The profile of natural antibodies present in human serum serves as an informative indicator of various diseases, vaccination history, immune system status, and even dietary habits of patients. The ability to provide detailed and precise interpretation of this data opens opportunities for the development of new diagnostic methods and vaccines. The application of synthetic oligosaccharides as antigens with distinct structures appears promising for obtaining reproducible results in immunological screenings. In this study, we analyzed the immunochemical reactivity, NMR spectra, and conformational behavior of oligomeric β-(1→2)-, β-(1→3)-, and β-(1→6)-d-glucosides, which are structurally related to well-known pathogen-associated molecular glycopatterns. β-(1→2)-Glucooligosaccharides were found to differ fundamentally from the other two β-glucans, exhibiting unusual extremal length dependencies in immunochemical properties, nonequivalence of glycosidic linkages within the homo-β-(1→2)-glucoside chain in the NMR spectra, and the formation of stable helical conformational states within MD simulations. The immunochemical features of β-(1→2)-glucosides revealed in this study provide a basis for the development of highly specific diagnostic assays based on the detection of β-(1→2)-glucan antigens as well as the corresponding complementary antibodies.

This study investigated the dynamic changes in rice quality, microbial communities, and volatile compound profiles during simulated summer transportation (35 °C, 70% RH, 15 days). Indica rice samples were systematically collected every 3 days and analyzed using HS-SPME-GC-MS/MS, HS-GC-IMS, and metagenomic sequencing. Prolonged transportation significantly altered the physicochemical properties of the rice. Moisture content plateaued on day 12, while germination rates declined significantly starting from day 6. Furthermore, fatty acid values increased continuously due to accelerated lipid hydrolysis and oxidation. Visible mold growth became evident on day 12, marking a critical tipping point for quality deterioration. The odor activity value (OAV) and relative odor activity value (ROAV) analyses revealed that the decline in unsaturated fatty aldehydes such as (E)-2-nonenal and the significant accumulation of alcohols, ketones, and short-chain esters, including 1-octen-3-ol and ethyl acetate, drove the transition from a “fresh and fatty” aroma to one characterized by moldy, fermented, and pungent notes. Metagenomic analysis demonstrated a profound ecosystem shift from bacterial dominance (Proteobacteria, Actinobacteria) to fungal dominance. Notably, Lichtheimia surged from <0.01% to 23.95%, becoming the dominant genus, while Aspergillus increased from 0.03% to 4.57%. Correlation analysis indicated that while Pseudomonas was associated with elevated fatty acid levels, the flavor shift was primarily linked to microbial succession. These findings provide insights into the synergistic mechanisms of rice spoilage and suggest that specific volatile markers could serve as early warning indicators for quality control in real-world grain logistics.

Abstract Immune assays are widely used in various fields of medicine, including enzyme-linked immunosorbent assays, fluorescence polarization immunoassay (FPIA), and immunochromatographic analysis. FPIA allows for high specificity and sensitivity detection of low-molecular-weight analytes in a homogeneous medium without separation; it has high accuracy and label stability, and analysis is fast, simple, and can be automated. A fluorescently labeled tracer for erythromycin determination with a new boron-dipyrromethene (BODIPY) dye was developed in this study. A pair of immunoreagents (tracers and antibodies) for detecting erythromycin was selected and characterized, and FPIA conditions were optimized for an analysis time of 5 min. Calibration curves were obtained, and FPIA analytical characteristics were determined. The limit of detection was 1.6 ng/mL with a detection range at pH 7.4 of 5 − 300 ng/mL. The developed FPIA was successfully used to determine erythromycin concentration in water and milk, with a recovery of 80% – 120%. Overall, an FPIA method for erythromycin determination was developed, the advantages of using the new BODIPY fluorescent label were shown, the accuracy of the method was verified by an recovery test, and real water samples were tested.

The detection of biological markers is critical not only for the early diagnosis of cancer but also for adjustments in antitumor therapy. Rapid, sensitive, and selective detection and monitoring of the content of specific biomarkers in real time are key to point-of-care testing diagnostics. We report the detection of heat shock proteins by fluorescence immunoassay with the appropriate phage antibodies, with a minimum detection limit of 1 ng/mL. The fluorescence immunoassay data were confirmed by dot immunoassay and by circular dichroism studies. The results of the study may help in the adaptation of the fluorescence immunoassay to cancer diagnostics.

Traditional fluorescence polarization immunoassay (FPIA) requires costly monoclonal antibodies. This work develops a cost-effective fluorescence polarization enzyme assay (FPEA) for quercetin, employing α-amylase as a significantly cheaper recognition agent. Based on competitive binding with a fluorescent zearalenone tracer, the assay achieves a detection limit of 1.7 mg/mL, a working range of 2.3–6.4 mg/mL, and completes analysis within 5 min, offering a substantial time saving versus typical HPLC runs ( 20 min). It demonstrates high specificity, with only minimal cross-reactivity to rutin (0.1

4-Chlorophenoxyacetic acid (4-CPA), a synthetic auxin analog, is employed in agriculture both as a plant growth regulator and as a constituent of herbicide formulations. Consequently, the establishment of simple and rapid detection methods is essential for effective environmental monitoring. This study reports the first development of a homogeneous fluorescence polarization immunoassay (FPIA) for the determination of 4-CPA. The monoclonal antibody (M1), raised against 4-CPA, was evaluated as a recognition element. Furthermore, two fluorescently labeled 4-CPA tracers-with ethylenediamine fluorescein thiocarbamate and aminohexylaminocarbonylfluorescein-were synthesized and purified, and their structures were unequivocally confirmed by high-performance liquid chromatography coupled with high-resolution mass spectrometric detection (HPLC-HRMS). Optimal concentrations of monoclonal antibodies and tracers were established, yielding a limit of detection of 1.2 ng/mL. The assay demonstrated a broad dynamic range of 2.3-300 ng/mL and a rapid analysis time of 15 min. Validation via the standard addition method in authentic open water samples resulted in recovery rates of 98-112%. To address the cross-reactivity with the prevalent herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), two novel strategies were devised and successfully implemented. The first approach involves the concurrent execution of two separate FPIAs-one for 2,4-D and one for 4-CPA-followed by the mathematical resolution of two analyte concentrations from the two measured binding values. The second strategy entails the preliminary selective removal of 2,4-D from sample matrices using affinity chromatography columns with immobilized anti-2,4-D antibodies prior to FPIA for 4-CPA. These proposed methodologies appear highly promising for overcoming the inherent limitations of traditional immunoassays when faced with significant cross-reactivity among structurally analogous compounds.

Protein-based APIs represent a big group of modern therapeutics. Their characterization involves complex analytical protocols which require special methods, especially in the case when the protein drug is included into tablet dosage forms. Although the fluorescence polarization assay (FPA) is not currently regulated by many national Pharmacopeias, it represents a promising approach for protein drug standardization, considering their rapid, sensitive, and automatable detection suitable for high-throughput analysis and real-time quality control. To evaluate the applicability of FPA for the analysis of protein drugs in tablets, the quantifying of lysozyme in tablet dosage forms was studied by this method with the use of a fluorescently labeled synthetic chitooligosaccharide tracer. It was shown that this approach overcomes the limitations of the conventional turbidimetric assay of lysozyme determination, which is labor-intensive and relies on unstable reagents. Measurements were performed with both portable and stationary fluorescence polarization readers. Commercial tablets from five manufacturers containing lysozyme (20 mg) and pyridoxine hydrochloride (10 mg) together with other excipients were analyzed. The FPIA method showed a linear range of 5.0–70 µg/mL, with specificity confirmed by the absence of interference from excipients. Accuracy, evaluated by standard addition (10–20 mg), yielded recoveries of 100.2–106.0%. Placebo spiked with lysozyme at 80–120% of nominal content demonstrated recoveries of 98.0–100.1%, with RSD (n = 6) not exceeding 13.7%, indicating good precision. The developed method enables reliable lysozyme quantification in tablets, offering speed, simplicity, and robustness, and shows its suitability for the routine quality control of protein-containing dosage forms including the enzyme ones.

Pathogenic bacteria of the genus Brucella cause a severe threat for public health and agricultural economics. The World Health Organization considers brucellosis to be one of the most serious and also neglected zoonotic diseases. The use of traditional whole-cell brucellosis vaccines complicates the differentiation between infected and vaccinated animals (DIVA). Moreover, diagnostics based on lipopolysaccharide of Brucella are susceptible to false positive results. Structural features of Brucella O-antigens make synthetic oligosaccharides promising agents for the development of diagnostic tools and vaccines against brucellosis. Here we report the synthesis of spacer-armed di-, tri-, tetra- and penta-4,6-dideoxy-4-formamido-α-(1→2)-d-mannopyranosides which are related to the A-epitope of Brucella O-antigen. The key α-(1→2)-linked disaccharide thioglycoside donor was synthesized by employing the strategy of orthogonal glycosylation of thioglycoside acceptor with trichloroacetimidate donor. Sequential block-wise assembly yielded a series of desired compounds, which were subsequently deprotected and converted into target molecules and then into their fluorescein-labeled conjugates. The obtained conjugates were employed as tracers in a fluorescence polarization assay (FPA) to detect anti-Brucella immunoglobulins. Among the studied compounds, the trisaccharide conjugate showed the greatest difference in median FP signals between Brucella-positive and Brucella-negative sera samples making it a promising candidate for developing FP diagnostic assays. The decreased FP signal in the cases of tetra- and pentasaccharide tracers can be associated with the known “propeller-effect” due to the rotational mobility of the part bearing the fluorescent label and of the fluorescein itself and/or the enlarging of the distance between the fluorescein part and the antibody-oligosaccharide complex. This observation demonstrates the advantages of using synthetic relatively small synthetic tracers with well-defined structure in comparison with heterogeneous fluorescein-labelled O-polysaccharides which are in use today in spite of the fact that they contain poorly characterized amounts of label attached along the polysaccharide chains.

The storage duration and hatchability of eggs largely depend on the lysozyme content in egg whites; therefore, determining the lysozyme status is important for characterizing their quality. For the first time, a fast and accurate method for determining the active lysozyme in egg whites has been proposed to establish the lysozyme status of eggs using the fluorescence polarization assay and synthetic chitooligosaccharide conjugates with a fluorescent label without sample preparation. The egg whites of hens, black hens, chukars, quails, ducks, geese, turkeys, peacocks, and ostriches were studied. Samples of egg whites from hens, black hens, chukars, and quails demonstrate the possibility of measuring the lysozyme activity. Samples of hen and black hen eggs from a farm showed approximately the same enzymatic activity of lysozyme. A relatively higher enzymatic activity was demonstrated by the samples from quail egg whites; however, a wide range of data was observed among the eggs. Chitooligosaccharide conjugates demonstrate that they bind only to C-type lysozyme, and no interaction with G-type lysozyme has been shown. Lysozyme activity in the egg whites of duck, goose, turkey, peacock, and ostrich eggs has not been detected by using the obtained chitooligosaccharide tracers, which may be related to the structural features of lysozyme in different bird species. Thus, the method of fluorescence polarization (FP), using fluorescently labeled chitopentaoside to determine the lysozyme status, can be used to characterize hen, black hen, chukar, and quail eggs, which will allow for the selection of a batch of eggs with a high content of active lysozyme, for example, for long-term storage.

The current work presents comparative assessment of affinity of the designed DNA aptamers for extracellular domain of the human epidermal growth factor receptor (EGFR*). The affinity data of the 20 previously published aptamers are summarized. Diversity of the aptamer selection methods and techniques requires unification of the comparison algorithms, which is also necessary for designing aptamers used in the post-selection fitting to the target EGFR* protein. In this study affinities of the DNA aptamers from two families, U31 and U2, previously obtained by Wu et al. from the same selection [Wu et al. (2014) PLoS One, 9, e90752] and their derivatives – GR20, U2s, and Gol1 obtained by us through rational design, were compared. Affinity of the aptamers to EGFR* was measured by two different methods: a solution-phase technique – fluorescence polarization of FAM-labeled aptamers, and by a kinetic method using biolayer interferometry technique with aptamers immobilized on the surface. Unlike the values of equilibrium dissociation constants obtained through titration and expressed in units of protein concentration, analysis of the titration curve profiles themselves and kinetics of interaction proved to be more informative. This allowed us to identify how even subtle changes in the aptamers and their structures affect affinity. Hypotheses regarding the “structure–function” relationships and recognition mechanisms were formulated. The data obtained for the set of aptamer constructs are critical for moving forward to examination of aptamer interactions with EGFR on the cell surface.

Zearalenone (ZEN) poses serious risks to human and animal health. Compared with physical and chemical methods, microbial transformation offers a safer and more sustainable strategy for ZEN detoxification. The yeast Hannaella zeae, isolated from the Qinghai–Tibet Plateau, showed the highest ZEN removal efficiency among 11 strains, achieving an 85.87% transformation rate within 36 h. Optimal conditions for ZEN transformation were determined by varying culture time, temperature, and pH. The products were putatively identified as zearalenone-14-β-D-glucopyranoside (C24H32O10) and zearalenone-16-β-D-glucopyranoside (C24H32O10) by UHPLC-Q-Orbitrap-HRMS. The safety of the mixed culture medium extract was further evaluated using a Caenorhabditis elegans model, showing significantly lower toxicity than untreated ZEN. H. zeae maintained high transformation efficiency under low temperature (57.48%) and acidic stress (47.10%), supported by active antioxidant enzymes (SOD, CAT, APX, GPx) and stress metabolites (trehalose, proline). Overall, this study identifies H. zeae as a promising, stress-tolerant biocontrol agent and elucidates its glycosylation-based detoxification mechanism, providing a foundation for future application in real food and feed systems.

Phthalate acid diesters (PAEs) are used as plasticizers in various applications and have accumulated in the environment, where they pose a risk to human health. In the body, PAEs are metabolized to toxic monophthalates. Currently, there are few immunoassays available for the determination of monophthalates. In this study, a fluorescence polarization immunoassay (FPIA) for determining monobutyl phthalate (MBP) was developed. This assay was validated by recovery tests of MBP in artificial urine buffer and wastewater samples. A conjugate of MBP with an amino derivative of fluorescein was synthesized and confirmed to bind to polyclonal antibodies. Two protocols for determining the MBP were developed, using different sample volumes depending on the complexity of the matrix. For urine, the FPIA method had a limit of detection of 100 ng/mL and a detection range of 200–500,000 ng/mL. For water, the limit of detection was 5 ng/mL and the detection range was 30–50,000 ng/mL. The analysis time was 5 min. The developed FPIA is suitable for determination of MBP in river water samples. Because the matrix did not affect the results for water samples, no pretreatment was required. Therefore, water samples can be tested on-site using a small portable FPIA analyzer. This method could be used to determine monophthalates in environmental waters or human biological fluids.

2,4-Dichlorophenoxyacetic acid (2,4-D) is one of the popular herbicides that is widely used in agriculture and can be found in food and water. A rapid and sensitive fluorescence polarization immunoassay (FPIA) was proposed for the detection of 2,4-D in juice and water. New tracers, 2,4-D-buthylenediamin fluoresceinthiocarbamyl (2,4-D-BDF) and 2,4-D-glycine aminofluorescein (2,4-D-GAF), were obtained and characterized. Monoclonal antibodies (MAb) obtained against 2,4-D were used as a recognition reagent. The kinetics of the interaction of MAb and tracers were studied, and the kinetic parameters of their binding were calculated. High specificity of binding of tracers and MAb was shown. In this work, an approach was elaborated on to reduce the detection limit of 2,4-D by the FPIA method by changing the volume of the studied sample. The optimized FPIA in a competitive format was characterized by the LODs of 2,4-D 8 and 0.4 ng/mL and the working ranges 30-3000 ng/mL and 3-300 ng/mL for juice and water, respectively. The entire test cycle (from sample receipt to evaluation of the analysis results) took only 20 min. The test for the recovery of 2,4-D in juice and water gave values from 95 to 120%, which demonstrated the reliability of the herbicide determination in real samples.

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018–2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

ctive: The development of a fast and reliable method for the quantitative evaluation of steroid hormones is very relevant for scientific and clinical research. The goal of this study was to develop a new universal method for the synthesis of fluorescent tracers from carbonyl-containing biomolecules by oxime ligation. Methods: A method for the synthesis of ketosteroid oxime conjugates with a fluorescein (6-FAM) derivative connected via a short bifunctional aminooxypropyl linker is proposed. Oxime stereoisomers were separated by preparative RP TLC and HPLC. The conjugates were used as tracers for the fluorescence polarization immunoassay (FPIA) method with monoclonal antibodies. Results and Discussion: Parameters of the binding of the tracers with monoclonal antibodies (mAbs) to progesterone were studied. The tracers demonstrated high efficiency for the measurement of steroid hormone concentration, with the detection limit of progesterone being lower than that for a previously described analog. The Z -isomer was found to be more sensitive in FPIA compared to the E -isomer. Conclusions: Thus, we have developed an effective and universal approach to the synthesis of fluorescent conjugates with a linker length optimal for immunoassay, which improves their analytical characteristics and simplifies their preparation. The synthesized compounds have potential for practical use in clinical diagnostics and the analysis of environmental objects.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control.

A method for the determination of amitriptyline, a tricyclic antidepressant, in human urine by immunosensors has been developed using supramolecular systems based on hexamolybdenum cluster complexes. These complexes have electrochemical activity and give a stable analytical signal, which was used in the development of amperometric immunosensors. Luminescence and dynamic light scattering methods were used to demonstrate the formation of a supramolecular system of self-organized hexamolybdenum nanoparticles and chitosan molecules. A composite material based on hexamolybdenum cluster complexes in combination with reduced graphene oxide has been developed. The working range of amitriptyline concentrations to be determined by an amperometric immunosensor was 1 × 10–9–1 × 10–4 M, the limit of determination was at a level of 5 × 10–10 M, and the amitriptyline content of urine samples was at a level of (n – 7) × 10–8 M. A comparison of the results of analysis performed using an amperometric immunosensor and a fluorescence polarization immunoassay showed the absence of significant systematic errors. The ability to determine amitriptyline in biological fluids makes it possible to select an optimal therapeutic dose of the drug, that is, to develop approaches to creating personalized medicine.