Dr. Madalina BARSAN
Scientific Researcher I
Laboratory of Functional Nanostructures
madalina[DOT]barsan[AT]infim[DOT]roProjects
1. Disposable nanostructured biosensors based on metallic electrospun fibers for real time monitoring of superoxide in cell cultures
Project Type: TE, Start Date: 2020-09-15 End Date: 2022-09-14
Publications
1. Electrochemical assay for the quantification of anticancer drugs and their inhibition mechanism
Authors:
Leote, RJB; Sanz, CG; Diculescu, VC; Barsan, MM
Published: SEP 2025, METHODS, 241, DOI: 10.1016/j.ymeth.2025.05.002
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.
Open Access
2. Electrochemical bienzymatic biosensor for pyruvate kinase activity evaluation and inhibitor screening
Authors:
Leote, RJB; Barsan, MM; Sanz, CG; Diculescu, VC
Published: AUG 15 2025, TALANTA, 291, 127886, DOI: 10.1016/j.talanta.2025.127886
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.
3. Electrochemical detection of superoxide anion in living systems: Recent trends and clinical implications
Authors:
Sanz, CG; Aldea, A; Barsan, MM
Published: OCT 2025, BIOELECTROCHEMISTRY, 165, 108998, DOI: 10.1016/j.bioelechem.2025.108998
Superoxide plays a significant role in maintaining physiological states of living systems, with major roles in eradicating invading microorganisms and in cell signaling. It is regulated intricately by the enzyme superoxide dismutase (SOD), and when not properly regulated it can lead to cascade biological pathways with severe and irreversible damage to biofilms, tissue, and organs, being linked with many neurodegenerative diseases, atherosclerotic and cardiovascular diseases. Therefore, superoxide anion (O center dot-2 ) detection has a tremendous potential in clinical diagnostics to assess oxidative stress in living cells. This comprehensive review aims to explore, discuss, and analyze recent trends in the electrochemical detection of O center dot-2 in living systems, focusing not only on the recognition mechanism for in vitro assays (living cell cultures/tissues) but also on the importance of the electrode design and operational parameters for in vivo measurements (implantable sensors). By analyzing current in vitro/in vivo electrochemical strategies we gather information that is helpful to overcome existing limitations in the dynamic monitoring of O center dot-2 , and further improve electrochemical strategies that can be adopted and applied to prevent its negative effect, with an insight into the pathophysiology of neurodegenerative disorders and even cellular malignancies that derive from its accumulation in living systems.
5. Synthetic peptide array on gold for the electrochemical assessment of the 20S proteasome activity and effect of inhibitory compounds
Authors:
Barsan, MM; Serban, A; Onea, M; Wysocka, M; Lesner, A; Diculescu, VC
Published: FEB 1 2023, APPLIED SURFACE SCIENCE, 610, 155620, DOI: 10.1016/j.apsusc.2022.155620
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.
6. Novel cells integrated biosensor based on superoxide dismutase on electrospun fiber scaffolds for the electrochemical screening of cellular stress
Authors:
Sanz, CG; Aldea, A; Oprea, D; Onea, M; Enache, AT; Barsan, MM
Published: JAN 15 2023, BIOSENSORS & BIOELECTRONICS, 220, 114858, DOI: 10.1016/j.bios.2022.114858
A novel electrochemical biosensor was developed to monitor fibroblast cells stress levels for the first time in situ under external stimuli based on the recognition of superoxide anion released upon cell damage. The biosensor comprised metallized polycaprolactone electrospun fibers covered with zinc oxide for improved cell adhesion and signal transduction, whilst stable bioconjugates of mercaptobenzoic acid-functionalized gold nanoparticles/ superoxide dismutase were employed as recognition bioelements. Biosensors were first tested and optimized for in situ generated superoxide detection by fixed potential amperometry at +0.3 V, with minimal interferences from electroactive species in cell culture media. L929 fibroblast cells were then implanted on the optimized biosensor surface and the biosensor morphologically characterized by scanning electron microscopy (SEM) and fluorescence microscopy, which illustrated the network-type pattern of fibroblasts adjacent to the fiber scaffold. Fibroblast stress was induced by zymosan and monitored at the cells integrated biosensor using fixed potential amperometry (CA) with a sensitivity of 26 nA cm-2 mu g mL-1 zymosan and electrochemical impedance spectros-copy (EIS), with similar sensitivity of the biosensor considering the Rs and Z' parameters of around 0.13 omega cm2 mu g-1 mL and high correlation factors R2 of 0.9994. The obtained results underline the applicability of the here developed biosensor for the electrochemical screening of the fibroblast cells stress. The concept in using low-cost biocompatible polymeric fibers as versatile scaffolds for both enzyme immobilization and cell adhesion, opens a new path in developing biosensors for the in-situ investigation of a variety of cellular events.
Open Access
7. PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications
Authors:
Oprea, D; Sanz, CG; Barsan, MM; Enache, TA
Published: JUL 2022, BIOSENSORS-BASEL, 12, 500, DOI: 10.3390/bios12070500
PC-12 cells have been widely used as a neuronal line study model in many biosensing devices, mainly due to the neurogenic characteristics acquired after differentiation, such as high level of secreted neurotransmitter, neuron morphology characterized by neurite outgrowth, and expression of ion and neurotransmitter receptors. For understanding the pathophysiology processes involved in brain disorders, PC-12 cell line is extensively assessed in neuroscience research, including studies on neurotoxicity, neuroprotection, or neurosecretion. Various analytical technologies have been developed to investigate physicochemical processes and the biosensors based on optical and electrochemical techniques, among others, have been at the forefront of this development. This article summarizes the application of different biosensors in PC-12 cell cultures and presents the modern approaches employed in neuronal networks biosensing.
8. Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media
Authors:
Sanz, CG; Onea, M; Aldea, A; Barsan, MM
Published: MAY 1 2022, TALANTA, 241, 123255, DOI: 10.1016/j.talanta.2022.123255
A novel and disposable biosensor based on superoxide dismutase (SOD) immobilized on gold metallized polycaprolactone electrospun polymeric fibers (PCl/Au) has been developed for the determination of superoxide (O-2(center dot-)) in cell culture media. SOD biosensors were constructed employing three immobilization methods: crosslinking with EDC/NHS at a cysteine self-assembled monolayer (PCl/Au/SODCYS), biopolymer encapsulation with chitosan (PCl/Au/SODCHI) and cross-linking with glutaraldehyde (PCl/Au/SODGA). Scanning electron microscopy was performed at the three different biosensors to evaluate their surface morphologies. Biosensors were employed for the electrochemical detection of superoxide by fixed potential amperometry at different applied potentials, with two distinct enzymatic mechanisms being proposed: i) the reduction of the enzymatically generated peroxide, at -0.3 V, for which the PCl/Au/SODCHI biosensor presented the highest value of sensitivity of 40.1 mu A mM(-1) cm(-2), and ii) the regeneration of the enzyme catalytic copper centre, at +0.3 V, for which the PCl/Au/SODCYS biosensor had the highest sensitivity value of 16.1 mu A mM(-1) cm(-2). The proposed recognition mechanisms were further confirmed by cyclic voltammetric measurements, which enabled also to determine the amount of immobilized electroactive SOD, with highest value corresponding to the PCl/Au/SODCYS biosensor. The biosensors with best analytical performance, PCl/Au/SODCYS and PCl/Au/SODCHI, were further investigated for stability and selectivity, with best results for the PCl/Au/SODCYS, chosen for superoxide monitoring in cell culture media. The study is promising for future application of PCl/Au/SODCYS for the on-line superoxide monitoring of superoxide in cell cultures, grown directly on the biosensor itself.
9. Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring
Authors:
Sanz, CG; Crisan, DN; Leote, RJB; Onea, M; Barsan, MM
Published: JUL 2022, MICROCHIMICA ACTA, 189, 245, DOI: 10.1007/s00604-022-05352-z
The use of gold nanoparticles/superoxide dismutase (AuNP/SOD) bioconjugates is described as building blocks in SOD biosensor development for the quantification of superoxide in cell culture media. AuNP functionalization with 11-mercaptoundecanoic acid (MUA) and 4-mercaptobenzoic acid (MBA) (AuNPMUA and AuNPMBA) was used to improve SOD immobilization through EDC/NHS coupling using their -COOH terminus, leading to the formation of more stable bioconjugates. AuNP and AuNP/SOD bioconjugates were characterized by SEM to determine their size and morphology, UV-Vis for optical properties, FT-IR, and Raman spectroscopies for chemical functional group analysis and EDX for elemental analysis. Electrochemical methods were used to characterize the Au/AuNP-modified electrodes. For the optimization of the biosensor architecture, different AuNP/enzyme bioconjugates were prepared by varying the amount of both enzyme and AuNP, as well as their incubation time. Finally, the biosensors incorporating the bioconjugates were characterized by fixed potential amperometry and voltammetric analysis in order to establish the enzymatic mechanism and to elucidate the best biosensor architecture for monitoring superoxide in cell culture media. The best sensitivity value for superoxide detection corresponded to 41.2 nA mu M cm(-2), achieved by a biosensor based on AuNPMBA/SOD bioconjugates monitored through fixed potential amperometry at 0.3 V vs. Ag/AgCl, with a limit of detection of 1.0 mu M, and overall very good operational stability, maintaining 91% of the initial sensitivity after 30 days. Finally, the optimized biosensor was employed for the quantification of successive additions of superoxide in cell culture media, with excellent recovery values.
10. Quantification of cell oxygenation in 2D constructs of metallized electrospun polycaprolactone fibers encapsulating human valvular interstitial cells
Authors:
Sanz, CG; Mihaila, AC; Evanghelidis, A; Diculescu, VC; Butoi, E; Barsan, MM
Published: JAN 15 2022, JOURNAL OF ELECTROANALYTICAL CHEMISTRY, 905, 116005, DOI: 10.1016/j.jelechem.2021.116005
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.
Open Access
11. Hybrid Nanocomposite Platform, Based on Carbon Nanotubes and Poly(Methylene Blue) Redox Polymer Synthesized in Ethaline Deep Eutectic Solvent for Electrochemical Determination of 5-Aminosalicylic Acid
Authors:
Hosu, O; Barsan, MM; Sandulescu, R; Cristea, C; Brett, CMA
Published: FEB 2021, SENSORS, 21, 1161, DOI: 10.3390/s21041161
A novel hybrid composite of conductive poly(methylene blue) (PMB) and carbon nanotubes (CNT) was prepared for the detection of 5-aminosalicylic acid (5-ASA). Electrosynthesis of PMB with glassy carbon electrode (GCE) or with carbon nanotube modified GCE was done in ethaline deep eutectic solvent of choline chloride mixed with ethylene glycol and a 10% v/v aqueous solution. Different sensor architectures were evaluated in a broad range of pH values in a Britton-Robinson (BR) buffer using electrochemical techniques, chronoamperometry (CA), and differential pulse voltammetry (DPV), to determine the optimum sensor configuration for 5-ASA sensing. Under optimal conditions, the best analytical performance was obtained with CNT/PMBDES/GCE in 0.04 M BR buffer pH 7.0 in the range 5-100 mu M 5-ASA using the DPV method, with an excellent sensitivity of 9.84 mu A cm(-2) mu M-1 (4.9 % RSD, n = 5) and a detection limit (LOD) (3 sigma/slope) of 7.7 nM, outclassing most similar sensors found in the literature. The sensitivity of the same sensor obtained in CA (1.33 mu A cm(-2) mu M-1) under optimal conditions (pH 7.0, E-app = +0.40 V) was lower than that obtained by DPV. Simultaneous detection of 5-ASA and its analogue, acetaminophen (APAP), was successfully realized, showing a catalytic effect towards the electro-oxidation of both analytes, lowering their oxidation overpotential, and enhancing the oxidation peak currents and peak-to-peak separation as compared with the unmodified electrode. The proposed method is simple, sensitive, easy to apply, and economical for routine analysis.
Open Access
12. Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome
Authors:
Barsan, MM; Sanz, CG; Onea, M; Diculescu, VC
Published: APR 2021, SENSORS, 21, 2702, DOI: 10.3390/s21082702
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%.
13. An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening†
Authors:
Barsan, MM; Diculescu, VC
Published: MAY 21 2021, ANALYST, 146, DOI: 10.1039/d0an02426k
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.
Open Access
14. Electrospun conductive gold covered polycaprolactone fibers as electrochemical sensors for O2 monitoring in cell culture media
Authors:
Serban, A; Evanghelidis, A; Onea, M; Diculescu, V; Enculescu, I; Barsan, MM
Published: FEB 2020, ELECTROCHEMISTRY COMMUNICATIONS, 111, 106662, DOI: 10.1016/j.elecom.2020.106662
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.
15. Direct Immobilization of Biomolecules through Magnetic Forces on Ni Electrodes via Ni Nanoparticles: Applications in Electrochemical Biosensors
Authors:
Barsan, MM; Enache, TA; Preda, N; Stan, G; Apostol, NG; Matei, E; Kuncser, A; Diculescu, VC
Published: JUN 5 2019, ACS APPLIED MATERIALS & INTERFACES, 11, 19877, DOI: 10.1021/acsami.9b04990
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.
16. Electrochemical assay for 20S proteasome activity and inhibition with anticancer drugs
Authors:
de Jesus, CSH; Chiorcea-Paquim, AM; Barsan, MM; Diculescu, VC
Published: JUL 1 2019, TALANTA, 199, 39, DOI: 10.1016/j.talanta.2019.02.052
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.
17. New electrochemical sensor based on CoQ(10) and cyclodextrin complexes for the detection of oxidative stress initiators
Authors:
Barsan, MM; Diculescu, VC
Published: APR 10 2019, ELECTROCHIMICA ACTA, 302, 448, DOI: 10.1016/j.electacta.2019.02.060
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.
18. Nanostructured palladium doped nickel electrodes for immobilization of oxidases through nickel nanoparticles
Authors:
Barsan, MM; Matei, E; Enculescu, M; Costescu, R; Preda, N; Enache, TA; Enculescu, I; Diculescu, VC
Published: AUG 20 2019, ELECTROCHIMICA ACTA, 315, 113, DOI: 10.1016/j.electacta.2019.04.143
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.
19. Hydroxyapatite-iron oxide bioceramic prepared using nano-size powders
Authors:
Predoi, D; Barsan, M; Andronescu, E; Vatasescu-Balcan, RA; Costache, M
Published: NOV 2007, JOURNAL OF OPTOELECTRONICS AND ADVANCED MATERIALS, 9, 3613, DOI:
The hydroxyapatite (HAp) is an important material for orthopedics and dentistry applications. In the present study a simple method was tried for added the iron oxide nanoparticles into HAp so as to establish and induce a biocompatibility. In the research the mechanism of the coated of iron oxide with HAp would also be described by the analysis of X-ray diffractometer, scanning electron microscope and Fourier transformation infrared. Osteoblast cells were permanent monitored to detect any possible influence due to samples that might alter the cell growth, viability and proliferation.
20. An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening dagger
Authors:
Barsan, MM; Diculescu, VC
Published: , ANALYST, DOI: 10.1039/d0an02426k
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.
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