intracellular, extracellular, or membrane protein subdomains to modulate their functions, aggregation/misfolding of proteins, and protein degradation pathways. Then, drug metabolism assays that involve mimicking natural metabolic pathways to identify potential safety and efficacy issues related to a drug or its metabolites. The third was dedicated to electrochemical drug delivery systems with anchored drugs in the form of bioconjugates, while the fourth was dedicated to electroanalytical methodologies for quantitative drug assays, where the electroactivity of the target species is often used to correlate the electrochemical signal to their concentration.

A sensor for the enzymatic activity and inhibition of the 20S proteasome was developed by immobilizing the synthetic peptide ABZ-VVSYAMG-(O2Oc)2-OH at Au electrodes. The detection principle is based on the elec-troactivity of ABZ, part of the ABZ-VVSY-OH moiety released from the peptide upon 20S proteasome chymo-trypsin action. The peptide was immobilized on a para-amino thiophenol (PATP) self-assembled monolayer on Au electrode by cross-linking its amino group to the -(O2Oc)2-OH moiety of the peptide (Au/PATP/peptide). The immobilization of the peptide and its interaction with 20S proteasome was investigated by SEM, QCM, SPR, ATR-FTIR and electrochemistry. The activity of 20S proteasome was assessed electrochemically by cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) after the immersion Au/PATP/peptide in 20S proteasome solution. CV study showed a decrease in both capacitive and faradaic currents corresponding to the ABZ-VVSY-OH removal, allowing the quantification of the 20S proteasome activity. The EIS study revealed that the resis-tance corresponding to charge transfer reactions at the peptide/solution interface correlated to the ABZ redox reaction, decreased linearly with increasing the incubation time in 20S proteasome solution. The perfected assay was applied for the investigation of the inhibitory effect of one synthetic, bortezomib, and two naturally occurring, epoxomicin, and lactacystin inhibitors.

Hematologic malignancies represent cancer diseases that affect the bone marrow and blood cells and include various subtypes depending mostly on the morphology of the cells. It is well known that an early diagnosis could be very useful for increasing survival rates in cancer, especially in the aggressive forms which can quickly progress to untreatable forms. The development of an easy, fast, and sensitive analytical tool with indicative applications in diagnosis and follow-up care, that could bring benefits in the discovery of new malignant diseases or relapses is reported. Oncofetal antigen/immature laminin receptor protein (OFA/iLRP) is an immunogenic protein found in fetal cells as well as overexpressed on the surface of some malignant tumors, including some hematologic malignancies. An aptamer, AB3, was selected and reported in the literature, having as a target the immature laminin receptor protein. Using the AB3 aptamer and its affinity to immature laminin receptor protein-positive cells an aptasensor was developed and tested on Jurkat cells. For the immobilization of the aptamer, graphene oxide modified screen printed electrodes were used and subjected to an activation procedure. The aptasensor development and cell caption were evaluated using electrochemical methods as well as microscopic techniques. The limit of detection of the developed aptasensor was 3.3 x 10(3) cells mL(-1), meaning 16 cells in the 5 mu L of suspension tested.

Due to the great significance of amino acids, a substantial number of research studies has been directed toward the development of effective and reliable platforms for their evaluation, detection, and identification. In order to support these studies, a new electrochemical platform based on PANI/ZnO nanowires' modified carbon inks screen-printed electrodes was developed for qualitative analysis of electroactive amino acids, with emphasis on tyrosine (Tyr) and tryptophan (Trp). A comparative investigation of the carbon ink before and after modification with the PANI/ZnO was performed by scanning electron microscopy and by Raman spectroscopy, confirming the presence of PANI and ZnO nanowires. Electrochemical investigations by cyclic voltammetry and electrochemical impedance spectroscopy have shown a higher charge-transfer rate constant, which is reflected into lower charge-transfer resistance and higher capacitance values for the PANI/ZnO modified ink when compared to the simple carbon screen-printed electrode. In order to demonstrate the electrochemical performances of the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes for amino acids analysis, differential pulse voltammograms were obtained in individual and mixed solutions of electroactive amino acids. It has been shown that the PANI/ZnO nanowires' modified carbon inks screen-printed electrodes allowed for tyrosine and tryptophan a peak separation of more than 100 mV, enabling their screening and identification in mixed solutions, which is essential for the electrochemical analysis of proteins within the proteomics research field.

A nanostructured samarium oxide electrode was constructed by electrodeposition onto the surface of a gold electrode on SiO2/Si wafer. The samarium oxides electrode's surface morphology was investigated by scanning electron microcopy showing a quasi-monodispersed nanoplatelets like structure. X-rays diffraction analysis demonstrated a mixture of monoclinic and hexagonal phases while the X-rays photoelectron spectroscopy indicated the co-existence of both Sm2+ and Sm3+ species in a 1:3 proportion. Cyclic voltammetry and electrochemical impedance spectroscopy were used to investigate the charge transfer processes at the surface of the samarium oxide electrode in the absence and in the presence of redox probes. A roughness factor of 2.5 was determined from the samarium oxide electrode while the charge transfer constant was almost double when compared to the planar gold electrode. Then, the samarium oxide electrode was used for the H2O2 detection by fixed potential amperometry at -0.20 V (vs. Ag/AgCl) with a linear region between 0.01 and 1.00 mM, a sensitivity of 153 mu A cm(-2) mM(-1) and a LoD = 2.70 mu M. Glucose oxidase was used as a model enzyme in order to test the capacity of the samarium oxides electrode for biosensing. The enzyme was immobilized by physical adsorption and the optimum conditions for glucose analysis investigated. The biosensor showed a linear range for glucose detection between 0.10 and 1.20 mM with a sensitivity of 8.40 mu A cm(-2) mM(-1) and a LoD = 8.00 mu M. Selectivity was tested toward common interfering species, and the results revealed the lack of biosensor response. The glucose biosensor on samarium oxides was tested for glucose detection in serum samples with a recovery factor of 90%, and the result validated with a commercial glucometer. (C) 2021 Elsevier Ltd. All rights reserved.

Electrospun polymeric fibers present an emerging alternative for the development of flexible electronics, enabling applications in wearable sensors and biosensors for continuous monitoring, and actuators for tissue engineering. The possibility to prepare sub-micrometric polymeric scaffolds, their processing for increasing the conductivity, their modification with different materials, conductive polymers and biomolecules in order to obtain functional flexible electrodes, allows the development of innovative devices for healthcare, and biomedical applications. In this review, the impact of metallized electrospun polymeric fibers in electrochemical (bio)sensors and actuators is discussed. A relation between their structure and functionality is provided, alongside with an overview of the different methods to obtain functional conductive fibers.

Azathioprine (AZA) is a pharmacologic immunosuppressive agent administrated in various conditions such as autoimmune disease or to prevent the rejection of organ transplantation. The mechanism of action is based on its biologically active metabolite 6-mercaptopurine (6-MP), which is converted, among others, into thioguanine nucleotides capable of incorporating into replicating DNA, which may act as a strong UV chromophore and trigger DNA oxidation. The interaction between azathioprine and DNA, before and after exposure to solar simulator radiation, was investigated using UV-vis spectrometry and differential pulse voltammetry at a glassy carbon electrode. The results indicated that the interaction of AZA with UV radiation was pH-dependent and occurred with the formation of several metabolites, which induced oxidative damage in DNA, and the formation of DNA-metabolite adducts. Moreover, the viability assays obtained for the L929 cell culture showed that both azathioprine and degraded azathioprine induced a decrease in cell proliferation.

Shikonin, a natural compound with pharmaceutical properties, has attracted interest due to its anti-oxidant properties, potential anti-cancer activity and activity over several biological pathways, such as dsDNA transcription/replication of cancer cells and inhibition of pyruvate kinase M2. The electrochemical behavior of shikonin in aqueous media was investigated at glassy carbon electrodes by cyclic and differential pulse voltammetry. The reduction involves the quinone moiety and formation of a semiquinone intermediate. In the absence of dissolved oxygen, the reduction is reversible while in normal atmosphere leads to formation of superoxide cation. The oxidation occurs at the dihydroxy moiety and reversibility was only observed in acid electrolytes. A redox mechanism was proposed. The interaction between shikonin and dsDNA was evaluated in incubated solutions and in situ with the dsDNA-electrochemical biosensor. The mechanism of interaction is time-dependent and follows an initial binding step at the grooves of the double strand leading to conformational modifications recognized through the variation of guanine and adenine oxidation peaks. The in-situ electrochemical production of the semiquinone intermediate leads to a preferential interaction with guanine residues, promoting their oxidation and consequently occurrence 8-oxo-guanine. An interaction mechanism was proposed.

The present study brings developments in the area of tissue engineering of heart valves, with the design and application of a new 2D model for human heart valve. This model contains valvular interstitial cells (VIC) encapsulated in gold covered electrospun polycaprolactone (PCL/Au) fibers, the latter serving also as an electrochemical sensor that enables the monitoring of oxygen levels of cellular media. The biocompatibility of PCL and PCL/Au with VIC was evaluated after the encapsulation of VIC in both scaffolds using the lactate dehydrogenase assay. Fluorescence microscopy of the encapsulated cells was used for the investigation of cell morphology, by employing phalloidin labelled F-actin (red) and DAPI nuclear staining (blue). The electrochemical techniques allowed to investigate the effect of cell encapsulation within the PCL/Au 2D constructs and to quantify the dissolved oxygen levels in cell culture media at both PCL/Au and PCL/Au with encapsulated cells (PCL/ Au/cells). Experiments were performed in a hypoxistation, which allowed a fine control on the cellular media oxygenation. Electrochemical impedance spectroscopy (EIS) was used to evaluate the electrochemical behavior of the 2D constructs, PCL/Au and PCL/Au/cells, in cellular media at different O-2 concentration. Results were analyzed and both impedance values at a fixed frequency and values of the equivalent circuit elements were used to construct calibration curves for dissolved O-2 determination. The second electrochemical technique used to monitor the levels of O-2 at PCL/Au and PCL/Au/cells in the cellular media was square wave voltammetry with sensitivities of 1.5 and 2.5 mu A cm(-2) mu M-1, for PCL/Au and PCL/Au/cells, respectively.

A dual strategy for the electrochemical detection for 20S proteasome (20S) is proposed, based on the oriented immobilization of a capture monoclonal antibody (Ab beta) on a self-assembled monolayer of 4-mercaptophenylboronic acid (4-MPBA) on gold electrodes, which led to the Au/4-MPBA/Ab beta immunosensor. The methodology comprises the correlation of 20S concentration with (i) its proteolytic activity toward the Z-LLE-AMC substrate, using the Au/4-MPBA/Ab beta/20S, and (ii) the enzymatic activity of an alkaline phosphatase (AlkP) from the AlkP-labeled secondary antibody (Ab(core)-AlkP), which involves the conversion of aminophenylphosphate to the electroactive aminophenol using Au/4-MPBA/Ab beta/20S/Ab(core)-AlkP. The step-by-step construction of the immunosensor and the interactions at its surface were evaluated by surface plasmon resonance and gravimetric analysis with quartz crystal microbalance, showing a high affinity between both antibodies and 20S. Morphological analysis by scanning electron microscopy demonstrated a pattern of parallel lines upon immobilization of Ab beta on 4-MPBA and morphological changes to a well-organized granular structure upon binding of 20S. A voltametric and impedimetric characterization was performed after each step in the immunosensor construction. The two detection strategies were evaluated. It was shown that the immunosensor responds linearly with 20S concentration in the range between 5 and 100 mu g mL(-1), which corresponds to proteasome levels in serum in the case of diverse pathological situations, and LoD values of 1.4 and 0.2 mu g mL(-1) were calculated for the detection strategies. The immunosensor was applied to the detection of 20S in serum samples with recovery values ranging from 101 to 103%.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 mu A cm(-2) mM(-1) and a LOD of 0.4 mu M. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K-0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Fibres of poly(methyl methacrylate) were obtained by electrospinning, subjected to coating with a gold layer and then attached on a thin polyethylene terephthalate substrate in order to obtain flexible electrodes for biosensing applications. The morphology of these electrodes, investigated by scanning electron microscopy showed multilayers of random oriented fibres of approx. 400 nm diameter. The electrochemical characterization of these flexible electrodes was performed by cyclic voltammetry and electrochemical impedance spectroscopy in acid and neutral media, in the absence and in the presence of redox probes, proving their superior performance (e.g. 5fold current density value) when compared to planar gold electrodes obtained on silicon wafers. The electrodes obtained from conductive electrospun polymeric fibres nets were tested by cyclic voltammetry and amperometry for the detection of hydrogen peroxide with a sensitivity of 0.84 mA cm-2 mM-1 and a detection limit of 20.40 ?M. The immobilization of the model enzyme glucose oxidase at the surface of the gold-coated electrospun polymeric fibres electrode was investigated and the obtained biosensor was applied for glucose determination in aqueous solutions by fixed potential amperometry with a sensitivity of 3.10 ?A cm-2 mM-1, a detection limit of 0.33 mM, and reduced interferences. Also, the practical applicability of the biosensor was tested for the detection of glucose in artificial sweat and serum samples.

Peppers are consumed all over the world, have several benefits to human health, such as antioxidant properties that can prevent diseases related to free radicals such as cardiovascular, inflammatory diseases, cancer, among others. This work aimed to evaluate the antioxidant capacity (AOC) of 36 varieties of peppers through ABTS and DPPH radical scavenging, electroanalytical assays, and to verify the vasorelaxant properties of selected samples. The greater the amount of capsaicin found in the extracts, the higher the AOC the greater the vasorelaxation. Naga had the highest scoring for antioxidant capacity, pout showed the lowest antioxidant capacity, vase pyramid intermediate level, whereas the capsaicin content followed the same trend. Extracts from all pepper varieties studied presented vasorelaxant properties in independent and dependent endothelial pathways.

The electrochemical behavior and the interaction of the immunosuppressive drug azathioprine (AZA) with deoxyribonucleic acid (DNA) were investigated using voltammetric techniques, mass spectrometry (MS), and scanning electron microscopy (SEM). The redox mechanism of AZA on glassy carbon (GC) was investigated using cyclic and differential pulse (DP) voltammetry. It was proven that the electroactive center of AZA is the nitro group and its reduction mechanism is a diffusion-controlled process, which occurs in consecutive steps with formation of electroactive products and involves the transfer of electrons and protons. A redox mechanism was proposed and the interaction of AZA with DNA was also investigated. Morphological characterization of the DNA film on the electrode surface before and after interaction with AZA was performed using scanning electron microscopy. An electrochemical DNA biosensor was employed to study the interactions between AZA and DNA with different concentrations, incubation times, and applied potential values. It was shown that the reduction of AZA molecules bound to the DNA layer induces structural changes of the DNA double strands and oxidative damage, which were recognized through the occurrence of the 8-oxo-deoxyguanosine oxidation peak. Mass spectrometry investigation of the DNA film before and after interaction with AZA also demonstrated the formation of AZA adducts with purine bases.

The ubiquitin-proteasome system regulates the level of proteins within cells through controlled proteolysis. In some diseases, the system function is dysregulated turning the ubiquitin-proteasome complex into a target for drug development. The redox behavior of proteasome inhibitors, epoxyketone and boronated peptides carfilzomib, oprozomib and delanzomib was investigated by voltammetric methods using glassy carbon and boron doped diamond electrodes. It was showed that the oxidation of epoxyketone peptides carfilzomib and oprozomib occurred in one step at glassy carbon electrode surface while at boron doped diamond two consecutive charge transfer reactions due to different adsorption orientation at the electrode surface were observed. The moieties of these peptides, involved in the oxidation process, were morpholine for carfilzomib and thiazole for oprozomib. For the boronated peptide delanzomib, two irreversible and independent redox processes, oxidation at +0.80 V and reduction at -1.40 V were identified in neutral media at both electrodes. The oxidation reaction occurred at the amino group close to the pyridine moiety of delanzomib with the transfer of one electron and one proton whereas the reduction process takes place at pyridine ring in a two-electrons two-protons mechanism. Redox mechanisms were proposed and the implications on the proteasome inhibition discussed.

This work reports the use of electrospun conductive gold covered polycaprolactone fibers for the quantification of dissolved O-2. The morphologies of the electrospun fibers obtained at a static and a dynamic drum collector were investigated by scanning electron microscopy. The reduction process of O-2 at negative potentials is analyzed by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) in sodium phosphate buffer (NaPB) pH 7.0 and in cellular media pH 7.4. The electrochemical sensing performance of Au/PCL towards O-2 quantification in NaPB and cellular media is compared by using three electrochemical techniques: cyclic and linear sweep voltammetry and EIS. Measurements are done in a two electrode configuration, using a silver wire as reference, to show the applicability of the method for O-2 quantification in cellular culture media.

In this study, the synergistic behavior of Ni species and bimodal mesoporous undoped SnO2 is investigated in oxygen evolution reactions (OERs) under alkaline conditions without any other modification of the compositional phases or using noble metals. An efficient and environmentally friendly hydrothermal method to prepare bimodal mesoporous undoped SnO2 with a very high surface area (>130 m(2) g(-1)) and a general deposition-precipitation method for the synthesis of well-dispersed Ni species on undoped SnO2 are reported. The powders were characterized by adsorption-desorption isotherms, TG-DTA, XRD, SEM, TEM, Raman, TPRH2, and XPS. The best NiSn composite generates, under certain experimental conditions, a very high TOF value of 1.14 s(-1) and a mass activity higher than 370 A g(-1), which are remarkable results considering the low amount of Ni deposited on the electrode (3.78 ng). Moreover, in 1 M NaOH electrolyte, this material produces more than 24 mA cm(-2) at an overpotential value of approximately +0.33 V, with only 5 wt % Ni species. This performance stems from the dual role of undoped SnO2, on the one hand, as a support for active and well-dispersed Ni species and on the other hand as an active player through the oxygen vacancies generated upon Ni deposition.

The present work describes the electrochemical properties of ionophores immobilized at the surface of electrodes obtained from electrospun polymeric fibers, in order to develop sensors for the analysis of electrolytes. Poly(methyl methacrylate) submicrometer fibers were prepared by electrospinning, coated with a gold layer by magnetron sputtering and then transferred on polyethylene terephthalate (PET) in order to obtained flexible electrodes. The ionophores were immobilized at the surface of these electrodes by drop-casting a ionophore-Nafion mixed solution. The sensor surface was investigated by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy in order to understand the morphology and distribution of a model Ca2+ ionophore over the electrode surface. Also, Fourier-transformed infrared spectroscopy was performed and demonstrated that the model Ca2+ ionophore can be immobilized in the nafion matrix maintaining its conformation, while cyclic voltammetry and electrochemical impedance spectroscopy demonstrated that the Ca2+ ionophore allows the diffusion of target ions through this this type of membrane. In order to prove the concept of ionophore-based sensors for the analysis of some electrolytes, Ca2+, NH4+, Cl- and H+ ionophore immobilized in a nafion matrix at the surface of these flexible electrodes were tested and the determination of the target ions performed by potentiometry in different media including artificial sweat. Finally, sensitivities, limits of detection, selectivity coefficients were determined. (C) 2020 Elsevier Ltd. All rights reserved.

The first electrochemical immunosensor for the determination of the 20S proteasome (P20S) was developed, entailing the immobilization of an antibody on an aminophenylboronic/poly-indole-6-carboxylic acid-modified electrode. The proposed electrochemical bioplatform is a simple and feasible analytical tool applicable for the determination of P20S in human plasma, considering its high clinical and biological relevance. Cyclic voltammetry, electrochemical impedance spectroscopy, and square wave voltammetry (SWV) were used to determine the optimal step-by-step process to obtain the electrochemical immunosensor. The interaction of P20S with the recognition layer of the immobilized antibody on the nanostructured surface took place by incubating the electrode in a P20S solution at 20 degrees C for 2 h. Using SWV as an electro-analytical technique, this immunosensor can quantify P20S. The current was linear with the P20S concentration within two dynamic concentration ranges from 20.0 to 80.0 and 80.0 to 200.0 ng.mL(-1) (r(2) = 0.992 and 0.98, respectively) with a limit of detection and quantification of 6 and 18 ng.mL(-1), respectively. Moreover, the immunosensor showed considerable repeatability and reproducibility, when the determination was done in human serum, which confirms that it is a promising alternative for direct detection of P20S in biological fluids with minimal interference.

The interaction of proteins with free radicals leads, among other types of damages, to the production of stable carbonyl groups, which can be used as a quantification of oxidative stress at proteins level. The aim of this study was the development of an electrochemical sensor for the detection of carbonyl groups in proteins oxidized by reactive oxygen species. Its working principle is based on the redox properties of dinitrophenylhydrazine (DNPH). BSA was used as a model protein and its oxidation achieved through Fenton reactions. Using differential pulse voltammetry at glassy carbon electrode, the electrochemical behavior of DNPH and of the native and oxidized BSA was investigated in solution. It has been shown that the hydrazine moiety of the DNPH is the electroactive center and is responsible for carbonyl complexation. Special attention was paid to the immobilization of the DNPH in order to retain its redox properties, and this was achieved on a mixed 4-styrenesulfonic acid-nafion matrix. The sensor's surface characterization and the detection of carbonyl groups in oxidized protein were performed by voltammetry, Fourier-transformed infrared spectroscopy and scanning electron microscopy while the voltammetric results were confirmed by surface plasmon resonance measurements. It has been shown that upon interaction with carbonyl groups of the oxidized protein, the oxidation peak of the hydrazine moiety of DNPH decreases as a function of incubation time and protein concentration. The sensor sensitivity was 0.015 nmol carbonyl per mg of oxidized protein and detection limits of 50 mu g oxidized BSA and 0.75 pmol carbonyls.

The present work describes a new simple procedure for the direct immobilization of biomolecules on Ni electrodes using magnetic Ni nanoparticles (NiNPs) as biomolecule carriers. Ni electrodes were fabricated by electroplating, and NiNPs were chemically synthesized. The chemical composition, crystallinity, and granular size of Ni electrodes, NiNP, and NiNP-modified Ni electrodes (NiNP/Ni) were determined by X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy (XPS). The electrochemical characterization of Ni electrodes by cyclic voltammetry and electrochemical impedance spectroscopy confirmed the existence of nickel oxides, hydroxides, and oxohydroxide films at the surface of Ni. Magnetic characterization and micromagnetic simulations were performed in order to prove that the magnetic force is responsible for the immobilization process. Further, Ni electrodes were employed as amperometric sensors for the detection of hydrogen peroxide because it is an important performance indicator for a material to be applied in biosensing. The working principle for magnetic immobilization of the enzyme-functionalized NiNP, without the use of external magnetic sources, was demonstrated for glucose oxidase (GOx) as a model enzyme. XPS results enabled to identify the presence of GOx attached to the NiNP (GOx-NiNP) on Ni electrodes. Finally, glucose detection and quantification were evaluated with the newly developed GOx-NiNP/Ni biosensor by amperometry at different potentials, and control experiments at different electrode materials in the presence and absence of NiNP demonstrated their importance in the biosensor architecture.

The majority of eukaryotic regulated protein turnover is performed by the proteasome, a multi-catalytic enzyme. Due to the fact that proteasome enzyme abnormal functioning was observed in different malignant cells, the proteasome is becoming a target for medical treatment. In order to evaluate the mechanisms of action of pharmaceutical compounds on proteasome enzyme inhibition, detecting and characterizing its activity is essential. An electrochemical assay that allows the monitoring of the chymotrypsin-like activity and inhibition of the 20S proteasome enzyme, based on the electrochemical detection of an electroactive compound released upon proteolysis of an adequate chymotrypsin-substrate is described. By employing differential pulse voltammetric measurement, the activity of the 20S proteasome enzyme was investigated for different incubation times of 20S with oligopeptide substrate as well as for different concentrations of substrate. Enzyme kinetic parameters were determined by voltammetry and the electrochemical assay compared with fluorescence spectroscopy. Electrochemical quartz crystal microbalance and atomic force microscopy were also used to investigate substrate interaction with the 20S proteasome and their adsorption at the electrode surface. Finally, the new electrochemical assay allowed to investigate the mechanisms of two different proteasome inhibitor drugs, bortezomib and oprozomib, underlying the applicability of the assay for understanding proteasome inhibitor action.

Staggered gap radial heterojunctions based on ZnO-CuxO core-shell nanowires are used as water stable photocatalysts to harvest solar energy for pollutants removal. ZnO nanowires with a wurtzite crystalline structure and a band gap of approximately 3.3 eV are obtained by thermal oxidation in air. These are covered with an amorphous CuxO layer having a band gap of 1.74 eV and subsequently form core-shell heterojunctions. The electrical characterization of the ZnO pristine and ZnO-CuxO core-shell nanowires emphasizes the charge transfer phenomena at the junction and at the interface between the nanowires and water based solutions. The methylene blue degradation mechanism is discussed taking into consideration the dissolution of ZnO in water based solutions for ZnO nanowires and ZnO-CuxO core-shell nanowires with different shell thicknesses. An optimum thickness of the CuxO layer is used to obtain water stable photocatalysts, where the ZnO-CuxO radial heterojunction enhances the separation and transport of the photogenerated charge carriers when irradiating with UV-light, leading to swift pollutant degradation.

The electro-oxidation mechanism of free methionine and bound within different peptide sequences was investigated by voltammetry, at glassy carbon electrode, and mass spectrometry. It is proposed that the electro-oxidation of free methionine occurs in two steps, each involving the transfer of one electron and turns pH-independent from mild acid to mild alkaline electrolytes. The first oxidation reaction leads to the formation of a cation radical stabilized either through the amino group resulting in the dehydromethionine intermediate, or by interaction with a neutral methionine molecule leading to production of a dimer cationic radical. The dehydromethionine hydrolysis gave methionine sulfoxide as final oxidation product, whereas a future oxidation of methionine dimer cation radical, i.e. the second electro-oxidation step, results in a methionine dimer dication. Moreover, at high acid media, the protonated amino group influence the electro-oxidation process to take place via proton transfer mechanism. The presence of methionine sulfoxide and of the dimer cationic radical as oxidation products of methionine was confirmed by mass spectroscopy.

The work describes the development of a flexible, hydrogel embedded pH-sensor that can be integrated in inexpensive wearable and non-invasive devices at epidermal level for electrochemical quantification of H+ ions in sweat. Such a device can be useful for swift, real time diagnosis and for monitoring specific conditions. The sensors' working electrodes are flexible poly(methyl methacrylate) electrospun fibers coated with a thin gold layer and electrochemically functionalized with nanostructured palladium/palladium oxide. The response to H+ ions is investigated by cyclic voltammetry and electrochemical impedance spectroscopy while open circuit potential measurements show a sensitivity of aprox. -59 mV per pH unit. The modification of the sensing interface upon basic and acid treatment is characterized by scanning and transmission electron microscopy and the chemical composition by X-ray photoelectron spectroscopy. In order to demonstrate the functionality of the pH-sensor at epidermal level, as a wearable device, the palladium/palladium oxide working electrode and silver/silver chloride reference electrode are embedded within a pad of polyacrylamide hydrogel and measurements in artificial sweat over a broad pH range were performed. Sensitivity up to -28 mV/pH unit, response time below 30 s, temperature dependence of approx. 1 mV/degrees C as well as the minimum volume to which the sensor responses of 250 nanoliters were obtained for this device. The proposed configuration represents a viable alternative making use of low-cost and fast fabrication processes and materials.

The development of coenzyme Q(10) (CoQ(10)) based electrochemical sensor for the detection of oxidative stress initiators is described for the first time. The sensor relies on CoQ(10) redox properties. CoQ(10) was immobilized at the surface of glassy carbon electrodes (GCE) in combination with cyclodextrins (CD), alpha-CD or beta-CD, that ensure the formation of a well dispersed CoQ(10) film. Nanostructured thin films of CoQ(10) alone and in complexes with alpha-CD or beta-CD at the electrode surface were characterized by scanning electron microscopy (SEM) and Fourier-transformed infrared spectroscopy (FTIR), enabling to identify the morphology of the films and the interactions between the CoQ(10) and CD. Nafion (R) was used to ensure sensor stability. The optimization of the CoQ(10) sensor configuration was made by assessing CoQ(10) redox properties through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), correlated with the results obtained from SEM and FTIR characterization. Next, the sensor in the optimized configuration, GCE/alpha-CD + CoQ(10)/Nafion (R), was applied for the detection of oxidant molecules, hydrogen peroxide and the superoxide radical, through the evaluation of the CoQ(10) redox properties monitored by fixed potential chronoamperometry and square wave voltammetry (SWV). (C) 2019 Elsevier Ltd. All rights reserved.

The present investigation deals with the development, characterization and application of nano-structured Pd doped Ni electrodes (Pd@Ni), which uses the electrochemical properties of Pd in synergy with the magnetic properties of Ni for biosensors development. The Pd@Ni electrodes have been characterized by X-ray diffraction, scanning electron microscopy with energy dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy. It has been shown that palladium presented spherical assemblies ranging 150-200 nm medium diameter size that covers large areas of the electrode surface while metallic nickel, which confers magnetic properties, showed a uniform granular structure with sizes between 20 and 50 nm. Cyclic voltammetry and electrochemical impedance spectroscopy were performed to understand the electrochemical process at the Pd@Ni electrodes in neutral media. The Pd@Ni electrodes were applied for the electrochemical detection of H2O2. Finally, Ni nanoparticles (NiNP) functionalized with the model enzyme glucose oxidase (GOx-NiNP) have been attached to the Pd@Ni electrode solely through magnetic interactions, and the obtained GOx-NiNP/Pd@Ni biosensor applied for glucose determination in aqueous solutions by fixed potential amperometry at -0.05 V (vs Ag/AgCl) with reduced interferences. (C) 2019 Elsevier Ltd. All rights reserved.

Proteasome is a multicatalytic enzyme complex responsible for proteolysis of damaged proteins and an important target for drug discovery in the pharmaceutical industry. Development of fast and economic strategies for detection of proteasome activity and inhibition is a topic of intensive research. The activity of the 20S proteasome was investigated by voltammetry and atomic force microscopy. The hydrolysis of peptide bonds was studied in incubated solutions of proteasome with oligopeptide sequences specific to each chymotrypsin, trypsin and caspase activity of proteasome, before and after inhibition with epoxomicin. The time-dependence of the proteolysis and the effect of substrate and inhibitor concentrations on the rate of enzymatic reaction were investigated. Different interaction mechanisms were characterized and enzyme kinetic parameters determined. The adsorption patterns of reaction mixture components were characterized by atomic force microscopy in order to understand the processes when saturation of enzyme catalytic centres occurs for high substrate concentrations. (C) 2017 Elsevier B.V. All rights reserved.

This paper proposes a novel, flexible, low cost administration patch which could be used as a non-invasive, controlled transdermal drug delivery system. The fabricated device consists in a flexible microfiber architecture heater covered with a thermoresponsive hydrogel, namely poly(N-isopropylacrylamide), as a matrix for the incorporation of active molecules. The manufacturing process consists of two main steps. First, the electrospun poly(methyl methacrylate) fiber networks are sputter coated with a thin gold layer and attached to flexible poly(ethylene terephthalate) substrates to obtain the heating platforms. Second, the heaters are encapsulated in poly(ethylene terephthalate) foils and covered with poly(N-isopropylacrylamide) hydrogel sheets. In order to illustrate the functionality of the fabricated patch, the hydrogel layer is loaded with methylene blue aqueous solution and is afterwards heated via Joule effect, by applying a voltage on the metalized fibers. The methylene blue releasing profiles of the heated patch are compared with those of the unheated one and the influence of parameters such as hydrogel composition and morphology, as well as the applied voltage values for microheating are investigated. The results indicate that the fabricated patch can be used as a drug administration instrument, while its performance can be tuned depending on the targeted application.

The development of soft actuators by using inexpensive raw materials and straightforward fabrication techniques, aiming at creating and developing muscle like micromanipulators, represents an important challenge nowadays. Providing such devices with biomimetic qualities, for example, sensing different external stimuli, adds even more complexity to the task. We developed electroactive polymer coated microribbons that undergo conformational changes in response to external physical and chemical parameters. These were prepared following three simple steps. During the first step nylon-6/6 microribbons were fabricated by electrospinning. In a second step the microribbons were one side coated with a metallic layer. Finally, a conducting layer of polypyrrole was added by means of electrochemical deposition. Strips of polypyrrole-coated aligned microribbon meshes were tested as actuators responding to current, pH, and temperature. The electrochemical activity of the microstructured actuators was investigated by recording cyclic voltammograms. Chronopontentiograms for specific current, pH, and temperature values were obtained in electrolytes with different compositions. It was shown that, upon variation of the external stimulus, the actuator undergoes conformational changes due to the reduction processes of the polypyrrole layer. The ability of the actuator to hold and release thin wires, and to collect polystyrene microspheres from the bottom of the electrochemical cell, was also investigated.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 mu A cm(-2) mM(-1) and a LOD of 0.4 mu M. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K-0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.