Search Results (631)

Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light and heavy chain coding genes, employing a wild-type Encephalomyocarditis virus (EMCV) IRES functional element to drive expression of the second gene. Methods: Using two neutralizing anti-SARS-CoV-2 IgG1 antibodies as model molecules, we conducted transient transfections in the commercially available ExpiCHO^TM platform. Following protein A affinity purification and quantification, vectors positioning the light chain as the first cistron consistently yielded higher expression levels than those with the heavy chain upstream. To confirm the quality attributes of the mAbs, we applied a comprehensive analytical workflow, including SDS-PAGE and Western blot for molecular mass and purity, circular dichroism for secondary structure, intrinsic tryptophan fluorescence for tertiary structure, and SEC-HPLC for quaternary structure and aggregate detection. Additionally, we assessed binding affinity to the target using spot blot and surface plasmon resonance, analyzed N-glycosylation profiles by HILIC-HPLC and mass spectrometry, and examined molecular structure by transmission electron microscopy. Results and Conclusions: Together, these results provide insight into the impact of gene positioning within bicistronic vectors on mAb expression efficiency and quality, supporting optimization strategies for scalable recombinant antibody production. Full article

Most biomarkers exhibit abnormal expression in more than one disease, making conventional single-biomarker detection strategies prone to false-negative results. Detecting multiple biomarkers associated with a single disease can therefore substantially improve diagnostic accuracy. Accordingly, recent research has focused on precise multiplex detection, leading to the development of sensors employing various readout methods, including electrochemical, fluorescence, Raman, and colorimetric approaches. This review focuses on optical sensing applications, such as fluorescence, Raman spectroscopy, and colorimetry, which offer rapid and straightforward detection and are well suited for point-of-care testing (POCT). These optical sensors exploit nanoscale phenomena derived from the intrinsic properties of nanomaterials, including metal-enhanced fluorescence (MEF), Förster resonance energy transfer (FRET), and surface-enhanced Raman scattering (SERS), which can be tailored through modifications in material type and structure. We summarize the types and properties of commonly used nanomaterials, including plasmonic and carbon-based nanoparticles, and provide a comprehensive overview of recent advances in multiplex biomarker detection. Furthermore, we address the potential of these nanosensors for clinical translation and POCT applications, highlighting their relevance for next-generation disease diagnostic platforms. Full article

The increasing prevalence of antimicrobial resistance and the persistent challenge of infectious diseases highlight the critical necessity for novel approaches that integrate pathogen management with swift detection methods. Carbon dots (CDs) are a versatile class of fluorescent nanomaterials that have garnered increasing attention owing to their tunable surface chemistry, strong photoluminescence, high stability, and biocompatibility. Recent studies demonstrate that CDs possess broad-spectrum antibacterial and antifungal activities via multiple mechanisms, including the generation of reactive oxygen species, disruption of membranes, inhibition of biofilms, and synergistic interactions with conventional antimicrobials. The performance is significantly affected by precursor selection, heteroatom doping, and surface functionalization, with minimum inhibitory concentrations reported to range from highly potent at the microgram level to moderate at elevated concentrations. The intrinsic fluorescence of CDs, in addition to their antimicrobial activity, facilitates their use as sensitive and selective probes for microbial detection, allowing for rapid and real-time monitoring in biomedical, food safety, and environmental settings. This review summarizes recent advancements in the antimicrobial properties of carbon dots (CDs) and their fluorescence-based applications in microbial detection. It emphasizes their theranostic potential and future prospects as multifunctional nanomaterials for combating infectious diseases and ensuring microbial safety. Full article

Two fluorescent ureido-dihomooxacalix[4]arene derivatives containing naphthyl residues at the lower rim (1 and 2) were studied for the detection of nitroaromatic compounds (NACs) in solution and in vapour phases. Their affinity in solution was determined by UV-Vis absorption, fluorescence and NMR spectroscopy. For NAC vapour sensing, calixarenes were dispersed in a polytetrafluoroethylene (PTFE) matrix. Four new solvated crystals of dihomooxacalix[4]arene 2 were obtained and the solvent’s influence on its structural characteristics was investigated. The solvent-dependent structural variations observed in the crystal structures highlight the intrinsic flexibility of the calixarene framework. Such conformational adaptability, evident in the disruption and reorganization of hydrogen bonding and π–π interactions, is directly relevant to nitroaromatic sensing, where a rapid and reversible host response is crucial for effective detection. Theoretical calculations were also performed to provide further insights on the binding process. The corrected Stern–Volmer constants (K_SV) obtained showed that both receptors present selectivity for TNP and follow the same quenching order (TNP > NT > NB > DNT > TNT > DNB). Factors other than electron density distribution should dominate the quenching extent and therefore the values of the SV constants, which will be greatly overestimated if no correction to the inner filter effect is applied. Detection of NB and NT and vapours by both calixarenes produced a complete, very fast (2 to 5 s), and reversible quenching, indicating the potential use of this porous PTFE–calixarene matrix for the sensing of volatile NACs. Full article

Diabetes mellitus is a characteristic metabolic disorder with diverse complications. α-Amylase and α-glucosidase, as key digestive enzymes regulating blood glucose, are important targets for diabetes prevention and management through their inhibition. This study investigated the inhibitory effects of six porphyrin compounds (TAPP, TCPP, THPP, Cu–TCPP, Fe–TCPP, Ni–TCPP) on two enzymes through in vitro inhibition assays, spectroscopic experiments, and molecular docking techniques. All six compounds effectively inhibited the activities of both enzymes. For α-amylase, the inhibitory potency (IC₅₀ = 13.03–245.04 μg/mL) followed the order TAPP > THPP > TCPP > Fe–TCPP > Ni–TCPP > Cu–TCPP. All six compounds exhibited more potent inhibitory activity against α-glucosidase (IC₅₀ = 0.24–25.43 μg/mL), with potency in the order of THPP > Ni–TCPP > Fe–TCPP > TCPP > Cu–TCPP > TAPP. Fluorescence quenching experiments revealed that all compounds statically quenched the intrinsic fluorescence of both enzymes (with Fe–TCPP exhibiting static-dominant mixed quenching against α-amylase), indicating complex formation. These interactions significantly altered the enzymes’ conformations, the microenvironments of Tyr/Trp residues, and secondary structure content, consequently reducing their catalytic activity. By examining the inhibitory impact of porphyrin compounds on α-amylase and α-glucosidase, this research establishes a vital experimental and theoretical basis for diabetes therapeutics. Full article

Eutectogels are advanced gel-based systems that integrate deep eutectic solvents (DES) into polymer networks. In this study, we report the first detailed characterization of an enzyme-containing eutectogel, representing a significant step toward advanced biosensing and biocatalytic applications. Specifically, we have incorporated pancreatic lipase, one of the main target enzymes in the treatment of obesity, in eutectogels via UV-induced radical polymerization of suitable precursors in appropriate DESs. Prior to immobilization, the enzyme was solubilized in selected DESs and its activity and conformational stability were evaluated using colorimetry and intrinsic fluorescence. Combinations of choline chloride/glycerol and tetramethylammonium chloride/glycerol were shown to be effective media for preserving and enhancing enzymatic function and conformational stability. The enzyme immersed in eutectogel exhibited high structural integrity and excellent thermal stability, maintaining its activity over several weeks. The ability of this new material to screen enzyme inhibitors was assessed using orlistat, a well-established anti-obesity agent. The results demonstrated clear detection of the drug’s inhibitory effect, even at nanomolar concentrations, highlighting the material’s potential as a screening platform for novel inhibitors with prospective anti-obesity activity. Furthermore, the device proved effective in quantifying drug presence, offering a promising and highly sensitive tool for pharmaceutical quality control applications. Full article

The rational design of advanced functional materials with tailored fluorescence hinges on a profound understanding of the complex interplay between a molecule’s intrinsic structure and its local solid-state environment. This work systematically investigates these factors by employing a dual approach that combines targeted molecular synthesis with the subsequent modulation of the fluorophore’s properties within polymer matrices. First, a series of phenylhydrazone derivatives was synthesized, providing compounds with intense, solid-state fluorescence in the blue spectrum (421–494 nm). It was demonstrated that their photophysical properties were intricately linked to the substituent’s nature, which simultaneously modulated their intramolecular electron density and conformational rigidity while also governing their specific intermolecular packing in the solid state. Subsequently, we investigated the role of the supramolecular environment by embedding two fluorophores with distinct electronic profiles into electrospun poly (N-vinylpyrrolidone) (PVP) and polystyrene (PS) matrices. Our results reveal that the polymer matrix is not a passive host but an active component; it governs dye aggregation, induces significant blue shifts, and most critically, can impart exceptional thermal stability. Specifically, the PVP matrix shielded the embedded dyes from thermal quenching, maintaining robust fluorescence up to 100 °C. By combining molecular-level synthesis with matrix-level engineering, this work demonstrates a powerful strategy for the rational design of emissive materials, where properties like color and operational stability can be deliberately tuned for demanding applications in optoelectronics and sensing. Full article

Hybrid systems combining animal and plant proteins are promising for developing sustainable, high-protein foods. However, structural incompatibility between proteins like casein and pea protein hinders the formation of stable systems such as gels. This study explores pH-shifting (alkalization at pH 12 followed by neutralization) as an innovative strategy to improve pea protein functionality and compatibility in hybrid gels. Modified pea protein showed increased solubility, reduced particle size, higher zeta potential, and decreased intrinsic fluorescence intensity, indicating conformational changes and exposure of buried tryptophan residues. These structural changes influenced gel behavior depending on the protein ratio (casein/pea—80:20, 50:50, 20:80). Gels with higher pea content showed increased hardness and water-holding capacity, while in casein-rich gels, hardness decreased, likely due to altered protein–protein interactions. This is the first study to systematically apply pH-shifting to enhance the compatibility between pea protein and casein in high-protein gels, integrating structural and functional analyses. The results demonstrate the potential of pH-shifting as a sustainable and effective approach for improving plant protein performance in hybrid formulations. Full article

Fenpropathrin (FPT) residues in tea pose a threat to consumers’ health. Lateral flow immunoassay (LFIA) offers a rapid and convenient approach for FPT detection. However, existing LFIA formats, particularly those employing fluorescent nanoparticles, are susceptible to interference from the tea matrix’s endogenous fluorescence, limiting their accuracy for FPT analysis. Here, two complementary LFIAs based on gold nanoparticle (AuNP) and carbon nanoparticle (CNP) tags were developed, both of which avoid matrix fluorescence effects due to their intrinsic coloration under ambient light. We systematically evaluated five cleanup materials and identified polyvinylpolypyrrolidone as the optimal cleanup material. Following PVPP-based purification, green tea extracts required only a four-fold dilution prior to LFIA analysis, effectively minimizing matrix interferences, such as tea polyphenols, and enhancing assay robustness and sensitivity. The visual limit of detection was 0.64 μg/g and a quantitative limit of detection (qLOD) was 0.11 μg/g for FPT in green tea using the AuNP-based LFIA. The CNP-based LFIA further improved sensitivity, with a visual limit of detection of 0.16 μg/g and a qLOD of 0.017 μg/g, a 6.4-fold enhancement in qLOD compared to the AuNP assay. Together, these two assays provide an efficient and accurate strategy for on-site screening of FPT residues in green tea. Full article

We present a dual-mode optical sensing strategy for selective and sensitive detection of sulfide ions (S²⁻), employing copper-anchored nitrogen-doped graphene quantum dots (Cu@N-GQDs) as bifunctional nanozymes. The Cu@N-GQDs were synthesized via citric acid pyrolysis in the presence of ammonium hydroxide (serving as both nitrogen source and reductant) and copper chloride, leading to uniform incorporation of copper oxide species onto the N-GQD surface. The resulting nanohybrids exhibit two synergistic functionalities: intrinsic fluorescence comparable to pristine N-GQDs, and significantly enhanced peroxidase-like catalytic activity attributed to the anchored copper species. Upon interaction with sulfide ions, the system undergoes a dual-optical response: (i) fluorescence quenching via Cu-S complexation, and (ii) inhibition of peroxidase-like activity due to the deactivation of Cu catalytic centers via the interaction with S²⁻. This dual-signal strategy enables sensitive quantification of S²⁻, achieving detection limits of 0.5 µM (fluorescence) and 3.5 µM (colorimetry). The sensor demonstrates excellent selectivity over competing substances and high reliability and precision in real tap water samples. These findings highlight the potential of Cu@N-GQDs as robust, bifunctional, and field-deployable nanozyme probes for environmental and biomedical sulfide ion monitoring. Full article

The overexpression of atypical protein kinase C-iota (PKC-ι) is a biomarker for carcinogenesis in various cell types, such as glioma, ovarian, renal, etc., manifesting as a potential drug target. In previous in vitro studies, ICA-1S and ICA-1T, experimental candidates for inhibiting PKC-ι, have demonstrated their specificity and promising efficacy against various cancers. Moreover, the in vivo studies have demonstrated low toxicity levels in acute and chronic murine models. Despite these prior developments, the binding affinities of the inhibitors were never thoroughly explored from a biophysical perspective. Here, we present the biophysical characterizations of PKC-ι in combination with ICA-1S/1T. Various methods based on molecular docking, light scattering, intrinsic fluorescence, thermal denaturation, and heat exchange were applied. The biophysical characteristics including particle sizing, thermal unfolding, aggregation profiles, enthalpy, entropy, free energy changes, and binding affinity (K_d) of the PKC-ι in the presence of ICA-1S were observed. The studies indicate the presence of domain-specific stabilities in the protein–ligand complex. Moreover, the results indicate a spontaneous reaction with an entropic gain, resulting in a possible entropy-driven hydrophobic interaction and hydrogen bonds in the binding pocket. Altogether, these biophysical studies reveal important insights into the binding interactions of PKC-ι and its inhibitors ICA-1S/1T. Full article

Liposomes (LPs) represent one of the most effective nanoscale platforms for drug delivery in cancer therapy due to their favorable pharmacokinetic and various body tissue compatibility profiles. Building on recent findings showing that carbon dots derived from N-hydroxyphthalimide (CDs-NHF) possess intrinsic antitumor activity, herein, we investigate the possibility of preparing complex nano-platforms composed of LPs encapsulating CDs-NHF and/or doxorubicin (DOX) for breast and lung cancer. Various LP formulations were prepared and characterized using Cryo-TEM and Cryo-SEM for morphological analysis, while zeta potential and fluorescence assessments confirmed their stability and optical properties. Cellular effects were evaluated through immunofluorescence microscopy and proliferation assays. LPs-CDs-NHF significantly reduced cancer cell viability at lower concentrations compared to free CDs-NHF, and this effect was further amplified when combined with doxorubicin. Mechanistically, the liposomal formulations downregulated key signaling molecules including pAKT, pmTOR, and pERK, indicating the disruption of cancer-related pathways. These findings suggest that LPs containing CDs-NHF, either alone or in combination with DOX, exhibit synergistic antitumor activity and hold strong promise as multifunctional nanocarriers for future oncological applications. Full article

The search for sustainable, economically viable, and effective plant protection strategies against pathogenic bacteria, fungi, and viruses is a major challenge in modern agricultural practices. Chitosan (CS) is an abundant cationic natural biopolymer known for its biocompatibility, low toxicity, and antimicrobial properties. Its potential use in agriculture for pathogen control is a promising alternative to traditional chemical fertilisers and pesticides, which raise concerns regarding public health, environmental protection, and pesticide resistance. This study focused on the preparation of chitosan nanoparticles (CS-NPs) through cross-linking with organic molecules, such as tannic acid (TA). Various formulations were explored for the development of stable nanoscale particles having encapsulation capabilities towards low compounds of varying polarity and with potential agricultural applications relevant to plant health and growth. The solution properties of the NPs were assessed using dynamic and electrophoretic light scattering (DLS and ELS); their morphology was observed through atomic force microscopy (AFM), while analytical ultracentrifugation (AUC) measurements provided insights into their molar mass. Their properties proved to be primarily influenced by the concentration of CS, which significantly affected its intrinsic conformation. Additional structural insights were obtained via infrared and UV–Vis spectroscopic measurements, while detailed fluorescence analysis with the use of three different probes, as model cargo molecules, provided information regarding the hydrophobic and hydrophilic microdomains within the particles. Full article

Electrochemical biosensors are revolutionizing food testing by addressing critical limitations of conventional strategies that suffer from cost, complexity, and field-deployment challenges. Emerging fluorescence and Raman techniques, while promising, face intrinsic drawbacks like photobleaching and matrix interference in opaque or heterogeneous samples. In contrast, electrochemical biosensors leverage electrical signals to bypass optical constraints, enabling rapid, cost-effective, and pretreatment-free analysis of turbid food matrices. This review highlights their operational mechanisms, emphasizing nano-enhanced signal amplification (e.g., Au nanoparticles and graphene) and biorecognition elements (antibodies, aptamers, and molecularly imprinted polymers) for ultrasensitive assay of contaminants, additives, and adulterants. By integrating portability, scalability, and real-time capabilities, electrochemical biosensors align with global food safety regulations and sustainability goals. Challenges in standardization, multiplexed analysis, and long-term stability are discussed, alongside future directions toward AI-driven analytics, biodegradable sensors, and blockchain-enabled traceability, ultimately fostering precision-driven, next-generation food safety and quality testing. Full article

The EGCG/PPN composite, prepared by combining pea protein nanofibrils (PPNs) with epigallocatechin gallate (EGCG), could be used as a multifunctional nanocarrier. Compared to pea protein isolate (PPI), EGCG/PPN composites exhibited remarkably higher turbidity and zeta potential, along with similar UV spectra. Intrinsic fluorescence spectroscopy, ThT fluorescence spectroscopy, and surface hydrophobicity analysis suggested that the interactions between EGCG and PPN were primarily driven by hydrophobic forces. UV spectra indicated that the microenvironment of amino acid residues in the tertiary structure of the protein changes upon complexation, and circular dichroism (CD) revealed that the incorporation of EGCG increases the β-sheet content in the protein’s secondary structure. Analyses of DPPH and ABTS radical scavenging activity, as well as reducing power, demonstrated that the synergistic effect between EGCG and PPN did not hinder the inherent antioxidant properties of EGCG but rather enhanced them significantly. Transmission electron microscopy (TEM) images showed that the addition of EGCG reconstructed the fibril morphology, thereby affecting the properties of PPNs. Overall, the composite fabricated through the interaction between PPN and EGCG shows great potential as a nanocarrier in the processing of functional foods. Full article