Antibacterial Properties of Rose Bengal Conjugated to Hyaluronic Acid.

Dental diseases, including conditions affecting oral structures, have become more common due to unhealthy lifestyle choices. Traditional antibiotic treatments face challenges related to the development of antibiotic resistance in bacteria. Photodynamic antibacterial chemotherapy is emerging as a promising alternative using photosensitizers to generate reactive oxygen species upon exposure to light. This article examines the photosensitizer Rose Bengal (RB) immobilized in hyaluronic acid (HA) for prolonged antibacterial action. The RB-HA conjugate demonstrated a molar ratio of approximately three RB residues to each of the ten units of HA. RB-HA exhibited a high singlet oxygen quantum yield (ΔΦ = 0.90), suggesting its efficacy in photodynamic treatment. A photostability analysis revealed slower photobleaching of RB-HA, which is essential for prolonged application. Under visible light and ultrasonic treatment, RB-HA exhibited effective antibacterial activity against Gram-positive S. aureus and Gram-negative E. coli bacteria for at least 80 days. The gradual release of RB ensured sustained bactericidal concentration. The study establishes RB-HA as a promising candidate for antimicrobial photodynamic and sonodynamic therapy in dental and other medical fields, providing enhanced stability and prolonged antibacterial efficacy.

Photo- and Sono-Active Food Colorants Inactivating Bacteria.

Food colorants are commonly used as excipients in pharmaceutical and nutraceutical fields, but they have a wide range of other potential applications, for instance, as cytotoxic drugs or mediators of physical antimicrobial treatments. The photodynamic antibacterial activity of several edible food colorants is reported here, including E127, E129, E124, E122, E133, and E150a, alongside Rhein, a natural lipophilic antibacterial and anticancer compound found in medicinal plants. Minimal inhibitory concentration (MIC) values for S. aureus and E. coli showed that E127 and Rhein were effective against both bacteria, while other colorants exhibited low activity against E. coli. In some cases, dark pre-incubation of the colorants with Gram-positive S. aureus increased their photodynamic activity. Adding Rhein to E127 increased the photodynamic activity of the latter in a supportive mode. Optional sensing mechanism pathways of combined E127/Rhein action were suggested. The antibacterial activity of the studied colorants can be ranged as follows: E127/Rhein >> E127 >> E150a > E122 > E124 >> E129 ≈ E133. E127 was also found to exhibit photodynamic properties. Short ultrasonic treatment before illumination caused intensification of E127 photodynamic activity against E. coli when applied alone and especially in combination with Rhein. Food colorants exhibiting photo- and sonodynamic properties may have good potential in food preservation.

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M-1·m-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M-1·m-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Antimicrobial and Antiviral Compounds of Phlomis viscosa Poiret.

Phlomis viscosa Poiret (an evergreen shrub) represents a valuable source of medicinal compounds. In this study, we discovered compounds with antimicrobial and antiviral properties. The aim of this study was to identify compounds of P. viscosa and estimate the antimicrobial and antiviral activity of its phytochemicals. The volatile compounds were identified using gas chromatography/mass spectrometry (GC/MS) analysis. For the identification of nonvolatile components of the extracts, high-performance liquid chromatography (HPLC), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) were applied. Quercetin 3-O-rutinoside and hesperidin caused a significant decrease in the bacterial concentration of Agrobacterium tumefaciens, Xylella fastidiosa and Pseudomonas syringae (p < 0.001). The growth of drug-resistant microorganisms (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Serratia marcescens and Salmonella enteritidis) was inhibited by quercetin 3-O-rutinoside, quercetin 3-O-arabinoside and hesperidin. In addition, these compounds demonstrated antiquorum-sensing properties. Diosmin, hesperidin and quercetin 3-O-arabinoside significantly inhibited varicella zoster virus (VZV) (p < 0.001). Quercetin 3-O-rutinoside and quercetin 3-O-arabinoside were effective against herpes simplex virus 1 (HSV-1), including mutant strains.

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay.

Laccase is a copper-containing enzyme that does not require hydrogen peroxide as a co-substrate or additional cofactors for an enzymatic reaction. Nanomaterials of various chemical structures are usually applied to the construction of enzyme-based biosensors. Metals, metal oxides, semiconductors, and composite NPs perform various functions in electrochemical transformation schemes as a platform for the enzyme immobilization, a mediator of an electron transfer, and a signal amplifier. We describe here the development of amperometric biosensors (ABSs) based on laccase and redox-active micro/nanoparticles (hereafter-NPs), which were immobilized on a graphite electrode (GE). For this purpose, we isolated a highly purified enzyme from the fungus Trametes zonatus, and then synthesized bi- and trimetallic NPs of noble and transition metals, as well as hexacyanoferrates (HCF) of noble metals; these were layered onto the surfaces of GEs. The electroactivity of many of the NPs immobilized on the GEs was characterized by cyclic voltammetry (CV) experiments. The most effective mediators of electron transfer were selected as the platform for the development of laccase-based ABSs. As a result, a number of catechol-sensitive ABSs were constructed and characterized. The laccase/CuCo/GE was demonstrated to possess the highest sensitivity to catechol (4523 A·M-1·m-2) among the tested ABSs. The proposed ABSs may be promising for the analysis of phenolic derivatives in real samples of drinking water, wastewater, and food products.

Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles.

Metallic nanoparticles potentially have wide practical applications in various fields of science and industry. In biosensorics, they usually act as catalysts or nanozymes (NZs) and as mediators of electron transfer. We describe here the development of amperometric biosensors (ABSs) based on purified oxidases, synthesized nanoparticles of CuCe (nCuCe), and micro/nanoporous gold (pAu), which were electro-deposited on a graphite electrode (GE). As an effective peroxidase (PO)-like NZ, nCuCe was used here as a hydrogen-peroxide-sensing platform in ABSs that were based on glucose oxidase, alcohol oxidase, methylamine oxidase, and L-arginine oxidase. At the same time, nCuCe is an electroactive mediator and has been used in laccase-based ABSs. As a result, the ABSs we constructed and characterized were based on glucose, methanol, methyl amine, L-arginine, and catechol, respectively. The developed nCuCe-based ABSs exhibited improved analytical characteristics in comparison with the corresponding PO-based ABSs. Additionally, the presence of pAu, with its extremely advanced chemo-sensing surface layer, was shown to significantly increase the sensitivities of all constructed ABSs. As an example, the bioelectrodes containing laccase/GE, laccase/nCuCe/GE, and laccase/nCuCe/pAu/GE exhibited sensitivities to catechol at 2300, 5055, and 9280 A·M-1·m-2, respectively. We demonstrate here that pAu is an effective carrier of electroactive nanomaterials coupled with oxidases, which may be promising in biosensors.

Antibacterial Activity of Rose Bengal Entrapped in Organically Modified Silica Matrices.

Photosensitizers (PSs) are known as powerful antibacterial agents that are activated by direct exposure to visible light. PSs can be noncovalently entrapped into the silica gel network for their controlled release into a contaminated area. The immobilization of PS-containing gel matrices on a polymer support expands their possible applications, such as antibacterial surfaces and coatings, which can be used for the disinfection of liquids. In the current study, we report the use of Rose Bengal (RB) incorporated into organically modified silica matrices (RB@ORMOSIL matrices) by the sol-gel technique. The RB matrices exhibit high activity against Gram-positive and Gram-negative bacteria under illumination by white light. The amount and timing of solidifier addition to the matrix affected the interaction of the latter with the RB, which in turn could affect the antibacterial activity of RB. The most active specimen against both Gram-positive and Gram-negative bacterial cells was the RB6@ORMOSIL matrix immobilized on a linear low-density polyethylene surface, which was prepared by an easy, cost-effective, and simple thermal adhesion method. This specimen, RB6@OR@LLDPE, showed the low release of RB in an aqueous environment, and exhibited high long-term antibacterial activity in at least 14 rounds of recycled use against S. aureus and in 11 rounds against E. coli.

"Green" Prussian Blue Analogues as Peroxidase Mimetics for Amperometric Sensing and Biosensing.

Prussian blue analogs (PBAs) are well-known artificial enzymes with peroxidase (PO)-like activity. PBAs have a high potential for applications in scientific investigations, industry, ecology and medicine. Being stable and both catalytically and electrochemically active, PBAs are promising in the construction of biosensors and biofuel cells. The "green" synthesis of PO-like PBAs using oxido-reductase flavocytochrome b2 is described in this study. When immobilized on graphite electrodes (GEs), the obtained green-synthesized PBAs or hexacyanoferrates (gHCFs) of transition and noble metals produced amperometric signals in response to H2O2. HCFs of copper, iron, palladium and other metals were synthesized and characterized by structure, size, catalytic properties and electro-mediator activities. The gCuHCF, as the most effective PO mimetic with a flower-like micro/nano superstructure, was used as an H2O2-sensitive platform for the development of a glucose oxidase (GO)-based biosensor. The GO/gCuHCF/GE biosensor exhibited high sensitivity (710 A M-1m-2), a broad linear range and good selectivity when tested on real samples of fruit juices. We propose that the gCuHCF and other gHCFs synthesized via enzymes may be used as artificial POs in amperometric oxidase-based (bio)sensors.

Photodynamic Eradication of Trichophyton rubrum and Candida albicans.

Conventional methods of onychomycosis treatment are ineffective in some cases because the cure of onychomycosis very often depends on the patient's individual response to the treatment; therefore, there is a crucial need to research and develop new methods of onychomycosis therapy. One of the most innovative treatments is photodynamic therapy (PDT) using photosensitizers (PSs). However, effective treatment depends on the correct choice of photosensitizer and substances that improve the characteristics of the final formulation. The aim of our work was to find an effective formulation for the treatment of onychomycosis. To achieve this goal, we tested the effect of three types of PSs, rose Bengal (RB), malachite green oxalate (MGO), and methylene blue (MB), on Candida albicans. The most effective PS was RB, and so the study was continued with Trichophyton rubrum. Additional comparative studies were carried out on substances included in the formulation (urea and thiourea), focusing on their antifungal activity, which can improve penetration through the nail plate. The composition of the formulation that achieved 100% eradication of Trichophyton rubrum under our conditions consisted of 150 µM RB, 5% urea, and 0.5% thiourea in glycerol/water (70/30%, w/w) solution. A white luminescent lamp was used as a light source (1.9 ± 0.1 mW cm-2). Stability of the formulation was checked. The selected formulation shows potential for future simplification and acceleration of PDT treatment of onychomycosis.

Sol-Gel Entrapped Lewis Acids as Catalysts for Biodiesel Production.

Replacing fossil fuels with biodiesel enables the emission of greenhouse gases to be decreased and reduces dependence on fossil fuels in countries with poor natural resources. Biodiesel can be produced by an esterification reaction between free fatty acids (FFAs) and methanol or by transesterification of triglycerides from oils. Both reactions require homogeneous or heterogeneous catalysis. Production of biodiesel catalyzed by heterogeneous catalysts seems to be the preferred route, enabling easy product separation. As we have previously shown, the Lewis acids AlCl3 and BF3 can serve as highly efficient catalysts under ultrasonic activation. The present study focused on the development of oleic acid (OA) esterification with methanol by the same catalysts immobilized in silica matrices using the sol-gel synthesis route. During the course of immobilization, AlCl3 converts to AlCl3 × 6H2O (aluminite) and BF3 is hydrolyzed with the production of B2O3. The immobilized catalysts can be reused or involved in a continuous process. The possibility of biodiesel production using immobilized catalysts under ultrasonic activation is shown for the conversion of FFAs into biodiesel in batch and continuous mode.

Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review.

The current review is devoted to nanozymes, i.e., nanostructured artificial enzymes which mimic the catalytic properties of natural enzymes. Use of the term "nanozyme" in the literature as indicating an enzyme is not always justified. For example, it is used inappropriately for nanomaterials bound with electrodes that possess catalytic activity only when applying an electric potential. If the enzyme-like activity of such a material is not proven in solution (without applying the potential), such a catalyst should be named an "electronanocatalyst", not a nanozyme. This paper presents a review of the classification of the nanozymes, their advantages vs. natural enzymes, and potential practical applications. Special attention is paid to nanozyme synthesis methods (hydrothermal and solvothermal, chemical reduction, sol-gel method, co-precipitation, polymerization/polycondensation, electrochemical deposition). The catalytic performance of nanozymes is characterized, a critical point of view on catalytic parameters of nanozymes described in scientific papers is presented and typical mistakes are analyzed. The central part of the review relates to characterization of nanozymes which mimic natural enzymes with analytical importance ("nanoperoxidase", "nanooxidases", "nanolaccase") and their use in the construction of electro-chemical (bio)sensors ("nanosensors").

LLDPE Composites with Nanosized Copper and Copper Oxides for Water Disinfection.

Consumption of contaminated water may lead to dangerous and even fatal water-borne diseases. Disinfection of drinking water is the most effective solution for this problem. The most common water treatment methods are based on the use of toxic disinfectants. Composites of polymers with nanosized metals and their oxides may become a good alternative to the existing methods. Expanding the scope of our previous publication, copper, cuprous, and copper oxide nanoparticles were immobilized onto linear low-density polyethylene by a simple thermal adhesion method. The antibacterial efficiency of the immobilized nanoparticles was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in batch experiments and for the first time the efficiency of these composites is reported for continuous flow regime. Immobilized copper and cuprous oxide nanoparticles demonstrated a high ability to eradicate bacteria after 30 min. These composites showed no or very limited leaching of copper ions into the aqueous phase both in the presence and in the absence of a bacterial suspension. Immobilized copper and cuprous oxide nanoparticles can be used for batch or continuous disinfection of water.

Keratin Biomembranes as a Model for Studying Onychomycosis.

Difficulties in obtaining human nails that are large enough for examining the penetration of drug formulations led us to produce keratin films regenerated from human hair. We assume that these films can simulate human nail plates in drug penetration and permeation tests and can serve as a biological model for studying onychomycosis. The films were formed from keratin extracted from human hair using dithiothreitol, urea and thiourea. The obtained keratin extract was dispensed into Teflon rings and dried at 40 °C and then cured at 110 °C. The structure, surface morphology, chemical characterization and thermal stability of the films were characterized and were compared to those of human nail, hair and bovine hoof samples using SDS-electrophoresis, scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The structure of the obtained films was found to be closer to human nails than to hair or bovine hooves. The keratin films were infected with Trichophyton rubrum and were proven to be appropriate for serving as a model for studying onychomycosis.

Dark Antibacterial Activity of Rose Bengal.

The global spread of bacterial resistance to antibiotics promotes a search for alternative approaches to eradication of pathogenic bacteria. One alternative is using photosensitizers for inhibition of Gram-positive and Gram-negative bacteria under illumination. Due to low penetration of visible light into tissues, applications of photosensitizers are currently limited to treatment of superficial local infections. Excitation of photosensitizers in the dark can be applied to overcome this problem. In the present work, dark antibacterial activity of the photosensitizer Rose Bengal alone and in combination with antibiotics was studied. The minimum inhibitory concentrations (MIC) value of Rose Bengal against S. aureus dropped in the presence of sub-MIC concentrations of ciprofloxacin, levofloxacin, methicillin, and gentamicin. Free Rose Bengal at sub-MIC concentrations can be excited in the dark by ultrasound at 38 kHz. Rose Bengal immobilized onto silicon showed good antibacterial activity in the dark under ultrasonic activation, probably because of Rose Bengal leaching from the polymer during the treatment. Exposure of bacteria to Rose Bengal in the dark under irradiation by electromagnetic radio frequency waves in the 9 to 12 GHz range caused a decrease in the bacterial concentration, presumably due to resonant absorption of electromagnetic energy, its transformation into heat and subsequent excitation of Rose Bengal.

Antibacterial Composites of Cuprous Oxide Nanoparticles and Polyethylene.

Cuprous oxide nanoparticles (Cu2ONPs) were used for preparing composites with linear low-density polyethylene (LLDPE) by co-extrusion, thermal adhesion, and attachment using ethyl cyanoacrylate, trimethoxyvinylsilane, and epoxy resin. The composites were examined by Scanning electron microscope and tested for their antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. All of these composites-except for the one obtained by extrusion-eradicated cells of both bacteria within half an hour. The composite prepared by thermal adhesion of Cu2ONPs on LLDPE had the highest external exposure of nanoparticles and exhibited the highest activity against the bacteria. This composite and the one obtained using ethyl cyanoacrylate showed no leaching of copper ions into the aqueous phase. Copper ion leaching from composites prepared with trimethoxyvinylsilane and epoxy resin was very low. The antibacterial activity of the composites can be rated as follows: obtained by thermal adhesion > obtained using ethyl cyanoacrylate > obtained using trimethoxyvinylsilane > obtained using epoxy resin > obtained by extrusion. The composites with the highest activity are potential materials for tap water and wastewater disinfection.

Effect of Photodynamic Antibacterial Chemotherapy Combined with Antibiotics on Gram-Positive and Gram-Negative Bacteria.

The well-known and rapidly growing phenomenon of bacterial resistance to antibiotics is caused by uncontrolled, excessive and inappropriate use of antibiotics. One of alternatives to antibiotics is Photodynamic Antibacterial Chemotherapy (PACT). In the present study, the effect of PACT using a photosensitizer Rose Bengal alone and in combination with antibiotics including methicillin and derivatives of sulfanilamide synthesized by us was tested against antibiotic-sensitive and antibiotic-resistant clinical isolates of Gram-positive S. aureus and Gram-negative P. aeruginosa. Antibiotic-sensitive and resistant strains of P. aeruginosa were eradicated by Rose Bengal under illumination and by sulfanilamide but were not inhibited by new sulfanilamide derivatives. No increase in sensitivity of P. aeruginosa cells to sulfanilamide was observed upon a combination of Rose Bengal and sulfanilamide under illumination. All tested S. aureus strains (MSSA and MRSA) were effectively inhibited by PACT. When treated with sub-MIC concentrations of Rose Bengal under illumination, the minimum inhibitory concentrations (MIC) of methicillin decreased significantly for MSSA and MRSA strains. In some cases, antibiotic sensitivity of resistant strains can be restored by combining antibiotics with PACT.

A Reagentless Amperometric Formaldehyde-Selective Chemosensor Based on Platinized Gold Electrodes.

Fabrication and characterization of a new amperometric chemosensor for accurate formaldehyde analysis based on platinized gold electrodes is described. The platinization process was performed electrochemically on the surface of 4 mm gold planar electrodes by both electrolysis and cyclic voltamperometry. The produced electrodes were characterized using scanning electron microscopy and X-ray spectral analysis. Using a low working potential (0.0 V vs. Ag/AgCl) enabled an essential increase in the chemosensor's selectivity for the target analyte. The sensitivity of the best chemosensor prototype to formaldehyde is uniquely high (28180 A·M-1·m-2) with a detection limit of 0.05 mM. The chemosensor remained stable over a one-year storage period. The formaldehye-selective chemosensor was tested on samples of commercial preparations. A high correlation was demonstrated between the results obtained by the proposed chemosensor, chemical and enzymatic methods (R = 0.998). The developed formaldehyde-selective amperometric chemosensor is very promising for use in industry and research, as well as for environmental control.

Increased copper bioremediation ability of new transgenic and adapted Saccharomyces cerevisiae strains.

Environmental pollution with heavy metals is a very serious ecological problem, which can be solved by bioremediation of metal ions by microorganisms. Yeast cells, especially Saccharomyces cerevisiae, are known to exhibit a good natural ability to remove heavy metal ions from an aqueous phase. In the present work, an attempt was made to increase the copper-binding properties of S. cerevisiae. For this purpose, new strains of S. cerevisiae were produced by construction and integration of recombinant human MT2 and GFP-hMT2 genes into yeast cells. The ySA4001 strain expressed GFP-hMT2p under the constitutive pADH1 promoter and the ySA4002 and ySA4003 strains expressed hMT2 and GFP-hMT2 under the inducible pCUP1 promoter. An additional yMNWTA01 strain was obtained by adaptation of the BY4743 wild type S. cerevisiae strain to high copper concentrations. The yMNWTA01, ySA4002, and ySA4003 strains exhibited an enhanced ability for copper ion bioremediation.

Polymer-immobilized photosensitizers for continuous eradication of bacteria.

The photosensitizers Rose Bengal (RB) and methylene blue (MB), when immobilized in polystyrene, were found to exhibit high antibacterial activity in a continuous regime. The photosensitizers were immobilized by dissolution in chloroform, together with polystyrene, with further evaporation of the solvent, yielding thin polymeric films. Shallow reservoirs, bottom-covered with these films, were used for constructing continuous-flow photoreactors for the eradication of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and wastewater bacteria under illumination with visible white light using a luminescent lamp at a 1.8 mW·cm⁻² fluence rate. The bacterial concentration decreased by two to five orders of magnitude in separate reactors with either immobilized RB or MB, as well as in three reactors connected in series, which contained one of the photosensitizers. Bacterial eradication reached more than five orders of magnitude in two reactors connected in series, where the first reactor contained immobilized RB and the second contained immobilized MB.