Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide, with critical implications for agricultural nitrogen losses and clinical infections caused by Helicobacter pylori. Despite the relevance of monitoring urease activity in both contexts, no paper-based platform has yet systematically compared multiple acid-base indicators combined with polymer surface modifiers across analytically distinct biological and environmental matrices. To address this gap, this study introduces a paper-based analytical device (PAD) that, for the first time, systematically evaluates five acid-base indicators (phenol red (PR), bromothymol blue (BTB), bromocresol purple (BCP), m-cresol purple (mCP), and neutral red (NR)) combined with PSS and PVP surface modifiers on CHR 3MM chromatographic paper for colorimetric urease activity determination. Following multiparameter optimization, the PAD achieved detection limits of 0.15–0.20 U mL−1 and a linear range of 0.25–5.0 U mL−1, with analysis completed within 5 min and RSD ≤ 5.31% (at 0.25 U mL−1) and ≤ 2.28% (at 4.0 U mL−1). Results were validated against the indophenol reference method, with analytical recoveries of 85–103% in saliva matrices, urease quantification in soil extracts (4.0–8.9 μmol N-NH₄+ g−1), and IC₅₀ determination for four classical inhibitors (NBPT, AHA, HU, and TU). As a proof of concept, the PAD differentiated urease activity in saliva between H. pylori-positive and -negative individuals (n = 8) and detected ureolytic activity in Proteus mirabilis and Cryptococcus neoformans. The results demonstrate that a single, low-cost PAD platform can cover the analytical needs of urease activity screening across soil, clinical, and microbiological matrices, offering a meaningful advance over existing single-indicator or single-matrix paper-based approaches.

The identification of biological fluids is a crucial step in forensic investigations. Semen is the most relevant bodily fluid in cases of sexual assault. Semen composition includes (bio)chemical components, such as the enzyme acid phosphatase (AP) and reduced ascorbic acid (AAᵣ), both of which are considered important markers of seminal fluid. In this work, a colorimetric kit was developed for the dual detection of AP and AAᵣ. For AP detection, ascorbic acid 2-phosphate (AAP) was employed as a substrate; in the presence of the enzyme (pH 5), it is hydrolyzed to inorganic phosphate and AAᵣ, which were subsequently used to monitor enzymatic activity. The AAᵣ, in the presence of Fe(III) and K₃Fe(CN)₆ under acidic conditions, leads to the formation of Prussian Blue (PB, Fe₄[Fe(CN)₆]₃), a dark blue colloid (λ = 710 nm). Linear range of 0.25-3.0 U L-1 was obtained for AP detection, with a limit of detection (LOD) of 0.04 U L-1. Similarly, AAᵣ could be quantified, yielding a linear range of 5-40 μM and LOD of 0.29 μM. Recovery assays (AAᵣ and AP) demonstrated quantitative performance, with recoveries ranging from 81 to 101% (RSD < 4%). The kit was qualitatively evaluated against potential interferents (biological fluids and food samples), with sensitivity, specificity, and efficiency exceeding 88%. The proposed kit demonstrated applicability both at crime scenes (in loco) and in laboratory environments, enabling rapid screening and supporting forensic decision- making.

Contamination by potentially toxic metals, such as mercury (Hg) and its derivatives, poses a serious risk to human health and the environment. Phenylmercury (PM) is an organometallic compound used as a fungicide, the toxicity of which has been little explored. This study investigated the effects of PM at three levels of complexity: protein (human hemoglobin [Hb]), cell (human erythrocytes [Ery]), and animal (zebrafish [Danio rerio]). Theoretical and biophysical studies indicated that PM interacts with the heme group and cysteine residues of Hb, with a binding constant (Kb) of 1.47(± 0.22)× 10⁴ M⁻¹ , suggesting the formation of a stable Hb-PM complex. The PM capacity to bind Hb cysteines, changes to the Soret band, and reduced protein electrophoretic mobility. In Ery exposed to PM (0.125-1.0 μM), oxygen uptake was reduced by 48%. The production of reactive oxygen species (ROS) increased 2.2-fold, with a 2-fold increase in H2O2 levels and a 90% increase in •NO production. Catalase and glutathione peroxidase activities decreased by 25% and 49%, respectively, while superoxide dismutase increased by 13%. The GSH/GSSG ratio and total sulfhydryl group content concentration in 34% and 44%, respectively, while carbonylated content increased 2.2-fold. PM caused structural changes in the Ery membrane, morphology, and elasticity. Finally, in vivo zebrafish assays demonstrated that PM has teratogenic effects and reduced heart rate. Therefore, PM exhibits toxicity at the three complexity levels evaluated: it compromises the structure and function of Hb and the redox system of Ery and causes marked changes in zebrafish development.

Urease, a metalloenzyme that catalyzes urea hydrolysis, is associated with nitrogen losses in soils. In agricultural settings, although the commercial inhibitor N-(butyl)thiophosphoric triamide (NBPT) is widely used, its low stability under adverse environmental conditions has prompted the search for new, more selective, and stable inhibitors. In this study, triphenylphosphine derivatives functionalized with chalcogens (SF1-SF3) and the corresponding precursor (SF4) were investigated for the ability to transiently inhibit urease catalytic activity. These compounds were tested against urease from Canavalia ensiformis and against soil with varying physicochemical properties. Classic kinetic assays and biophysical studies of urease-ligand interactions were carried out to investigate the mechanisms of urease inhibition. Even in the presence of humic substances, the selenium-containing derivative SF3 was the most effective urease inhibitor among the tested compounds, regardless of soil type. SF3 works as a typical uncompetitive inhibitor, likely by interacting with free cysteine residues located in the flap region near the active site. In situ spectroscopic evidence shows that SF3 may react with cysteine residues to form SF1 and H2Se. Molecular fluorescence approaches demonstrated that SF3 spontaneously interacts with urease and with urease-SF3 via static quenching, driven by electrostatic interactions. These findings highlight SF3 as a promising candidate for application as a urease inhibitor in enhanced-efficiency fertilizers.

Acid phosphatase (AP) is an enzyme present in various tissues of the human body and is found in high concentrations in seminal fluid. Therefore, it can be easily used as a forensic marker for the presence of semen, assisting criminal investigations and clinical diagnostic tests. This work developed a simple colorimetric method for detecting AP in semen using a probe derived from an azo dye. The proposed method utilizes (E)-4-(phenyldiazenyl)phenyldihydrogen phosphate (AZO1-P) as a substrate, which, upon enzymatic hydrolysis, yields the yellow-colored (E)-4-(phenyldiazenyl)phenol (AZO1) (lambda max = 423 nm). However, for applications involving complex matrices, this coloration is not ideal due to low colorimetric contrast. Thus, a coupling reaction was performed between AZO1 and 4-aminoantipyrine (4-AAP) to improve colorimetric resolution. The product generated provided greater colorimetric contrast (red color, lambda max = 516 nm) and was easily visualized. Under ideal conditions, the method exhibited a linear range of 1-6 U L- 1 for AP, with a detection limit of 0.42 U L- 1 and a relative standard deviation (RSD) of 3 %. Classical AP inhibitors, such as sodium tungstate and molybdate, were evaluated and showed IC50 values of 11.7 +/- 0.5 and 2.9 +/- 0.7 mM, respectively. In addition, the method was applied to real samples and against possible interferents that can be found at sex crime scenes, proving to be sensitive and selective for detecting AP in semen (from 1000-fold dilution). Finally, the proposed method can be used for the quantitative and qualitative detection of AP in the laboratory and the field as an alternative to presumptive tests in forensic sciences associated with sex crimes.

Assessing antibiotic contamination presents significant challenges, as the distribution and concentration of these compounds can be altered upon interaction with various chemical entities in the environment. Organic matter (OM) fractions are ubiquitous in the biosphere and play a crucial role in the biogeochemistry of ecosystems. This study, therefore, investigates the interaction between sulfaquinoxaline (SQX) and OM fractions using spectroscopic and theoretical approaches, simulating environmental conditions. The species under investigation formed a supramolecular complex in the ground state, with a binding constant (K-b) of 2.97 x 10(3) M-1(37 degrees C). SQX predominantly interacts with humic substances (HS) through hydrophobic forces (Delta H > 0, Delta S > 0), which agrees with theoretical predictions. Moreover, electrostatic interactions were influenced by ionic strength during the process. Structural alterations in native HS were observed through 3D and synchronized fluorescence spectroscopy, primarily attributed to changes in the protein-like region of the superstructure. The impact of metal ions on the interaction was also evaluated, with the ions Cu(II), Al(III), and chromium species influencing the complex formation. 1H NMR results facilitated the proposal of the ligand epitope associated with the interaction process. Overall, this work contributes to a deeper understanding of the environmental implications of antibiotic contamination in soils, particularly concerning the behavior of SQX in the presence of organic matter and metal ions.

Tetracycline (TC) is a widely used antibiotic, and evaluating its interaction with humic substances (HS) that act as a complexing agent in the environment is essential to understanding the availability of this contaminant in the environment. This study evaluated the interaction between HS and TC using different spectroscopic techniques, theoretical studies, and biological assays simulating environmental conditions. TC interacts with HS, preferably by electrostatic forces, with a binding constant of 9.2 × 103 M−1 (30 °C). This process induces conformational changes in the superstructure, preferably in the HS, like protein fraction. Besides, studies using the 8-anilino-1-naphthalene sulfonate (ANS) probe indicated that the antibiotic alters the hydrophobicity degree on HS’s surface. Synchronized fluorescence shows that the TC interaction occurs preferentially with the protein-like fraction of soil organic matter (KSV = 26.28 ± 1.03 M−1). The TC epitope was evaluated by 1H NMR and varied according to the pH (4.8 and 9.0) of the medium, as well as the main forces responsible for the stabilization of the HS-TC complex. The molecular docking studies showed that the formation of the HS-TC complex is carried out spontaneously (ΔG = −7.1 kcal mol−1) and is stabilized by hydrogen bonds and electrostatic interactions, as observed in the experimental spectroscopic results. Finally, biological assays indicated that HS influenced the antimicrobial activity of TC. Thus, this study contributed to understanding the dynamics and distribution of TC in the environment and HS’s potential in the remediation of antibiotics of this class in natural systems, as these can have adverse effects on ecosystems and human health.

In this study, we report the synthesis of mixed metal Eu3+/Sb3+ layered europium hydroxides (LEuH:x%Sb (with x ranging from 0 to 30%)) using the hydrothermal route. Our research provides a detailed analysis of the chemical, structural, thermal, and morphological characteristics of this class of materials. The results of this study demonstrated slight changes in the structure for antimony fractions greater than 20%, modification in thermal stability, and significant morphological modification due to the increase in the incorporation of Sb3+ into the LEuH-Cl host. Furthermore, we comprehensively investigated the photophysical properties of the material, describing its unique luminescent characteristics. These results showed increased luminescence of Eu3+ ions promoted by the energy transfer between Sb3+ → Eu3+. This study contributes valuable information on the structural, thermal, and photophysical properties of layered hydroxide materials containing europium and antimony, offering insights into the design of functional materials of this class for use as efficient chemical sensors, catalysis systems, and platforms for drug delivery.

The current investigation encompasses the structural planning, synthesis, and evaluation of the urease inhibitory activity of a series of molecular hybrids of hydroxamic acids and Michael acceptors, delineated from the structure of cinnamic acids. The synthesized compounds exhibited potent urease inhibitory effects, with IC50 values ranging from 3.8 to 12.8 mu M. Kinetic experiments unveiled that the majority of the synthesized hybrids display characteristics of mixed inhibitors. Generally, derivatives containing electron-withdrawing groups on the aromatic ring demonstrate heightened activity, indicating that the increased electrophilicity of the beta carbon in the Michael Acceptor moiety positively influences the antiureolytic properties of this compounds class. Biophysical and theoretical investigations further corroborated the findings obtained from kinetic assays. These studies suggest that the hydroxamic acid core interacts with the urease active site, while the Michael acceptor moiety binds to one or more allosteric sites adjacent to the active site.

In this study, HSA was used as a transport protein model to simulate the transport dynamics and availability of cocaine (COC) and its pharmacologically active metabolite, cocaethylene (CE). In the interaction studies between the alkaloids and HSA, the binding constant (Kb) b ) values were higher for CE (2.92 to 8.33 x 104 4 M- 1 ) than COC (1.15 to 3.77 x 104 4 M- 1 ) at different temperatures. Thermodynamic parameter calculations indicate that COC preferentially interacts through van der Waals forces and hydrogen bonds, while CE interacts hydrophobically. Competition studies using the ANS probe confirmed the more hydrophobic characteristics of CE. Synchronized fluorescence indicated that both compounds preferentially interact in the microregion of the Trp214 residue. Based on 3D fluorescence, UV-vis, and circular dichroic results, changes in the secondary structure of the protein were confirmed. Studies to assess the HSA binding site were carried out using warfarin (site I), diazepam (site II), and digitoxin (subdomain IIIB) as markers, and it was verified that both compounds interact preferentially at the site I. Finally, based on the 1 H NMR and theoretical studies, it was possible to propose the alkaloid epitope in the interaction with HSA. In conclusion, when compared to COC, CE presented greater HSA protein affinity, justifying its longer plasma lifetime and increasing the drug's effects on the body.

Background: Thimerosal (TM) is a toxic, organometallic mercury compound (which releases ethyl-mercurycontaining compounds in aqueous solutions) used as a preservative in vaccines. Mitochondria are organelle which are highly vulnerable to many chemical compounds, including mercury (Hg) and its derivatives. Method: Wistar rats (at 21 days of age) were used to model a child's TM exposure following childhood vaccination, divided in two groups: TM exposed (20 mu g/kg/day) and unexposed controls (saline solution), both for 24 h. Atomic Fluorescence Spectrometry was used to quantify the amounts of mercury in tissues. The electron transport chain (ETC) from isolated mitochondria was evaluated using an oxygen electrode. The mitochondrial membrane potential and H2O2 production were analyzed using selective fluorescence probes. The activity of some enzymes (SOD, CAT, GPx, and AChE) and secondary markers of oxidative stress (GSH, GSSG, total free thiol) were also examined in tissues. Results: Hg accumulation in the brain and liver was higher in exposed animals when compared to the control. Liver-isolated mitochondria showed that TM improved respiratory control by 23%; however, states 3 and 4 of the ETC presented a decrease of 16% and 37%, respectively. Furthermore, brain-isolated mitochondria presented an improvement of 61% in respiratory control. Brain enzyme activities were significantly impacted in TM-exposed rats compared to unexposed rats as follows: decreases in SOD (32%) and AChE (42%) and increases in GPx (79%) and CAT (100%). GPx enzyme activity in the liver was significantly increased (37%). Among secondary oxidative stress markers, the brain's total reduced thiol (SH) concentration was significantly increased (41%). Conclusion: Acute TM treatment exposure in a Wistar rat model mimicking TM exposure in an infant following childhood vaccination significantly damaged brain bioenergetic pathways. This study supports the ability of TM exposure to preferentially damage the nervous system.

Thimerosal, a preservative commonly used in the pharmaceutical and cosmetic industry, has raised concerns regarding its potentially toxic effects as an organic mercury compound. Within this context, using an NMR-based metabolomics profile and chemometric analysis, zebrafish embryos were used as an in vivo model to study the effects of thimerosal in metabolic profiles after exposure to sublethal concentrations of the mercury compound. The thimerosal concentrations of 40 and 80 nM were employed, corresponding to 40% and 80% of the LC50, respectively, for zebrafish embryos. The most significant alterations in the metabolic profile included changes in carbohydrates, amino acids, nucleotides, trimethylamine-N-oxide, ethanolamine, betaine, and ethanol. Furthermore, thimerosal exposure affects various metabolic pathways, impairing the nervous system, disrupting protein metabolism, and potentially causing oxidative damage. Therefore, adopting a metabolomics approach in this investigation provided insights into the potentially implicated metabolic pathways contributing to the deleterious effects of thimerosal in biological systems.

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Hemoglobin (Hb) is a hemeprotein found inside erythrocytes and is crucial in transporting oxygen and carbon dioxide in our bodies. In erythrocytes (Ery), the main energy source is glucose metabolized through glycolysis. However, a fraction of Hb can undergo glycation, in which a free amine group from the protein spontaneously binds to the carbonyl of glucose in the bloodstream, resulting in the formation of glycated hemoglobin (HbA1c), widely used as a marker for diabetes. Glycation leads to structural and conformational changes, compromising the function of proteins, and is intensified in the event of hyperglycemia. The main changes in Hb include structural alterations to the heme group, compromising its main function (oxygen transport). In addition, amyloid aggregates can form, which are strongly related to diabetic complications and neurodegenerative diseases. Therefore, this chapter discusses in vitro protocols for producing glycated Hb, as well as the main techniques and biophysical assays used to assess changes in the protein's structure before and after the glycation process. This more complete understanding of the effects of glycation on Hb is fundamental for understanding the complications associated with hyperglycemia and for developing more effective prevention and treatment strategies.

A new automated method was developed combining the features of the salting-out effect, single-phase liquid–liquid extraction (SPLLE), and flow-batch analysis to determine Cr( vi ) in river water samples by ETAAS.

Herein, we report the synthesis and evaluation of four aminoguanidine hydrazone derivatives with different aromatic moieties. This class of compounds presents a series of biological applications. Derivative AGH-3 with an indole nucleus offered the highest antioxidant capacity with results comparable to Trolox in 2,2-diphenyl-2-picrylhydrazyl radical (DPPH• ), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+), FeIII reduction assay (FRAP), and nitric oxide (•NO) radical scavenging assays. Furthermore, AGH-3 showed the highest antiproliferative activity against human kidney cancer cells (786-0) with concentration necessary to inhibit 50% cell growth (GI50) = 6.3 µM; additionally, in biophysical studies, AGH-3 interacted with ctDNA (biological target model) forming a fluorescent supramolecular complex with a binding constant (Kb) of 2.89 × 103 M-1 with preferentially an intercalator mechanism. The tested compounds revealed the potential of aminoguanidine hydrazones as a strategic class of compounds with multitarget biological activity.

Urease is a metalloenzyme that contains two Ni(II) ions in its active site and catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The development of effective urease inhibitors is crucial not only for mitigating nitrogen losses in agriculture but also for offering an alternative treatment against infections caused by resistant pathogens that utilize urease as a virulence factor. This study focuses on synthesizing and investigating the urease inhibition potential of Biginelli Adducts bearing a boric acid group. An unsubstituted or hydroxy-substituted boronic group in the Biginelli adducts structure enhances the urease inhibitory activity. Biophysical and kinetics studies revealed that the best Biginelli adduct (4e; IC50 = 132 +/- 12 mu mol/L) is a mixed inhibitor with higher affinity to the urease active site over an allosteric one. Docking studies confirm the interactions of 4e with residues essential for urease activity and demonstrate its potential to coordinate with the nickel atoms through the oxygen atoms of carbonyl or boronic acid groups. Overall, the Biginelli adduct 4e shows great potential as an additive for developing enhanced efficiency fertilizers and/or for medical applications.

The Mundaú lagoon in Maceió (Alagoas, Brazil) is a crucial resource for the local population, particularly fishing communities. Recent studies have revealed potential toxic metal contamination in the lagoon, particularly with mercury (Hg) levels exceeding the maximum regulated values. This inorganic contaminant may be impacting the health of fishermen and the local population. In this context, metabolomics, a study of small-molecule metabolites, can offer insights into the physiological impact of environmental contamination on humans. Thus, volunteers from the control and exposed groups were selected, considering the main exposure criteria primarily defined by their proximity and interaction with the lagoon. Blood and urine samples were collected from the volunteers and subjected to analysis using NMR spectroscopy. The data underwent Principal Component Analysis (PCA) and Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA) based on metabolic patterns to establish group discrimination or identification. Metabolic pathways were assessed through enrichment analysis. The study revealed several metabolic disturbances in the exposed group's urine and plasma samples compared to control group. Noteworthy findings included arginine and proline metabolism disruptions, indicative of ammonia recycling and urea cycle impairment. These changes suggest compromised ammonia detoxification in the exposed group. Disturbances in the tricarboxylic acid (TCA) cycle and the transfer of acetyl groups into mitochondria suggested systemic metabolic stress in energy metabolism. Furthermore, elevated carnitine and ketone levels may indicate compensatory responses to low TCA cycle activity. Alterations in glutamate and glutathione metabolism and imbalances in glutathione levels indicate oxidative stress and impaired detoxification. This study highlights significant metabolic changes in fishermen exposed to contaminated environments, which can affect various metabolic pathways, including energy metabolism and antioxidant processes, potentially making individuals more vulnerable to the adverse effects of environmental contaminants. Finally, this work highlights insights into the relationship between environmental contamination and metabolic pathways, particularly in regions with limited studies.

Urea is the most widely used nitrogen fertilizer worldwide. However, ammonia volatilization, resulting from applying urea to the soil surface, causes economic and environmental losses; thus, urease inhibitors have been developed to mitigate these losses. In this work, the anti-ureolytic activity of Schiff’s base 4-(3-hydroxybenzylideneamino) phenol (3B4) and its amine-derived (3B4a) was evaluated. The most promising urease inhibitor in soil was 3B4 (55.0 ± 3.9% inhibition), with comparable results to N-(butyl) thiophosphoric triamide (p = 0.659). In the in vitro analysis (Canavalia ensiformis), the results of anti-ureolytic activity were similar, 22.6 ± 6.9% for 3B4 and 24.2 ± 9.6% for 3B4a. Biophysical interaction studies were also carried out through molecular docking studies and molecular fluorescence spectroscopy. These studies showed that both substances are preferentially competitive inhibitors, with the interaction between 3B4a and urease forming a more stable complex. In the analysis by Fourier transform infrared spectroscopy, no interaction was observed when 3B4 or 3B4a was mixed with urea (1:1) for 48 h, providing evidence of compatibility. Thus, the Schiff base 3B4 and its corresponding amine 3B4a may represent potential additives for urea fertilization aiming to assist in the urease inhibition process.

Developing fluorescent probes that exhibit high selectivity and sensitivity for detecting molecules in complex samples poses a significant challenge. In various scientific fields, fluorescent probes are widely employed to obtain qualitative and quantitative information from diverse systems and matrices. This chapter evaluates fluorescent probes used in determining and imaging H2S in samples. It explores a range of reaction mechanisms with H2S and investigates the spectroscopic properties of fluorophore systems. Analytical parameters, particularly sensitivity and selectivity, and potential applications are compared and assessed. Considering the limited availability of commercially viable fluorescent probes for H2S, especially with high selectivity, the scope of these molecular systems should be broadened, particularly for plant tissue applications. Consequently, this chapter also presents the most promising fluorescent probes and their respective organic synthetic routes.