AlSi10Mg-based composites were fabricated by laser powder bed fusion (LPBF) technique with the addition of aluminum oxide-carbon nanofiber (Al2O3-CNF) nanocomposite powder. Al2O3-CNF nanocomposite was prepared via a wet impregnation method followed by heat treatment. It consists of aggregated aluminum oxide nanoparticles with an average particle size of 8 nm uniformly distributed among the carbon nanofibers. A planetary ball mill was employed to modify AlSi10Mg powders with 0.1 wt %, 0.25 wt % and 0.5 wt % Al2O3-CNF additions in the AlSi10Mg matrix. The samples obtained with a scanning speed of 1050-1200 mm/s exhibited the highest relative density across all levels of laser power (300-375 W) while the hatch distance and layer thickness were kept constant at 120 and 40 mu m, respectively. A finer sub-cell size was observed only in the AlSi10Mg/Al2O3-CNF samples containing 0.1 wt % and 0.25 wt % of the modified additive. The microhardness of the samples reinforced with 0.1 wt % and 0.25 wt% Al2O3-CNF increased by 27.0 % and 17.5 %, respectively, compared to the values exhibited by AlSi10Mg without the additive. However, samples with higher additive content showed comparable or lower hardness values. A part of CNFs was reacted with aluminum during selective laser melting, transforming into Al4C3. The combination of CNFs, Al2O3 nanoparticles and formed Al4C3 contributed to improvements in the mechanical properties of LPBF fabricated AlSi10Mg/Al2O3-CNF composites through the fine grain strengthening, second phase strengthening and load transfer strengthening.

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.

Brucellosis is a dangerous zoonotic disease caused by bacteria of the genus Brucella. Diagnosis of brucellosis is based on the detection in animal and human sera of antibodies to the O-polysaccharide of Brucella lipopolysaccharide. The currently employed serodiagnosis of brucellosis relies on the use of the Brucella O-polysaccharide as a diagnostic antigen. However, the existence of bacterial species, which also express O-polysaccharides structurally similar to that of Brucella, may decrease the specificity of the brucellosis detection due to false-positive test results. It has been shown that the efficiency of the test can be significantly improved by using synthetic oligosaccharides that correspond to the so-called M epitope of the Brucella O-antigen. This epitope is characterized by an α-(1→3)-linkage between d-perosamine units and is unique to Brucella. Here we report on an efficient approach to the synthesis of oligosaccharides that model the M epitope of the Brucella O-polysaccharide. The approach is based on the use of the α-(1→3)-linked disaccharide thioglycoside as the key donor block. Its application allowed the straightforward assembly of a set of four protected oligosaccharides, which includes a disaccharide, two trisaccharides, and a tetrasaccharide, in five glycosylation steps. The synthesized oligosaccharides are planned to be used in the development of diagnostic tools for identifying brucellosis in humans and domestic animals, as well as a potential vaccine against it.

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.

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.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden’s method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

A fluorescence polarization immunoassay method has been developed for the determination of antibiotic tylosin in honey. For this purpose, rabbits were immunized with a conjugate of tylosin and bovine serum albumin, and antisera were obtained. The conditions for the interaction of antisera in a competitive format with tylosin in the sample and with tylosin labeled with fluorescein were optimized. The developed method is characterized by a limit of detection for tylosin of 34.7 ng/mL and an analytical range of 65.2 to 564.0 ng/mL. High reproducibility of measurements using a portable detector was demonstrated, with relative standard deviation (RSD) values of 1.5–2.5

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer–fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

A method for the synthesis of ketosteroid oxime conjugates with a fluorescein (6-FAM) derivative connected via a short bifunctional aminooxypropyl linker is proposed. The conjugates were used as tracers for fluorescence polarization immunoassay (FPIA) with monoclonal antibodies. Stereoisomers by the oxime group were separated by preparative RP TLC and HPLC and tested in the FPIA method. Binding parameters of of tracers with monoclonal antibodies (mAbs) to progesterone were studied. Tracers showed high efficiency in the analysis of steroid hormones, the detection limit of progesterone being lower than that for a previously described analog. The Z-isomer was found to be more sensitive in the FPIA analysis compared to the E-isomer.

Biosensor devices that include hybrid nanostructures as transducer surface modifiers meet current requirements for methods of research and determination of drugs, including antidepressants. Here, we consider the features of amperometric monoamine oxidase biosensors based on screen-printed graphite electrodes modified with nanocomposite consisting of C60/cobalt nanoparticles/amino derivative of a second-generation polyether polyol/chitosan in the determination of the tricyclic antidepressant amitriptyline. The best modifier was selected using transmission electron microscopy, scanning electron microscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry. In the biosensor development, the conditions for applying the composite based on cobalt nanoparticles/amino derivative of polyether polyol to the electrode surface were varied: electrochemical deposition, sequential deposition by the layer-on-layer method, and deposition of a mixture. As an analytical signal of the biosensor, we used the peak of the electrochemical oxidation of hydrogen peroxide, which is formed during the enzymatic oxidation of serotonin under the action of monoamine oxidase. The operating principle of the biosensor is based on the inhibitory effect of amitriptyline on the catalytic activity of immobilized monoamine oxidase. For the selected modifier, the determined concentration range of amitriptyline is 1 × 10–4–1 × 10–8 mol/L and the lower limit of the determined contents is 5 × 10–9 mol/L under optimal operating conditions. Comparison of the results of the amitriptyline determination in a pharmaceutical preparation and urine that were obtained using a monoamine oxidase biosensor and the method of fluorescence polarization immunoassay (dilution of the tracer of 1 : 32, dilution of antibodies of 1 : 128, range of working concentrations from 5 × 10–8 to 5 × 10–9 mol/L), which has proven itself in the determination of medicinal substances, confirmed the correctness of the developed method.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular reproductive functions and fetal development, the content of DBP must be controlled in food and the environment. The present study aims to develop a sensitive, fast and simple fluorescence polarization immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this chemical with antibodies are studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitivity analysis-16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (DBP concentration at which a 50% decrease in the fluorescence polarization signal occurs, 40 ng/mL) and the limit of detection (LOD, 7.5 ng/mL) values were improved by a factor of 45 over the previously described FPIA using polyclonal antibodies. This technique was tested by the recovery method, and the high percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS. Thus, the FPIA method developed in this work can be used to determine DBP in open-water reservoirs.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular, reproductive function and fetal development, the content of DBP must be controlled in food and environmental objects. The present study aims to develop a sensitive, fast and simple polarization fluorescent immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this drug with antibodies is studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitive analysis - 16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (40 ng/mL) and LOD (10 ng/mL) values were improved by a factor of 35 for the previously described FPIA using polyclonal antibodies. This technique was tested by the added/found method, the percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS/MS. Thus, the FPIA method developed in this work can be used to determine DBP in open water reservoirs.

Conditions are studied for the synthesis of carbon quantum dots (CQD) exhibiting green fluorescence by the ultrasonic treatment of multilayered carbon nanotubes in a mixture HNO3 with H2SO4 (CQD-1) and by the microwave decomposition of 9,10-dinitroanthracene in ethanol (CQD-2). The sizes of COD are estimated by atomic force microscopy, and their optical properties, by spectrophotometry and fluorescence spectrometry. Infrared spectrometry was used to identify active functional groups on the CQD surface, participating in the formation of stable bonds in the synthesis of tracers. A possibility of using CQD as labels in fluorescence polarization immunoassay (FPIA) is shown. Procedures are developed for determining aminoglycoside antibiotics gentamycin, streptomycin, and amikacin by FPIA using a TDx-analyzer (Abbott Diagnostics, United States); their performance characteristics are presented. The limit of detection is (ng/mL) 20, 10, and 3, and the analytical range (µg/mL) is 0.05–3.00, 0.02–6.00, and 0.01–3.00 for gentamycin, streptomycin, and amikacin respectively. The procedures are tested in the determination of gentamycin, streptomycin, and amikacin in dairy products.

In this study, a homogeneous fluorescence polarization immunoassay (FPIA) for the detection of hazardous aquatic toxin okadaic acid (OA) contaminating environmental waters was for the first time developed. A conjugate of the analyte with a fluorophore based on a fluorescein derivative (tracer) was synthesized, and its interaction with specific anti-OA monoclonal antibodies (MAbs) was tested. A MAbs-tracer pair demonstrated highly affine immune binding (KD = 0.8 nM). Under optimal conditions, the limit of OA detection in the FPIA was 0.08 ng/mL (0.1 nM), and the working range of detectable concentrations was 0.4-72.5 ng/mL (0.5-90 nM). The developed FPIA was approbated for the determination of OA in real matrices: river water and seawater samples. No matrix effect of water was observed; therefore, no sample preparation was required before analysis. Due to this factor, the entire analytical procedure took less than 10 min. Using a compact portable fluorescence polarization analyzer enables the on-site testing of water samples. The developed analysis is very fast, easy to operate, and sensitive and can be extended to the determination of other aquatic toxins or low-molecular-weight water or food contaminants.

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and antiLF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.