Electrochemical Impedance Spectroscopy for the Sensing of the Kinetic Parameters of Engineered Enzymes.

The study presents a promising approach to enzymatic kinetics using Electrochemical Impedance Spectroscopy (EIS) to assess fundamental parameters of modified enteropeptidases. Traditional methods for determining these parameters, while effective, often lack versatility and convenience, especially under varying environmental conditions. The use of EIS provides a novel approach that overcomes these limitations. The enteropeptidase underwent genetic modification through the introduction of single amino acid modifications to assess their effect on enzyme kinetics. However, according to the one-sample t-test results, the difference between the engineered enzymes and hEKL was not statistically significant by conventional criteria. The kinetic parameters were analyzed using fluorescence spectroscopy and EIS, which was found to be an effective tool for the real-time measurement of enzyme kinetics. The results obtained through EIS were not significantly different from those obtained through traditional fluorescence spectroscopy methods (p value >> 0.05). The study validates the use of EIS for measuring enzyme kinetics and provides insight into the effects of specific amino acid changes on enteropeptidase function. These findings have potential applications in biotechnology and biochemical research, suggesting a new method for rapidly assessing enzymatic activity.

Unveiling Versatile Applications and Toxicity Considerations of Graphitic Carbon Nitride.

Metal-free, low-cost, organic photocatalytic graphitic carbon nitride (g-C3N4) has become a promising and impressive material in numerous scientific fields due to its unique physical and chemical properties. As a semiconductor with a suitable band gap of ~2.7 eV, g-C3N4 is an active photocatalytic material even after irradiation with visible light. However, information regarding the toxicity of g-C3N4 is not extensively documented and there is not a comprehensive understanding of its potential adverse effects on human health or the environment. In this context, the term "toxicity" can be perceived in both a positive and a negative light, depending on whether it serves as a benefit or poses a potential risk. This review shows the applications of g-C3N4 in sensorics, electrochemistry, photocatalysis, and biomedical approaches while pointing out the potential risks of its toxicity, especially in human and environmental health. Finally, the future perspective of g-C3N4 research is addressed, highlighting the need for a comprehensive understanding of the toxicity of this material to provide safe and effective applications in various fields.

Monitoring of the Surface Charge Density Changes of Human Glioblastoma Cell Membranes upon Cinnamic and Ferulic Acids Treatment.

Cinnamic acid (CA) and ferulic acid (FA) are naturally occurring phenolic acids claimed to exert beneficial effects against disorders related to oxidative stress, including cancer. One such malignancy that still remains a therapeutic challenge mainly due to its heterogeneity and inaccessibility to therapeutic agents is Glioblastoma multiforme (GBM). Here, the influence of CA and FA on the surface charge density of human GBM cell line LN-229 was studied using the electrophoretic light scattering technique. Also, the cytotoxicity of both phenolic acids was determined by metabolic activity-assessing tetrazolium test (MTT) analysis after exposure to CA and FA for 24 h and 48 h. Results showed that both compounds reduced cell viability of LN-229 cells, with more pronounced effect evoked by CA as reflected in IC50 values. Further analyses demonstrated that, after treatment with both phenolic acids, the negative charge of membranes decreased at high pH values and the positive charge of the membranes increased at low pH values compared to the data obtained for untreated cells. Afterward, a four-equilibrium model was applied to estimate the total surface concentrations of both acidic and basic functional groups and their association constants with solution ions in order to calculate theoretical values of membrane surface charge densities. Then, the theoretical data were compared to the experimental data in order to verify the mathematical model. As such, our results indicate that application of electrochemical methods to determine specific drug-membrane interactions might be crucial for predicting their pharmacological activity and bioavailability.

The modulating effect of lipid bilayer/p-coumaric acid interactions on electrical properties of model lipid membranes and human glioblastoma cells.

Biological membranes are one of the most important elements of living cells determining their permeability to the active compounds. Still, little is known about the drug-membrane interactions in terms of pharmacological properties of potential drugs. Chemoprevention based on natural compounds is becoming a strong trend in modern oncopharmacology, and p-coumaric acid (p-CoA) is one such compound with tentative anticancer activity. The microelectrophoretic mobility measurements and electrochemical impedance spectroscopy were applied to study the effects of p-CoA on electrical properties of liposomes, spherical bilayers, and human glioblastoma cell membranes. Our results demonstrated that after treatment with p-CoA, the surface charge of LBC3, LN-229 and LN-18 cell lines was significantly changed in alkaline pH solutions, but not in acidic pH solutions. In contrast, no changes in surface charge density values were registered for phosphatidylethanolamine liposomal membranes and A172 cell membranes after p-CoA treatment. The impedance data showed an increase in values of both the electrical capacitance and the electrical resistance, indicating that p-CoA can be partially inserted into the phosphatidylcholine bilayers. The MTT assay showed cell line-dependent cytotoxic effect of p-CoA. Further molecular analyses revealed the ATP depletion and gene transcription modulation, which might indicate organelle membrane-crossing potential of p-CoA. These results suggest, that changes in surface charge of membranes of living cells not only might be potential predictor of membrane permeability, but also indicate differential composition of cell membranes in various cell lines. Thus further multidirectional analyses are required to implement electrochemical methods as standard testing procedures during drug development process.

The Modulating Effect of p-Coumaric Acid on The Surface Charge Density of Human Glioblastoma Cell Membranes.

p-Coumaric acid (p-CoA), a phenolic acid belonging to the hydroxycinnamic acids family, is a compound with tentative anticancer potential. Microelectrophoretic mobility measurements conducted at various pH values of electrolyte solution were applied to study p-CoA effects on electrical properties of human glioblastoma cell membranes. The obtained results demonstrated that after the p-CoA treatment, the surface charge density of cancer cells changed in alkaline pH solutions, while no noticeable changes were observed in cell membranes incubated with p-CoA compared to control at acidic pH solutions. A four-equilibrium model was used to describe the phenomena occurring on the cell membrane surface. The total surface concentrations of both acidic and basic functional groups and their association constants with solution ions were calculated and used to define theoretical curves of membrane surface charge density versus pH. The resulting theoretical curves and the experimental data were compared to verify the reliability and validity of the adopted model. The deviation of both kinds of data obtained at a higher pH may be caused by disregarding interactions between the functional groups of cancer cells. Processes occurring in the cell membranes after their incubation with p-CoA can lead to disorders of existing equilibria, which result in changes in values of the parameters describing these equilibria.

Photoisomerization of Bis(tridentate) 2,6-Bis(1H-pyrazol-1-yl)pyridine Ligands Exhibiting a Multi-anthracene Skeleton.

A novel molecular design is described where two peripheral moieties made of 2,6-bis(1H-pyrazol-1-yl)pyridine are linked through multi-1,8-diethynylanthracene moieties. The optimized synthesis of the three isostructural analogues 1 a, 1 b, and 1 c, containing the anthraquinone, anthracene, and 10-methoxyanthracene units, respectively, is reported. The resulting spatial face-to-face arrangement of the peripheral anthracene rings enables to trigger the intramolecular [4+4] photocycloaddition affording the isomers P1 b and P1 c, which can be thermally cleaved back to the original anthracene derivatives 1 b and 1 c, respectively. Single-crystal X-ray diffraction studies confirm the expected molecular structures of compounds 1 a-1 c as well as of their corresponding isomers P1 b and P1 c. The spectral, optical, and electrochemical properties of all synthesized compounds are investigated and discussed.

Wastewater analysis: the mean of the monitoring of frequently prescribed pharmaceuticals in Slovakia.

The estimation of medication use is based on the statistical data from pharmacies and hospitals. Excessive use or misuse of some compounds, especially psychoactive medications, has not yet been monitored in Slovakia. Wastewater analysis provides useful data about the medication use and misuse in individual regions. This study is focused on the analysis of 23 substances in the wastewaters of Slovakia. The monitoring programme has included stimulants, opioid and morphine derivatives, benzodiazepines, antidepressants, drug precursors and their metabolites. Urinary markers of these compounds were analysed at WWTP influent in seven regions (Bratislava, Kosice, Zvolen, Banská Bystrica, Trencín, Presov a Piestany) using LC-MS/MS technique. The analysis was performed from March to October 2013. The pattern in use of these compounds was also monitored. Tramadol and venlafaxine were found to be the most concentrated compounds among of all studied psychoactive pharmaceuticals. The highest specific loads of tramadol were detected in Piestany (409 mg/day/1000 inhabitants) and Zvolen (366 mg/day/1000 inhabitants). There is a considerable number of spa facilities (hotels) situated in these cities and this fact contributes to a higher occurrence of these psychoactive compounds in respective wastewaters.

Degradation of Chemical Warfare Agent Nitrogen Mustard Using Ferrate (VI).

Chemical warfare agents (CWAs) are one of the most toxic compounds. Degradation of CWAs using decontamination agents is one of the few ways to protect human health against the harmful effects of CWAs. A ferrate (VI)-based potential chemical warfare agent decontaminant was studied for the degradation of persistent nitrogen mustard (tris(2-chloroethyl)amine, HN3). By optimizing the reaction conditions, the complete degradation of HN3 was achieved in 4 min. The degradation products contained mostly reduced Fe species, which confirmed the environmental friendliness of the proposed decontamination solution.

Direct electrochemical determination of environmentally harmful pharmaceutical ciprofloxacin in 3D printed flow-through cell.

Rising amounts of antibiotic residues in wastewater cause serious problems including increased bacterial resistance. Wastewater treatment plants (WWTPs) do not, in the case of new, modern pharmaceuticals, ensure their complete removal. Ciprofloxacin (CIP) is one of many micropollutants that partially pass through WWTPs, implying that its monitoring is essential for the assessment of the water quality. In real sewage systems, the determination of CIP needs to be performed under flowing conditions, which calls for the deployment of inexpensive, robust, and easily integrable approaches such as electrochemical techniques. However, to the best of our knowledge, there is no report on the electrochemical determination of CIP in a flowing matrix. To bridge this gap, we perform here cyclic and square-wave voltammetric sensing study of CIP employing boron-doped diamond screen printed electrodes in a custom-made 3D printed flow-through cell to mimic conditions in real sewage systems. An irreversible two-step oxidation of CIP is demonstrated, with the first step providing clear Faradaic response as analytically relevant signal. This response was found to scale with the sample flow rate according to the prediction given by Levich equation. Our work provides an in-depth inspection of the electrochemical response of CIP under controlled-convection conditions, which is an essential prerequisite for monitoring this antibiotic in real flowing sewage systems.

On the stability of the bioactive flavonoids quercetin and luteolin under oxygen-free conditions.

The natural flavonoid compounds quercetin (3,3',4',5,7-pentahydroxyflavone) and luteolin (3',4',5,7-tetrahydroxyflavone) are important bioactive compounds with antioxidative, anti-allergic, and anti-inflammatory properties. However, both are unstable when exposed to atmospheric oxygen, which causes degradation and complicates their analytical determinations. The oxidative change of these flavonoids was observed and followed by UV-visible spectrophotometry, both in aqueous and ethanolic solutions. The distribution of the degradation products in aqueous media was monitored by LC-MS and LC-DAD analysis. The amounts of oxidative reaction products increase with the exposure time. The oxidative degradation reduces the pharmacological efficiency of these antioxidants and renders analytical determination inaccurate. The oxidative changes in flavonoid test solutions can explain the inconsistent dissociation constants reported in the literature. Dissociation constants of quercetin and luteolin were determined both by alkalimetric titration and by UV-visible spectrophotometry under deaerated conditions. The values pK(1) = 5.87 ± 0.14 and pK(2) = 8.48 ± 0.09 for quercetin, and pK(1) = 5.99 ± 0.32 and pK(2) = 8.40 ± 0.42 for luteolin were found.

The influence of the pH on the incorporation of caffeic acid into biomimetic membranes and cancer cells.

Caffeic acid (CA) is a phenolic compound synthesized by all plant species. It constitutes the main hydroxycinnamic acid found in human diet and presents a variety of beneficial effects including anticancer activity. Current data suggests essential role of the interplay between anticancer drugs and the cell membrane. Given this, biophysical interactions between CA and cancer cells or biomimetic membranes were investigated. Glioblastoma cell line U118MG and colorectal adenocarcinoma cell line DLD-1, as well as lipid bilayers and liposomes, were used as in vitro models. Electrophoretic light scattering was used to assess the effect of CA on the surface charge of cancer cells and liposomal membranes. Electrochemical impedance spectroscopy was chosen to evaluate CA-dependent modulatory effect on the electrical capacitance and electrical resistance of the bilayers. Our results suggest that CA fulfills physicochemical criteria determining drug-like properties of chemical compounds, and may serve as a potential cytostatic agent in cancer treatment.

Ferrate (VI), Fenton Reaction and Its Modification: An Effective Method of Removing SARS-CoV-2 RNA from Hospital Wastewater.

The outbreak of the coronavirus disease 2019 (COVID-19) raises questions about the effective inactivation of its causative agent, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in medical wastewater by disinfectants. For this reason, our study of wastewater from a selected hospital evaluated several different advanced oxidation methods (Fenton reaction and Fenton-like reaction and ferrate (VI)) capable of effectively removing SARS-CoV-2 RNA. The obtained results of all investigated oxidation processes, such as ferrates, Fenton reaction and its modifications achieved above 90% efficiency in degradation of SARS-CoV-2 RNA in model water. The efficiency of degradation of real SARS-CoV-2 from hospital wastewater declines in following order ferrate (VI) > Fenton reaction > Fenton-like reaction. Similarly, the decrease of chemical oxygen demand compared to effluent was observed. Therefore, all of these methods can be used as a replacement of chlorination at the wastewater effluent, which appeared to be insufficient in SARS-CoV-2 removal (60%), whereas using of ferrates showed efficiency of up to 99%.

Electron dopable molecular wires based on the extended viologens.

Facile electron transfer in molecules with one dimension greatly exceeding the other two is essential in the development of new molecular electronic devices as these molecules can serve as so-called molecular wires. In this communication the electrochemical behavior of a series of molecules with multiple extended viologen moieties has been studied. We show that the electron transfer in the shortest wire is due to reduction of two identical communicating pyridinium moieties leading to a full charge delocalization, whereas the electron transfer in molecules with n≥ 2 is due to reduction of initially non-communicating centers. This was confirmed by digital simulation of cyclic voltammograms. All studied molecules accept reversibly at least four and up to ten electrons without any long-term chemical changes, which is a prerequisite for their future application. Chemical stability of these molecules after multiple electron transfer was confirmed by in situ UV-Vis spectroelectrochemical detection.

Extended viologen as a source of electric oscillations.

A long organic molecule 1 with five bipyridinium functions separated by benzene rings (extended viologen) undergoes a reversible multi-step electron transfer. Here we show that this decacation accepts electrons at the heterogeneous interface with the occurrence of the periodically changing electric reduction currents. According to the applied bias voltage the observed current-time dependence changes from chaotic through periodic and irregular to sinusoidal and finally to monotonous. A careful choice of the controlling parameters yields the sustained periodic sinusoidal currents lasting for a prolonged time. Oscillations stem from a mutual interplay of the heterogeneous supply of electrons and the homogeneous redox reactions (disproportionation) between the transient redox forms. In difference to many other electrochemical oscillating systems the described oscillations do not require any additional external impedance. The principle of these oscillatory currents may serve as a model of a truly 'molecular oscillator'.

Wastewater-Based Epidemiology as an Early Warning System for the Spreading of SARS-CoV-2 and Its Mutations in the Population.

New methodologies based on the principle of "sewage epidemiology" have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.

Hospital Wastewater-Source of Specific Micropollutants, Antibiotic-Resistant Microorganisms, Viruses, and Their Elimination.

Municipal wastewaters can generally provide real-time information on drug consumption, the incidence of specific diseases, or establish exposure to certain agents and determine some lifestyle consequences. From this point of view, wastewater-based epidemiology represents a modern diagnostic tool for describing the health status of a certain part of the population in a specific region. Hospital wastewater is a complex mixture of pharmaceuticals, illegal drugs, and their metabolites as well as different susceptible and antibiotic-resistant microorganisms, including viruses. Many studies pointed out that wastewater from healthcare facilities (including hospital wastewater), significantly contributes to higher loads of micropollutants, including bacteria and viruses, in municipal wastewater. In addition, such a mixture can increase the selective pressure on bacteria, thus contributing to the development and dissemination of antimicrobial resistance. Because many pharmaceuticals, drugs, and microorganisms can pass through wastewater treatment plants without any significant change in their structure and toxicity and enter surface waters, treatment technologies need to be improved. This short review summarizes the recent knowledge from studies on micropollutants, pathogens, antibiotic-resistant bacteria, and viruses (including SARS-CoV-2) in wastewater from healthcare facilities. It also proposes several possibilities for improving the wastewater treatment process in terms of efficiency as well as economy.

Mathematical modeling based on RT-qPCR analysis of SARS-CoV-2 in wastewater as a tool for epidemiology.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges to scientific research and monitoring of wastewaters to predict the spread of the virus in the community. Our study investigated the COVID-19 disease in Bratislava, based on wastewater monitoring from September 2020 until March 2021. Samples were analyzed from two wastewater treatment plants of the city with reaching 0.6 million monitored inhabitants. Obtained results from the wastewater analysis suggest significant statistical dependence. High correlations between the number of viral particles in wastewater and the number of reported positive nasopharyngeal RT-qPCR tests of infected individuals with a time lag of 2 weeks/12 days (R2 = 83.78%/R2 = 52.65%) as well as with a reported number of death cases with a time lag of 4 weeks/27 days (R2 = 83.21%/R2 = 61.89%) was observed. The obtained results and subsequent mathematical modeling will serve in the future as an early warning system for the occurrence of a local site of infection and, at the same time, predict the load on the health system up to two weeks in advance.

Effervescent ferrate(VI)-based tablets as an effective means for removal SARS-CoV-2 RNA, pharmaceuticals and resistant bacteria from wastewater.

Waterborne pathogens including viruses, bacteria and micropollutants secreted from population can spread through the sewerage system. In this study, the efficiency of unique effervescent ferrate-based tablets was evaluated for total RNA and DNA removal, disinfection and degradation of micropollutants in hospital wastewater. For the purpose of testing, proposed tablets (based on citric acid or sodium dihydrogen phosphate) were used for various types of hospital wastewater with specific biological and chemical contamination. Total RNA destruction efficiency using tablets was 70-100% depending on the type of acidic component. DNA destruction efficiency was lower on the level 51-94% depending on the type of acidic component. In addition, our study confirms that effervescent ferrate-based tablets are able to efficiently remove of SARS-CoV-2 RNA from wastewater. Degradation of often detected micropollutants (antiepileptic, antidepressant, antihistamine, hypertensive and their metabolites) was dependent on the type of detected pharmaceuticals and on the acidic component used. Sodium dihydrogen phosphate based tablet appeared to be more effective than citric acid based tablet and removed some pharmaceuticals with efficiency higher than 97%. Last but not least, the disinfection ability was also verified. Tableted ferrates were confirmed to be an effective disinfectant and no resistant microorganisms were observed after treatment. Total and antibiotic resistant bacteria (coliforms and enterococci) were determined by cultivation on diagnostic selective agar growth media.

On the adsorption of extended viologens at the electrode|electrolyte interface.

Extended viologens represent a group of organic molecules intended to be used as molecular wires in molecular electronic devices. Adsorption properties of a novel series of extended viologen molecules were studied at the mercury electrode|electrolyte interface. These compounds form compact monolayers around the potential of zero charge with a constant differential capacitance value of 2.5 ± 0.2 µF cm(-2) independent of temperature, length of the molecule, and its bulk concentration. At more negative potentials their reduction in the adsorbed state takes place. We showed that the adsorption process is diffusion controlled and time needed to fully cover the electrode surface is independent of the electrode potential. A modified Koryta equation was employed for the calculation of the surface concentration of the adsorbates leading to the value of 5.3 × 10(-11) mol cm(-2) for the shortest wire and to 1.6 × 10(-11) mol cm(-2) for the longest one. Based on the space filling model and the differential capacitance value in the compact film region, it was postulated that these molecules lay flat on the electrode surface.