Low-Cost Plant-Based Metal and Metal Oxide Nanoparticle Synthesis and Their Use in Optical and Electrochemical (Bio)Sensors.

Technological progress has led to the development of analytical tools that promise a huge socio-economic impact on our daily lives and an improved quality of life for all. The use of plant extract synthesized nanoparticles in the development and fabrication of optical or electrochemical (bio)sensors presents major advantages. Besides their low-cost fabrication and scalability, these nanoparticles may have a dual role, serving as a transducer component and as a recognition element, the latter requiring their functionalization with specific components. Different approaches, such as surface modification techniques to facilitate precise biomolecule attachment, thereby augmenting recognition capabilities, or fine tuning functional groups on nanoparticle surfaces are preferred for ensuring stable biomolecule conjugation while preserving bioactivity. Size optimization, maximizing surface area, and tailored nanoparticle shapes increase the potential for robust interactions and enhance the transduction. This article specifically aims to illustrate the adaptability and effectiveness of these biosensing platforms in identifying precise biological targets along with their far-reaching implications across various domains, spanning healthcare diagnostics, environmental monitoring, and diverse bioanalytical fields. By exploring these applications, the article highlights the significance of prioritizing the use of natural resources for nanoparticle synthesis. This emphasis aligns with the worldwide goal of envisioning sustainable and customized biosensing solutions, emphasizing heightened sensitivity and selectivity.

In situ Electrochemical Evaluation of the Interaction of dsDNA with the Proteasome Inhibitor Anticancer Drug Bortezomib.

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.

Carbon Inks-Based Screen-Printed Electrodes for Qualitative Analysis of Amino Acids.

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.

Influence of the Photodegradation of Azathioprine on DNA and Cells.

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.

PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications.

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.

Electrochemical evaluation of proton beam radiation effect on the B16 cell culture.

The interaction of radiation with matter takes place through energy transfer and is accomplished especially by ionized atoms or molecules. The effect of radiation on biological systems involves multiple physical, chemical and biological steps. Direct effects result in a large number of reactive oxygen species (ROS) within and outside and inside of the cells as well, which are responsible for oxidative stress. Indirect effects are defined as alteration of normal biological processes and cellular components (DNA, protein, lipids, etc.) caused by the reactive oxygen species directly induced by radiation. In this work, a classical design of an electrochemical (EC) three-electrodes system was employed for analyzing the effects of proton beam radiation on melanoma B16 cell line. In order to investigate the effect of proton radiation on the B16 cells, the cells were grown on the EC surface and irradiated. After optimization of the experimental set-up and dosimetry, the radiobiological experiments were performed at doses ranging between 0 and 2 Gy and the effect of proton beam irradiation on the cells was evaluated by the means of cyclic voltammetry and measuring the open circuit potential between working and reference electrodes.

Redox Mechanism of Azathioprine and Its Interaction with DNA.

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.

Natural phenolic antioxidants electrochemistry: Towards a new food science methodology.

Natural phenolic compounds are abundant in the vegetable kingdom, occurring mainly as secondary metabolites in a wide variety of chemical structures. Around 10,000 different plant phenolic derivatives have been isolated and identified. This review provides an exhaustive overview concerning the electron transfer reactions in natural polyphenols, from the point of view of their in vitro antioxidant and/or pro-oxidant mode of action, as well as their identification in highly complex matrixes, for example, fruits, vegetables, wine, food supplements, relevant for food quality control, nutrition, and health research. The accurate assessment of polyphenols' redox behavior is essential, and the application of the electrochemical methods in routine quality control of natural products and foods, where the polyphenols antioxidant activity needs to be quantified in vitro, is of the utmost importance. The phenol moiety oxidation pathways and the effect of substituents and experimental conditions on their electrochemical behavior will be reviewed. The fundamental principles concerning the redox behavior of natural polyphenols, specifically flavonoids and other benzopyran derivatives, phenolic acids and ester derivatives, quinones, lignins, tannins, lignans, essential oils, stilbenes, curcuminoids, and chalcones, will be described. The final sections will focus on the electroanalysis of phenolic antioxidants in natural products and the electroanalytical evaluation of in vitro total antioxidant capacity.

Charge transfer reaction mechanisms of epoxyketone and boronated peptides at glassy carbon and boron doped diamond electrodes.

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.

Electrochemical Sensor for Carbonyl Groups in Oxidized Proteins.

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 µg oxidized BSA and 0.75 pmol carbonyls.

Electrochemistry of Alzheimer Disease Amyloid Beta Peptides.

Alzheimer's disease (AD) is a widespread form of dementia that is estimated to affect 44.4 million people worldwide. AD pathology is closely related to the accumulation of amyloid beta (Aß) peptides in fibrils and plagues, the small oligomeric intermediate species formed during the Aß peptides aggregation presenting the highest neurotoxicity. This review discusses the recent advances on the Aß peptides electrochemical characterization. The Aß peptides oxidation at a glassy carbon electrode occurs in one or two steps, depending on the amino acid sequence, length and content. The first electron transfer reaction corresponds to the tyrosine Tyr10 amino acid residue oxidation, and the second to all three histidine (His6, His13 and His14) and one methionine (Met35) amino acid residues. The Aß peptides aggregation and amyloid fibril formation are electrochemically detected via the electroactive amino acids oxidation peak currents decrease that occurs in a time dependent manner. The Aß peptides redox behaviour is correlated with changes in the adsorption morphology from initially random coiled structures, corresponding to the Aß peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aß peptides in a ß-sheet configuration, observed by atomic force microscopy. Electrochemical studies of Aß peptides aggregation, mediated by the interaction with metal ions, particularly zinc, copper and iron, and different methodologies concerning the detection of Aß peptide biomarkers of AD in biological fluids, using electrochemical biosensors, are also discussed.

Amyloid Beta Peptide VHHQ, KLVFF, and IIGLMVGGVV Domains Involved in Fibrilization: AFM and Electrochemical Characterization.

The time-dependent structural modifications and oxidation behavior of specifically chosen five short amyloid beta (Aß) peptides, Aß1-16, Aß1-28, Aß10-20, Aß12-28, and Aß17-42, fragments of the complete human Aß1-40 peptide, were investigated by atomic force microscopy (AFM) and voltammetry. The objective was to determine the influence of different Aß domains (VHHQ that contains electroactive histidine H residues, KLVFF that is the peptide hydrophobic aggregation core, and IIGLMVGGVV that is the C-terminus hydrophobic region), and of Aß peptide hydrophobicity, in the fibrilization mechanism. The short Aß peptides absence of aggregation or the time-dependent aggregation mechanisms, at room temperature, in free chloride media, within the time window from 0 to 48 h, were established by AFM via changes in their adsorption morphology, and by differential pulse voltammetry, via modifications of the amino acid residues oxidation peak currents. The first oxidation peak was of tyrosine Y residue and the second peak was of histidine H and methionine M residues oxidation. A correlation between the presence of an intact highly hydrophobic KLVFF aggregation core and the time-dependent changes on the Aß peptides aggregation was found. The hydrophobic C-terminal domain IIGLMVGGVV, present in the Aß1-40 peptide, also contributed to accelerate the formation of Aß1-40 peptide aggregates and fibrils.

Alzheimer's disease amyloid beta peptides in vitro electrochemical oxidation.

The oxidative behaviour of the human amyloid beta (Aß1-40 and Aß1-42) peptides and a group of similar peptides: control inverse (Aß40-1 and Aß42-1), mutants (Aß1-40Phe10 and Aß1-40Nle35), rat Aß1-40Rat, and fragments (Aß1-28, Aß1-16, Aß10-20, Aß12-28, and Aß17-42), in solution or adsorbed, at a glassy carbon electrode, by cyclic and differential pulse voltammetry, were investigated and compared. Structurally the Aß1-40 and Aß1-42 sequences contain five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). The Aß peptide 3D structure influenced the exposure of the redox residues to the electrode surface and their oxidation peak currents. Depending on the amino acid sequence length and content, the Aß peptides gave one or two oxidation peaks. The first electron transfer reaction corresponded to the tyrosine amino acid residue oxidation, and the second to both histidines and methionine amino acid residues. The highest contribution to the second oxidation peak current was from His13, followed by His14 and His6 residues, and Met35 residue had the lowest contribution. The Aß peptides electron transfer depended on peptide hydrophobicity and 3D structure, the redox residues position in the sequence, the redox residues close to N-termini giving the highest oxidation peak currents.

Amyloid-ß peptides time-dependent structural modifications: AFM and voltammetric characterization.

The human amyloid beta (Aß) peptides, Aß1-40 and Aß1-42, structural modifications, from soluble monomers to fully formed fibrils through intermediate structures, were investigated, and the results were compared with those obtained for the inverse Aß40-1 and Aß42-1, mutant Aß1-40Phe(10) and Aß1-40Nle(35), and rat Aß1-40Rat peptide sequences. The aggregation was followed at a slow rate, in chloride free media and room temperature, and revealed to be a sequence-structure process, dependent on the physicochemical properties of each Aß peptide isoforms, and occurring at different rates and by different pathways. The fibrilization process was investigated by atomic force microscopy (AFM), via changes in the adsorption morphology from: (i) initially random coiled structures of ∼0.6 nm height, corresponding to the Aß peptide monomers in random coil or in α-helix conformations, to (ii) aggregates and protofibrils of 1.5-6.0 nm height and (iii) two types of fibrils, corresponding to the Aß peptide in a ß-sheet configuration. The reactivity of the carbon electrode surface was considered. The hydrophobic surface induced rapid changes of the Aß peptide conformations, and differences between the adsorbed fibrils, formed at the carbon surface (beaded, thin, <2.0 nm height) or in solution (long, smooth, thick, >2.0 nm height), were detected. Differential pulse voltammetry showed that, according to their primary structure, the Aß peptides undergo oxidation in one or two steps, the first step corresponding to the tyrosine amino acids oxidation, and the second one to the histidine and methionine amino acids oxidation. The fibrilization process was electrochemically detected via the decrease of the Aß peptide oxidation peak currents that occurred in a time dependent manner.

Electrochemical evaluation of glutathione S-transferase kinetic parameters.

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/µM and a LOD of 6.4µM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis-Menten and Lineweaver-Burk double reciprocal plots, was determined. A value of KM~100µM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60µmol/min per mg of GST.

Virgin olive oil ortho-phenols--electroanalytical quantification.

An electroanalytical methodology was developed for the determination of the total ortho-phenol content of virgin olive oil (VOO) with high sensitivity and reproducibility. The VOO ortho-phenol content depends on its freshness and is normally expressed as HT equivalent. Screen-printed electrodes were used with cyclic voltammetry to investigate the oxidation of catechol, phenol, hydroxytyrosol (HT), tyrosol, caffeic acid and ferulic acid. The oxidation of ortho-phenols and mono-phenols occurs following different mechanisms, and at different potentials. Using screen-printed electrodes and square wave voltammetry, an HT detection limit of 0.40 µM, was obtained. The electroanalytical methodology developed was applied to the determination of ortho-phenol content in fresh and old VOO. The HT equivalent determined for a two-year-old VOO sample was 3mg/kg, for one-year-old samples was 6-7 mg/kg, and for a fresh VOO sample 30 mg/kg, recoveries in the range of 78-93% of HT standard being obtained. The effect of VOO matrix components on the HT standard response was investigated.

Redox behaviour of verbascoside and rosmarinic acid.

The electrochemical oxidation mechanisms of rosmarinic acid (RA) and verbascoside (VB), both caffeic acid esters with two catechol moieties, were investigated. The redox mechanism is associated with the oxidation of the catechol groups, and was studied over a wide pH range by cyclic, differential pulse and square wave voltammetry, using a glassy carbon electrode. The voltammetric study revealed that both molecules, RA and VB, are reversibly oxidized in two successive pH-dependent steps each with the transfer of two electrons and two protons. Moreover, it was found that the first oxidation step is associated with the caffeic acid moiety, whereas the second oxidation step corresponds to the oxidation in VB of the hydroxytyrosol group and in RA of the 3,4-dihydroxyphenyl lactic acid residue.

Electrochemical behavior of triflusal, aspirin and their metabolites at glassy carbon and boron doped diamond electrodes.

The electrochemical behavior of triflusal (TRF) and aspirin (ASA), before and after hydrolysis in water and in alkaline medium using two different electrode surfaces, glassy carbon and boron doped diamond, was study by differential pulse voltammetry over a wide pH range. The hydrolysis products are 2-(hydroxyl)-4-(trifluoromethyl)-benzoic acid (HTB) for triflusal and salicylic acid (SA) for aspirin, which in vivo represent their main metabolites. The hydrolysis processes were also followed by spectrophotometry. The UV results showed complete hydrolysis after one hour for TRF and after two hours for ASA in alkaline solution. The glassy carbon electrode enables only indirect determination of TRF and ASA through the electrochemical detection of their hydrolysis products HTB and SA, respectively. The oxidation processes of HTB and SA are pH dependent and involve different numbers of electrons and protons. Moreover, the difference between the oxidation peak potential of SA and HTB was equal to 100 mV in the studied pH range from 1 to 8 due to the CF3 of the aromatic ring of HTB molecule. Due to its wider oxidation potential range, the boron doped diamond electrode was used to study the direct oxidation of TRF and ASA, as well as of their respective metabolites HTB and SA.