Search Results (593)

Polyvinyl alcohol (PVA) hydrogels modified through radical polymerization under UV irradiation and Ce⁴⁺ ion treatment were investigated as a potential platform for developing highly sensitive biosensors for rapid biochemical oxygen demand analysis in water. These modifications enhance PVA physicochemical properties, including mechanical strength, stability, and biocompatibility, making it promising for immobilizing microorganisms in bioanalytical systems. A dual-mediator biosensor system using ferrocene (FC) and neutral red (NR) was developed with yeast Blastobotrys adeninivorans immobilized in modified PVA. The FC+NR–B. adeninivorans–PVA–Ce⁴⁺ system exhibited high sensitivity (linear range of 0.1–3.81 mgO₂/dm³), selectivity, and operational stability (up to 37 days service life), outperforming existing analogs. Testing with wastewater confirmed strong correlation with standard BOD₅, highlighting the potential for monitoring water quality. The described radical modification method is a simple and effective approach for creating sensitive and stable biosensors. It opens up new possibilities for environmental monitoring technology. Full article

Nanosurface energy transfer (NSET) has emerged as a pivotal mechanism in nanobiophotonics, facilitating the development of highly sensitive biosensors with extended dynamic ranges. Unlike conventional Förster resonance energy transfer, NSET exhibits an inverse fourth-power dependence on distance, enabling quantitative measurements over distances up to 40 nm. This review comprehensively explores the fundamental principles governing NSET, with particular emphasis on non-radiative coupling between fluorescent donors and metallic nanostructures such as gold nanoparticles. Additionally, the applications of these probes are surveyed across various bioanalytical domains, including nucleic acid assays, immunoassays, real-time intracellular monitoring, and various biomolecule detection. Additionally, the evolving integration of NSET, plasmonics, and nanophotonic architectures is discussed, focusing on emerging trends and the trajectory for developing next-generation, multiplexed, and point-of-care diagnostic platforms. Current challenges and prospective pathways for translating these advanced sensing systems into clinical and field-deployable solutions are also considered. Full article

The accurate determination and quantification of praziquantel are essential for optimizing its therapeutic effectiveness in treating schistosomiasis and neurocysticercosis, two significantly neglected tropical diseases. Its challenging physicochemical profile, extensive metabolism, and stereochemical complexity requires robust analytical methods for reliable quantification in clinical, veterinary, and pharmaceutical samples. This review provides a comprehensive and critical evaluation of analytical strategies used for PZQ determination, spanning fluorometric and radiometric assays, HPLC–UV, LC–MS, LC–MS/MS, and enantioselective chromatographic approaches. Particular emphasis is placed on the evolution toward highly sensitive LC–MS/MS methods and their alignment with contemporary regulatory expectations, including ICH M10 requirements. These advancements have significantly improved sensitivity, specificity, and reproducibility, which are crucial for pharmacokinetic, pharmacodynamic, and bioequivalence studies. Enantioselective methods for distinguishing PZQ enantiomers and metabolites are discussed. The aim of these innovations is to increase praziquantel bioavailability, improve patient adherence, and support its continued use in mass drug administration programs. Finally, the review highlights implementation challenges in resource-limited settings and proposes analytical models to expand global bioanalytical capacity. Together, these insights provide a structured foundation for selecting and developing high-quality, regulatory-compliant analytical methods for PZQ. Full article

In the last half-century, capillary gel electrophoresis (CGE) became a versatile and high-performance analytical platform for the separation of complex biomolecular mixtures featuring rapid separations, high efficiency, and small sample consumption. Integrating a pore-size gradient mechanism in CGE makes it possible to achieve enhanced selectivity of polyionic macromolecules such as SDS-proteins and nucleic acids. This review provides a comprehensive overview of the theoretical foundations and operational principles of capillary pore-size gradient gel electrophoresis (CGGE), including the physicochemical basis of gradient formation, the influence of pore-size distributions on analyte mobility, and the challenges of generating stable, reproducible gradients in narrow-bore capillaries. Instrumental considerations such as capillary surface treatment, gradient filling and polymerization strategies, temperature and voltage control, detection modalities, and method-development frameworks are discussed in detail, emphasizing their critical impact on analytical performance and reproducibility. Key application areas in bioanalytical chemistry are highlighted, covering nucleic acid analysis and peptide/protein characterization. CGGE offers unique analytical advantages where fine molecular discrimination, tunable selectivity, and high resolution in a broad molecular weight range are required. Full article

Background/Objectives: Given the clinical limitations of azathioprine (AZA) in treating inflammatory bowel disease, this study developed an AZA-loaded microbiota-modulating and colon-targeted nanoparticle constructed from pectin, Zein, and Eudragit^®S100 (APZE), which was hypothesized to enhance efficacy while reducing toxicity. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to simultaneously quantify AZA and its metabolites, enabling the investigation of the pharmacokinetic and microbial metabolism differences between APZE and AZA suspension (AZAS). Methods: APZE was characterized, and an LC-MS/MS method was developed for quantifying AZA and its metabolites in multiple matrices. Given the potential of APZE for colon targeting and modulation of the microbiota, which may affect drug absorption, distribution, and microbiota-mediated metabolism, we determined analyte concentrations in rat plasma, tissues, and microbial cultures at different time points following administration of APZE or AZAS. Results: AZA, 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanine (6-TG) were quantified in positive ion mode, and 6-thiouric acid (6-TU) in negative ion mode. The assay demonstrated excellent accuracy, precision, and stability over the concentration range of 5–1000 ng/mL. Orally administered APZE exhibited higher bioavailability, improved intestinal absorption, and reduced formation of the inactive metabolite 6-TU compared to AZAS. In microbial cultures, AZA was metabolized primarily to 6-MP, and APZE underwent more extensive metabolism to 6-MP than AZAS. Conclusions: This method provides accurate and precise quantification of physiologically relevant concentrations of AZA and its metabolites (6-MP, 6-MMP, 6-TG, and 6-TU), offering a bioanalytical tool for the pharmacokinetic and gut microbiota metabolism studies of AZA formulations. These findings suggest that APZE is a promising drug delivery formulation. Full article

Low-flow liquid chromatography has become the primary tool for advanced chromatographic analysis and is an indispensable technique for the sensitive detection of biomolecules. In this study, we developed a new 4-tritylphenyl methacrylate-based monolithic nano-column with an internal diameter of 20 µm for bioanalytical separations in nano-liquid chromatography (nano-LC). The composition of the monolith was optimized with regard to the monomer and porogenic solvent. The column was characterized using Fourier Transformed Infrared Spectroscopy (FT-IR) spectroscopy, scanning electron microscopy (SEM) and chromatographic analyses. Chromatographic characterization was performed using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), which facilitate hydrophobic and π–π interactions. Run-to-run and column-to-column reproducibility values were found to be <2.51% and 2.4–3.2%, respectively. The final monolith was then used to separate six standard proteins, including β-lactoglobulin A, carbonic anhydrase, ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C) and myoglobin (Myo), as well as three dipeptides: Alanine-tyrosine (Ala-Tyr), Glycine-phenylalanine (Gly-Phe) and L-carnosine. The nano-column was then applied to profiling peptides and proteins in the MCF-7 cell line, enabling high-resolution peptide analysis. Full article

There is a growing demand for biocompatible, non-toxic nanomaterials with specific functional properties, including catalytic activity. In this study, magnetic iron oxide nanoparticles were synthesized via chemical co-precipitation in the presence of polyethylene glycol (PEG). PEG was used as a coating agent to reduce particle agglomeration. Comprehensive characterization of the synthesized nanocomposites was performed using scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDX) and vibrating sample magnetometry (VSM). SEM studies confirmed the nanosized structure of the particles with an average diameter of 20–60 nm. The saturation magnetization values were 57.37 emu·g⁻¹ for nFe₃O₄-PEG6000, 11.95 emu·g⁻¹ for nFe₃O₄-PEG4000 and 3.97 emu·g⁻¹ for nCo_0.5Ni_0.5Fe₂O₄-PEG4000. In addition to their high magnetic properties, ferrofluids exhibited peroxidase-like activity, which makes them highly suitable for bioanalytical and biomedical use. The Michaelis–Menten constant (K_M) for hydrogen peroxide ranged from 1.15 to 4.98 mM. Transmission electron microscopy (TEM) proved the penetration of the nano-ferrofluids into the yeast cells of Ogataea polymorpha. The studied nano-ferrofluids were found to be non-toxic at concentrations up to 0.2 mg·mL⁻¹ for both prokaryotic and eukaryotic cells, showing no inhibitory effect on the growth of the bacterium Escherichia coli, the yeast Ogataea polymorpha, or animal and human cell lines. These results indicate that the advantages of synthetic nano-ferrofluids—including peroxidase-like activity, strong magnetic properties, cost-effective synthesis, stability, and low toxicity—make the synthesized nano-ferrofluids highly promising for future biomedical and bioanalytical applications. Full article

Background: Ovarian cancer remains one of the leading causes of cancer-related mortality among women and constitutes a major unmet medical need. A common treatment-limiting factor for ovarian cancer patients is resistance to Poly(ADP-ribose) polymerase (PARP) inhibitors such as olaparib. Resistance mechanisms include restoration of functional homologous recombination repair, replication fork protection, PARP1 mutations, and increased drug efflux or metabolism. Understanding these cellular and molecular mechanisms is essential for developing more effective therapeutic strategies and improving patient outcomes. Methods: In this study, patient-derived ovarian cancer cells (OC12) in which resistance to olaparib was induced by exposing the cells to increasing concentrations of the drug over multiple treatment cycles were investigated. To compare intracellular olaparib levels in sensitive and resistant cell lines, a UPLC-MS/MS method to quantify olaparib in the range of 1–300 ng/mL was developed. Results: The method was validated for selectivity, calibration curve performance, carryover, dilution integrity, precision, accuracy, matrix effect, and recovery in accordance with ICH M10 guidelines for bioanalytical method validation. Our findings revealed no significant difference in olaparib levels between resistant and sensitive OC12 cells, excluding the involvement of efflux transporters or enhanced metabolism of olaparib in the resistant OC12 ovarian cancer cells. Conclusions: These results shift the future focus toward pharmacodynamic factors as key drivers of olaparib resistance in OC12 cells. Taken together, the developed UPLC-MS/MS analytical method can be successfully applied to quantify intracellular olaparib levels and investigate the potential contribution of drug efflux mechanisms or increased metabolic activity in cells resistant to olaparib treatment. Full article

Background: Nintedanib (NIN), an intracellular inhibitor of tyrosine kinases that inhibits processes fundamental to the progression of pulmonary fibrosis (PPF), is used in the treatment of patients with PPF associated with systemic sclerosis. During NIN therapy, adverse events lead to a permanent dose reduction and treatment discontinuation. Therapeutic drug monitoring (TDM) can be used to manage and optimize drug administration based on the measurement of drug concentrations. Therefore, TDM can be helpful in minimizing the impact of adverse events and help patients remain in therapy. The aim of this study was to develop and validate a new bioanalytical UPLC-MS/MS method enabling the determination of NIN and its active metabolite in the plasma of patients with PPF associated with systemic sclerosis. Methods: Sample preparation was carried out using protein precipitation with an extraction mixture: acetonitrile neutralized with 2 M sodium carbonate. Analytes and the internal standard (intedanib-d3) were monitored using mass spectrometry (MS) and positive-ion-mode electrospray ionization by MRM. Chromatographic analysis was performed on a Zorbax SB-C18 column kept at 40 °C using isocratic elution. The mobile phase contained 0.1% formic acid in water; acetonitrile (35:65 v/v) was pumped at a flow rate of 0.3 mL/min. The analysis time was 5 min. Results: The method was verified according to the EMA guidelines over a concentration range of 2.00–200.00 ng/mL. The correlation coefficients for the calibration curves were found to be 0.9991 and 0.9957 for NIN and its metabolite BIBF 1202, respectively. The within- and between-run precision and accuracy of LLOQ were evaluated for NIN and BIBF 1202 to be within RSD 2.96%, 4.53%, 5.51%, and 6.72% and in the ranges of 102.2–107.3%, 98.0–101.8%, 104.3–114.2%, and 99.1–104.9, respectively. The stability of the analytes in plasma after 4 h at 30 °C was found to be satisfactory, meeting the assumed bias criteria below 15%. Conclusions: The proposed method was successfully applied to analyze two active compounds—NIN and BIBF 1202—in plasma samples at two time points: trough (pre-dose concentration) and 2–3 h (maximum concentration) after the administration of NIN. Full article

Accurate and sensitive determination of hormones in biological matrices is essential for clinical diagnostics, therapeutic monitoring, and endocrine research. However, hormone determination presents significant challenges due to their typically low concentrations, complex sample matrices, and structural diversity. In recent years, microextraction techniques have emerged as strategic tools in bioanalytical chemistry, offering advantages in terms of miniaturization, enhanced selectivity, and compatibility with the principles of green analytical chemistry (GAC). This review provides a comprehensive overview of green and emerging microextraction approaches for the determination of steroidal, thyroid, peptide, and other hormones in biological samples. Key techniques such as solid-phase microextraction (SPME) and dispersive liquid–liquid microextraction (DLLME), followed by high-performance liquid chromatography (HPLC) coupled to diode array detectors (DADs) or mass spectrometry (MS), are critically discussed. Special emphasis is placed on the use of environmentally friendly solvents, such as deep eutectic solvents (DESs), supramolecular solvents (SUPRASs), and advanced sorbents including molecularly imprinted polymers (MIPs) and nanostructured magnetic phases. Applications across various bioanalytical matrices (urine, plasma, serum, saliva, tissues…) are examined in terms of sensitivity, selectivity, and validation parameters. Finally, current challenges, method development gaps, and future directions are highlighted to support the continued advancement of sustainable hormone determination in complex biological systems. Full article

Background: Mycophenolic acid (MPA) is frequently used in pediatric renal transplantation as part of immunosuppressive therapy, yet therapeutic drug monitoring (TDM) remains challenging. Accurate monitoring is essential due to MPA’s narrow therapeutic window, variable pharmacokinetics, and high protein binding. This study examined whether saliva could serve as a non-invasive alternative to plasma for measuring MPA exposure. Methods and Results: Concentrations of MPA and its primary glucuronide metabolite (MPAG) were determined in plasma, capillary blood, plasma ultrafiltrate, wet saliva, and dried saliva collected using volumetric absorptive microsampling (VAMS). A novel LC–MS/MS method for quantifying MPA and MPAG in dried saliva collected with the Mitra™ device was developed and validated within a 1–700 μg/L calibration range, demonstrating robust analytical performance. Dried and wet saliva showed high correlation (r = 0.99 and 0.98 for MPA and MPAG, respectively). However, both salivary matrices—dried saliva collected with Mitra™ (vsMPA, vsMPAG) and wet saliva (sMPA, sMPAG)—exhibited poor correlation with unbound (fMPA, fMPAG) and total plasma concentrations (tMPA, tMPAG). A modest, yet positive, correlation was observed between the measured concentrations for the following pairs: sMPA versus fMPA (r = 0.376, p = 0.1036), sMPA versus tMPA (r = 0.305, p = 0.1904), sMPAG versus fMPAG (r = 0.205, p = 0.3851), and sMPAG versus tMPAG (r = 0.472, p = 0.0012). Pharmacokinetic parameters supported these findings, highlighting discrepancies between saliva and plasma. Conclusions: From a clinical perspective, saliva sampling—although minimally invasive and patient-friendly—does not offer a reliable substitute for plasma in routine TDM of MPA and MPAG. Capillary blood collected through VAMS remains a promising alternative for long-term monitoring of pediatric patients; however, several considerations still need to be addressed. Full article

The bioanalysis of body fluids plays a crucial role in clinical diagnostics, pharmaceutical research, forensic science, and biomarker discovery. Traditional chromatographic techniques are widely used in clinical laboratories but are often costly and time-consuming. Capillary electrophoresis (CE) and its modifications, such as capillary zone electrophoresis, isotachophoresis, and micellar electrokinetic chromatography, have emerged as efficient, cost-effective, and miniaturized alternatives for analyzing small organic and inorganic molecules in biological fluids. This paper deals with the applications of CE-based electromigration techniques in the determination of various analytes in urine, blood, saliva, and cerebrospinal fluid. The study further discusses the advantages and limitations of different detection methods, including ultraviolet-visible spectroscopy, laser-induced fluorescence, mass spectrometry, conductivity, and amperometric detection. A focus is given to the identification and quantification of amino acids and their metabolites as potential biomarkers for metabolic and degenerative disorders. The work highlights recent advancements in CE methodologies and their potential to enhance sensitivity and selectivity in bioanalytical applications. Full article

The contamination of ground and surface waters with micropollutants like estrogenic compounds and genotoxins is a major public health concern. Conventional wastewater treatment plants are currently not capable of completely removing those contaminants. In this study, we applied planar bioassays to investigate the genotoxicity and estrogenic activity of hospital and municipal wastewater from an Austrian treatment plant. Using the open-source 2LabsToGo platform in combination with the HPTLC-SOS-UmuC and HPTLC-YES assays, both genotoxic and estrogenic compound zones were detected in untreated wastewater. Genotoxic activity was found in sewage sludge filtrate and hospital wastewater, with bioanalytical concentrations ranging from 1.6 to 21.8 µg 4-NQO-EQ L⁻¹. Estrogenic responses were observed in the influent and hospital wastewater samples, with BEQ values between 3.5 and 16.0 µg E2-EQ L⁻¹. No activity was detected in the treated effluent, indicating efficient removal of these compounds during wastewater treatment. These results confirm the presence of biologically active micropollutants in hospitals and raw wastewater and demonstrate the suitability of planar bioassays for sensitive, spatially resolved detection. The use of portable equipment like the 2LabsToGo system suggests that on-site monitoring of estrogenic and genotoxic activities in wastewater is feasible and could support routine surveillance of treatment efficiency. Full article

Albendazole (ABZ) is a broad-spectrum anthelmintic drug whose residual presence in food and the environment raises public health concerns, requiring rapid and sensitive methods of detection. In this work, a sensitive Fabry–Pérot interferometer (FPI) probe was fabricated by realizing a cavity located at the tip of a single-mode optical fiber core with a molecularly imprinted polymer (MIP) for ABZ detection. The fabrication process involved the development of a photoresist-based micro-hole filled by the specific MIP via thermal polymerization. Interferometric measurements obtained using the proposed sensor system have demonstrated a limit of detection (LOD) of 27 nM, a dynamic concentration range spanning from 27 nM (LOD) to 250 nM, and a linear response at the nanomolar level (27 nM–100 nM). The selectivity test demonstrated no signal when interfering molecules were present, and the application of the sensor for ABZ quantification in a commercial pharmaceutical sample provided good recovery, in accordance with bioanalytical validation standard methods. These results demonstrate the capability of a MIP layer-based FPI probe to provide low-cost and selective optical-sensing strategies, proposing a competitive approach to traditional analytical techniques for ABZ monitoring. Full article

This work presents the development of carbon nanotubes functionalised with carboxylic acid groups (CNT-COOH) as an oxygen-sensitive electrochemical platform for parallel multi-analyte enzymatic biosensing. The platform was constructed by depositing carboxylic-acid-functionalised single-walled carbon nanotubes covalently onto nanostructured gold electrodes modified with a self-assembled monolayer of 4-aminothiophenol. Atomic force microscopy characterization revealed that the nanotubes attached via their ends to the surface and had a predominantly horizontal orientation. Glucose oxidase, lactate oxidase, glutamate oxidase, and tyrosinase were immobilised onto the electrodes to create selective biosensor for lactate, glucose, glutamate, and dopamine, respectively. A key finding is that incorporating catalase significantly extends the linear detection range for analytes by mitigating the accumulation of hydrogen peroxide. The resulting multifunctional biosensor demonstrated its capability for the simultaneous and independent measurement of glucose, lactate as the key bioanalytes under uniform conditions in blood plasma samples, highlighting its potential for applications in health and food technologies. Full article