Conformational Changes in the BSA-LT4 Complex Induced by the Presence of Vitamins: Spectroscopic Approach and Molecular Docking.

Levothyroxine (LT4) is known for its use in various conditions including hypothyroidism. LT4 interaction with serum albumin may be influenced by the presence of vitamins. For this reason, we investigated the effect of vitamin C, vitamin B12, and folic acid on the complex of Bovine Serum Albumin with LT4 (BSA-LT4). UV-Vis spectroscopy was used to monitor the influence of vitamins on the BSA-LT4 complex. Fluorescence spectroscopy revealed a static quenching mechanism of the fluorescence of BSA-LT4 complex by the vitamin C and folic acid and a combined mechanism for vitamin B12. The interaction of vitamin C and folic acid with BSA-LT4 was moderate, while the binding of vitamin B12 was much stronger, extending the storage time of LT4 in blood plasma. Synchronous fluorescence found that the vitamins were closer to the vicinity of Trp than to Tyr and the effect was more pronounced for the binding of vitamin B12. The thermal stability of the BSA-LT4 complex was more evident, but no influence on the stability of BSA-LT4 complex was obtained for vitamin C. Molecular docking studies showed that vitamin C and folic acid bound the same site of the protein, while vitamin B12 bonded to a different site.

Insight into dual fluorescence effects induced by molecular aggregation occurring in membrane model systems containing 1,3,4-thiadiazole derivatives.

This work reports on biophysical insights into the excited state intramolecular proton transfer (ESIPT) processes taking place in three 1,3,4-thiadiazole derivatives that served as model compounds, on which electronic absorption, fluorescence, Fourier-transform infrared spectroscopy (FTIR), surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies were performed. The fluorescence spectra recorded in various solvents revealed an interesting dual fluorescence effect. In molecules in their monomeric form, the effect is associated with the ESIPT phenomenon, and may be further enhanced by aggregation-related effects, such as aggregation-induced emissions. Other spectroscopic studies on the selected molecules in a liposomal medium as a model revealed that, in a biomimetic environment, they can exist in both monomeric and aggregated forms. In both cases, however, the effects observed are closely related to the lipid's main phase transition temperature and the structure of the molecule. Introduction of specific substituents to the phenyl moiety either allows or prevents proton transfer from occurring in the excited state. The hydrophobicity changes in a lipid environment may result in an emergence of specific molecular forms and therefore either facilitate or hinder ESIPT processes. SPR and EIS confirmed the significant hydrophobicity changes in the model lipid systems, while FTIR measurements revealed a notable influence of 1,3,4-thiadiazoles on the fluidity of liposomal membranes. The results obtained clearly show that the thiadiazole derivatives are very good model molecules for studying hydrophobic-hydrophilic environments, and particularly with polymers or liposomes used as drug delivery systems.

A Nanoparticle-Based Label-Free Sensor for Screening the Relative Antioxidant Capacity of Hydrosoluble Plant Extracts.

One of the most important aspects of the detection of antioxidant compounds is developing a fast screening method. The screening of the overall relative antioxidant capacity (RAC) of several Romanian hydrosoluble plant extracts is the focus of this work. This is important because of the presence of increasing levels of reactive oxygen species (such as H2O2) generates oxidative stress in the human body. The consequences are a large number of medical conditions that can be helped by a larger consumption of plant extracts as food supplements, which do not necessarily contain the specified antioxidant contents. By exploiting the catalytic properties of gold nanoparticles, a specific and sensitive nanoparticle-based label-free electrochemical sensor was developed, where the working parameters were optimized for RAC screening of hydrosoluble plant extracts. First, electrochemical measurements (cyclic voltammetry and amperometry) were used to characterize different nanoparticle-based sensors, revealing the best performance of gold nanoparticle-based sensors, obtaining a RAC of 98% for lavender extracts. The sensing principle is based on the quenching effect of antioxidants for H2O2 amperometric detection, where the decrease in electrical signal suggests an increasing antioxidant capacity. The obtained results were expressed in terms of ascorbic acid and Trolox equivalents in order to be able to correlate our results with classical methods like chemiluminescence and UV-Vis spectrophotometry, where a correlation coefficient of 0.907 was achieved, suggesting a good correlation between electrochemistry and spectrophotometry. Considering these results, the optimized gold nanoparticle-based label-free sensor can be used as a simple, rapid alternative towards classical methods for relative antioxidant capacity detection of hydrosoluble plant extracts.

Tyrosinase-Based Biosensors for Selective Dopamine Detection.

A novel tyrosinase-based biosensor was developed for the detection of dopamine (DA). For increased selectivity, gold electrodes were previously modified with cobalt (II)-porphyrin (CoP) film with electrocatalytic activity, to act both as an electrochemical mediator and an enzyme support, upon which the enzyme tyrosinase (Tyr) was cross-linked. Differential pulse voltammetry was used for electrochemical detection and the reduction current of dopamine-quinone was measured as a function of dopamine concentration. Our experiments demonstrated that the presence of CoP improves the selectivity of the electrode towards dopamine in the presence of ascorbic acid (AA), with a linear trend of concentration dependence in the range of 2-30 µM. By optimizing the conditioning parameters, a separation of 130 mV between the peak potentials for ascorbic acid AA and DA was obtained, allowing the selective detection of DA. The biosensor had a sensitivity of 1.22 ± 0.02 µA·cm-2·µM-1 and a detection limit of 0.43 µM. Biosensor performances were tested in the presence of dopamine medication, with satisfactory results in terms of recovery (96%), and relative standard deviation values below 5%. These results confirmed the applicability of the biosensors in real samples such as human urine and blood serum.

Optoelectronic microfluidic device for point-of-care blood plasma viscosity measurement.

Physical properties of blood plasma, such as viscosity, serve as crucial indicators of disease. The inherent capillary effect of paper microchannels, coupled with minimal sample requirement, stimulated the advancement of paper-based viscometers. This study presents a precise, non-contact optoelectronic system using a microfluidic platform for the measurement of blood plasma viscosity. Microchannels were defined onto the filter paper using an available and inexpensive wax crayon, without the need for conventional wax printing equipment. The time required for the 5 µL sample to pass a specific distance was measured using two pairs of infrared sensors. Subsequently, this data was sent to the microcontroller, which automatically calculated the viscosity. Throughout the measurements, sample temperature was maintained at a constant 37 °C through an integrated heater with automated control. The microfluidic platform successfully processed real samples, yielding viscosity measurements in under three minutes. Evaluation with fetal bovine serum, spiked with varying protein concentrations in both native and denatured states, demonstrated a precision exceeding 96% compared to conventional Ostwald viscometer readings. For human subjects exhibiting pathologies affecting serum and plasma viscosity compared to physiological norms, strong correlations were observed between resultant values and clinical diagnoses. The proposed device aims to replace expensive and complex optical equipment, offering a safer alternative for measuring plasma viscosity. Unlike similar devices, it eliminates the risk of component deformation due to chemical contact or unsafe irradiation.

Biologically Synthesized Gold Nanoparticles with Enhanced Antioxidant and Catalytic Properties.

Increasing levels of reactive oxygen species generate oxidative stress in the human body that can lead to various medical conditions. The use of nanomaterials exhibiting antioxidant properties may prevent these effects. The biological synthesis of metallic nanoparticles using plant extracts with antioxidant properties can offer benefits due to their active compounds. The used extracts contained reducing and stabilizing agents, which were shown to be transferred onto the gold nanoparticles, functionalizing them. Herin, we report a gold nanoparticle synthesis by eco-friendly biological methods (b-AuNPs) using extracts of sea buckthorn, lavender, walnuts, and grapes, obtained through ultrasound-assisted extraction and pressure-enhanced extraction. The obtained b-AuNPs were characterized by UV-Vis and FTIR spectroscopies and visualized using transmission electron microscopy. The catalytic and scavenging effect of the b-AuNPs towards H2O2 (as reactive oxygen species) was evaluated electrochemically, highlighting the protective behavior of b-AuNPs towards lipid peroxidation. All experiments demonstrated the stability and reproducibility of prepared b-AuNPs with enhanced antioxidant and catalytic properties, opening a new perspective for their use in biomedical applications.

Molecular insights into binding mechanism of rutin to bovine serum albumin - Levothyroxine complex: Spectroscopic and molecular docking approaches.

Bovine serum albumin (BSA) has been used as a transporter protein for levothyroxine (LT4) and rutin, due to its property of binding to various ligands. Rutin binding to the BSA-LT4 complex can bring many benefits due to its proven pharmacological properties. Using Fourier-Transform Infrared Spectroscopy (FT-IR) the changes induced by rutin in the structure of BSA-LT4 complex were determined. Fluorescence studies allowed us to determine the quenching mechanism and affinity of rutin to the BSA-LT4 complex. The thermodynamic parameters suggest the binding of rutin to BSA-LT4 is a spontaneous process, driven by enthalpy and electrostatic forces. Also, the second derivative of the emission spectra suggests the Trp's of BSA are located in two different microenvironments. Thermal and chemical denaturation of BSA-LT4-rutin complex presents similar behavior but with better stability of the complex in case of chemical denaturation. Molecular docking studies show the binding of the two ligands to the same BSA site, suggesting that rutin may influence the bond of LT4 with the protein. Studies on the antioxidant activity of the BSA-LT4-rutin complex suggest that the presence of LT4 decreases the antioxidant activity of the rutin, but even so this antioxidant activity can be used to bring benefits for medical purposes.

Evaluation of the interaction of levothyroxine with bovine serum albumin using spectroscopic and molecular docking studies.

Bovine serum albumin (BSA) acts as a carrier for many endogenous and exogenous compounds, such as thyroid hormones or corresponding drugs. Binding of the hydrophilic levothyroxine drug (LT4) to BSA is of significant pharmacological importance. In this work, UV-vis measurements were used to determine the pH value at which LT4 interacts optimally with proteins. The binding mechanism and affinity of the interaction between LT4 and BSA were investigated using Fourier-transform infrared spectroscopy (FT-IR), fluorescence, fluorescence resonance energy transfer (FRET), Surface Plasmon Resonance (SPR), supplemented by molecular docking analysis. Fluorescence measurements revealed the quenching effect of LT4 on the BSA intrinsic fluorescence and LT4 binding with BSA is driven by a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. The thermodynamic parameters correspond to an enthalpic process, driven mainly by hydrogen bonds and van der Waals forces. Using SPR, the adsorbed amount of biomolecules was calculated and the binding affinity of LT4 with confined-BSA was characterized, indicating that the BSA immobilization plays an important role in LT4 binding. Docking studies confirmed the formation of the LT4-BSA complex with LT4 bound to site I on the BSA structure mainly with amino acid residues Trp 213, Tyr 137, Tyr 147. The calculation of the apparent association constant confirms the result obtained in SPR.Communicated by Ramaswamy H. Sarma.

Biosensors for Antioxidants Detection: Trends and Perspectives.

Herein we review the recent advances in biosensors for antioxidants detection underlying principles particularly emphasizing advantages along with limitations regarding the ability to discriminate between the specific antioxidant or total content. Recent advances in both direct detection of antioxidants, but also on indirect detection, measuring the induced damage on DNA-based biosensors are critically analysed. Additionally, latest developments on (bio)electronic tongues are also presented.

Monitoring biomolecular interaction between folic acid and bovine serum albumin.

Folic acid is a bioactive food component whose deficiency can lead to a variety of health problems, while a high intake of folic acid can reduce the cytotoxicity of natural killer cells. The binding mechanism of folic acid to free bovine serum albumin (BSA) was studied using fluorescence, while the biomolecular interaction between confined-BSA and free folic acid was assessed by electrochemical methods and surface plasmon resonance. The fluorescence quenching mechanism of BSA by folic acid was found to have a static character. The thermodynamic parameters of the interaction were determined and indicated a spontaneous exothermic process with a binding constant of 8.72 × 104 M-1 at 25 °C. Confinement of BSA to gold surfaces occurred through different immobilization methods (static and hydrodynamic), inducing conformational changes, which influenced the orientation of BSA molecules binding sites towards free folic acid. The apparent binding constant using electrochemical methods (voltammetry and impedance spectroscopy) was only 5 times higher (41 and 37 × 104 M-1) compared to BSA free in solution, while for surface plasmon resonance, where the hydrodynamic immobilization method was used, the value was much higher (19 × 106 M-1). This work gives also an insight on the interaction of BSA with gold substrates, surface plasmon resonance enabling the calculation of the adsorbed amount. The obtained results help understanding the specific interaction between free and confined BSA with free folic acid.

Evaluation of Heat-Treated AISI 316 Stainless Steel in Solar Furnaces to Be Used as Possible Implant Material.

The appropriate selection of implant materials is very important for the long-term success of the implants. A modified composition of AISI 316 stainless steel was treated using solar energy in a vertical axis solar furnace and it was subjected to a hyper-hardening treatment at a 1050 °C austenitizing temperature with a rapid cooling in cold water followed by three variants of tempering (150, 250, and 350 °C). After the heat treatment, the samples were analyzed in terms of hardness, microstructure (performed by scanning electron microscopy), and corrosion resistance. The electrochemical measurements were performed by potentiodynamic and electrochemical impedance spectroscopy in liquids that simulate biological fluids (NaCl 0.9% and Ringer's solution). Different corrosion behaviors according to the heat treatment type have been observed and a passivation layer has formed on some of the heat-treated samples. The samples, heat-treated by immersion quenching, exhibit a significantly improved pitting corrosion resistance. The subsequent heat treatments, like tempering at 350 °C after quenching, also promote low corrosion rates. The heat treatments performed using solar energy applied on stainless steel can lead to good corrosion behavior and can be recommended as unconventional thermal processing of biocompatible materials.

Comment from the Editors on the Special Issue: Advanced Analytical Methods in Clinical Diagnosis and Therapy.

With this Editorial, we want to present the Special Issue, "Advanced Analytical Methods in Clinical Diagnosis and Therapy". The development of medicine is not possible without progress in the field of identifying different biomarkers or treatments using modern approaches, such as the analytical methods presented in articles that are part of this issue. Thus, with the support of experts, both aspects of theoretical and practical interest from different fields of pathologies have been addressed.

Bioelectrochemical evaluation of plant extracts and gold nanozyme-based sensors for total antioxidant capacity determination.

The antioxidant properties of different plant extracts are usually claimed and used by food, medicine and cosmetic industry due to their health promoting capacities. In this study the presence of antioxidant compounds and the total antioxidant capacity of water-soluble extracts, prepared using two extraction methods and a variety of solvents, have been determined and a rapid screening method has been developed. Plant extracts characterisation, composition and antioxidant properties were confirmed by FTIR and Raman spectroscopies. Voltammetry, amperometry and electrochemical impedance were used to highlight the total antioxidant capacity of each extract using an electrochemical gold nanozyme-sensor based on the enzyme-like catalytic activity of gold nanoparticles. Both anodic area of cyclic voltammograms (10.31 µA V) and electrochemical index (153) calculated using differential potential voltammetry show the total content of antioxidant compounds, allowing to discriminate between different extracts. Amperometric total antioxidant capacity measurements were associated with those from classical chemiluminescence and good correlation has been found (Pearson's correlation coefficient of 0.958).

Ti-Zr-Si-Nb Nanocrystalline Alloys and Metallic Glasses: Assessment on the Structural Development, Thermal Stability, Corrosion and Mechanical Properties.

The development of novel Ti-based amorphous or ß-phase nanostructured metallic materials could have significant benefits for implant applications, due to potentially improved corrosion properties, and mechanical characteristics (lower Young's modulus, better wear performance, improved fracture toughness) in comparison to the standardized α+ß titanium alloys. Moreover, the devitrification phenomenon, occurring during heating, could contribute to lower input power during additive manufacturing technologies. Ti-based alloy ribbons were obtained by melt-spinning, considering the ultra-fast cooling rates this method can provide. The titanium alloys contain Zr, Nb, and Si (Ti60Zr10Si15Nb15, Ti64Zr10Si15Nb11, Ti56Zr10Si15Nb19) in various proportions. These elements were chosen due to their reported biological safety, as in the case of Zr and Nb, and the metallic glass-forming ability and biocompatibility of Si. The morphology and chemical composition were analyzed by scanning electron microscopy and energy-dispersive X-ray spectroscopy, while the structural features (crystallinity, phase attribution after devitrification (after heat treatment)) were assessed by X-ray diffraction. Some of the mechanical properties (hardness, Young's modulus) were assessed by instrumented indentation. The thermal stability and crystallization temperatures were measured by differential thermal analysis. High-intensity exothermal peaks were observed during heating of melt-spun ribbons. The corrosion behavior was assessed by electrocorrosion tests. The results show the potential of these alloys to be implemented as materials for biomedical applications.

Insight into the interaction of human serum albumin with folic acid: A biophysical study.

Folic acid (FA) is a water soluble bioactive food constituent from the vitamin B-family complex (B9). FA deficiency can lead to a variety of human health problems, while a high intake of FA can reduce the cytotoxicity of natural killer cells. The main goal of this study was to investigate the interaction of FA with human serum albumin (HSA) at physiological pH using ATR-FTIR, fluorescence spectroscopy, cyclic voltammetry and electrochemical impedance spectroscopy in order to understand the role of HSA as a blood transporter for FA in aqueous solution that can be used in different therapies. The quenching of HSA in the presence of FA was followed and the binding constant (Kb) was determined. The variation of electrochemical parameters proved that the FA binds to immobilized HSA and the binding constant was ten times than the value obtained when the interaction takes place between free molecules in solution when studied by fluorescence quenching. The results can be used in future studies to improve drug delivery systems or cellular uptake of folic acid and food components conjugated to HSA nanoparticles or nanocapsules.

"Click" access to multilayer functionalized Au surface: A terpyridine patterning example.

This work is focused on self-assembled monolayers (SAMs) fabrication, using two types of Au surfaces, by subsequent attachment of different layers in order to develop a stable platform consisting of covalent multilayer functionalized gold surfaces. The key step in the construction of SAMs is the covalent linkage to the gold surface, via an amino-thiol derivative, of a cyclooctyne unit exhibiting strained triple bonds which react fast (catalysts are not needed) and quantitatively with organic azides and enable the introduction of various chemical functionalized entities on the gold surface. The versatility of the system is demonstrated by the reaction of the cyclooctyne decorated gold surface with an azide functionalized terpyridine followed by step by step complexation with Fe(II) and another terpyridine unit resulting into a multilayer covered gold surface. The Au surfaces were characterized by XPS to determine the chemical composition of the resulting SAMs. SPR was applied for real-time monitoring of the molecular interactions that occurred on the Au surface for each deposited layer. DPN was used to direct pattern the terpyridine-ink on a pre-functionalized AuIDE electrode. The AFM topology resulted from DPN and PEIS demonstrated metal-coordinating ligand of Fe(II)-Terpy.

A new self-assembled layer-by-layer glucose biosensor based on chitosan biopolymer entrapped enzyme with nitrogen doped graphene.

The layer-by-layer (LbL) technique has been used for the construction of a new enzyme biosensor. Multilayer films containing glucose oxidase, GOx, and nitrogen-doped graphene (NG) dispersed in the biocompatible positively-charged polymer chitosan (chit(+)(NG+GOx)), together with the negatively charged polymer poly(styrene sulfonate), PSS(-), were assembled by alternately immersing a gold electrode substrate in chit(+)(NG+GOx) and PSS(-) solutions. Gravimetric monitoring during LbL assembly by an electrochemical quartz microbalance enabled investigation of the adsorption mechanism and deposited mass for each monolayer. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the LbL modified electrodes, in order to establish the contribution of each monolayer to the overall electrochemical properties of the biosensor. The importance of NG in the biosensor architecture was evaluated by undertaking a comparative study without NG in the chit layer. The GOx biosensor's analytical properties were evaluated by fixed potential chronoamperometry and compared with similar reported biosensors. The biosensor operates at a low potential of -0.2V vs., Ag/AgCl, exhibiting a high sensitivity of 10.5 µA cm(-2) mM(-1), and a detection limit of 64 µM. This study shows a simple approach in developing new biosensor architectures, combining the advantages of nitrogen-doped graphene with the LbL technique for enzyme immobilization.

Development and characterization of a new conducting carbon composite electrode.

A new conducting composite flexible material prepared from cellulose acetate (CA) polymer and graphite has been developed and used for the fabrication of electrodes, which were then characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Scanning electron microscopy (SEM) was used to provide information concerning the morphology of the composite electrode surface. The potential window, background currents and capacitance were evaluated by cyclic voltammetry in the pH range from 4.6 to 8.2. The voltammetry of model electroactive species demonstrates a close to reversible electrochemical behaviour, under linear diffusion control. The electroactive area of the composite electrodes increases after appropriate electrode polishing and electrochemical pre-treatment. The electrodes were used as substrate for the electropolymerisation of the phenazine dye neutral red, for future use as redox mediator in electrochemical biosensors. The composite electrodes were also successfully used for the amperometric detection of ascorbate at 0.0 V vs. SCE, and applied to the measurement of ascorbate in Vitamin C tablets; the sensor exhibits high sensitivity and a low detection limit of 7.7 microM. Perspectives for use as a versatile, mechanically flexible and robust composite electrode of easily adaptable dimensions are indicated.

Development and evaluation of electrochemical glucose enzyme biosensors based on carbon film electrodes.

Electrochemical glucose enzyme biosensors have been prepared on carbon film electrodes made from carbon film electrical resistors. Evaluation and characterisation of these electrodes in phosphate buffer saline solution has been carried out with and without pretreatment by cycling in perchloric acid or at fixed applied potential. Both pretreatments led to a reduction in the carbon surface oxidation peak and enabled better detection of hydrogen peroxide in the pH range of 5-7. Glucose oxidase enzyme was immobilised on the carbon surface by mixing with glutaraldehyde, bovine serum albumin and with and without Nafion. The performance of these two types of electrode was similar, that containing Nafion being more physically robust. Linear ranges were up to around 1.5mM, with detection limits 60muM, and pretreatment of the carbon film electrode at a fixed potential of +0.9V versus SCE for 5min was found to be the most beneficial. Michaelis-Menten constants between 5mM and 10mM were found under the different experimental conditions. Coating the immobilised enzyme layer with a thin layer of Nafion was found to give similar results in the determination of glucose to mixing it but with benefits against interferences for the analysis of complex matrices, such as wine. Potentialities, for a short-term-use or disposable sensors, are indicated.