The use of small (with a diameter of 7–12 nm) superparamagnetic Fe3O4 nanoparticles as carriers for antibodies in lateral flow immunoassay is considered. Increased total surface area for such suspension provides a concentration of analytes with an increase in their concentration up to 50 times. When the concentrated complexes were redissolved, aggregates with diameters of 100–500 nm were obtained, serving as colored markers in the assay. Further, magnetite–antibodies complexes are more tolerant to methanol (up to 30%) than native antibodies, thus providing minimal dilution of tested extracts. Lateral flow tests for mycotoxins zearalenone, T2-toxin, and aflatoxin B1 were developed and demonstrated applicability to control food products and raw materials.

Maximum permissible levels of mycotoxins in baby food may be 1% of those in ordinary food. Therefore, highly sensitive methods of mycotoxin control are in demand. To detect such low amounts, expensive instrumental methods are commonly used. Advantages of immunochromatographic analyses are their low cost and simple sample preparation; however, their sensitivity needs to be increased to contend with instrumental methods. A scheme for competitive immunochromatography with indirect labelling was implemented and developed for the detection of mycotoxin zearalenone (ZEA). Two separate reagents were used for the assay, namely free specific antibodies and antispecies antibodies conjugated with gold nanoparticles. This made it possible to simultaneously increase the sensitivity of the assay and the reliability of measurements. The instrumental detection limit of ZEA in baby food was 5 pg/mL (100 pg/g). Thus, the sensitivity attained is comparable with liquid chromatography characteristics. The duration of the analysis was 17 min.

Objective: The study aimed at increasing the sensitivity of immunochromatographic tests for the control of toxic contaminants (on the examples of aflatoxin B1 and T-2 toxin) in agricultural products. Methods: For reliable immunochromatographic detection of low concentrations of analytes, a replacement of the (specific antibodies – gold nanoparticle) conjugate by a combination of native specific antibodies and anti-species antibodies conjugated with gold nanoparticles was proposed. Different variants of test systems based on the principle of indirect labeling were realized and compared. Results: Immunochromatographic assays with indirect labeling for aflatoxin B1 and T-2 toxin were implemented experimentally. A reduction in the detection limit by one to two orders of magnitude was demonstrated. Conclusion: The presented results confirm that indirect labeling of specific antibodies overcomes the limitations of the competitive immunochromatographic analysis and can be used to detect analytes of different chemical nature.

Point-of-care (POC) or bedside analysis is a global trend in modern diagnostics. Progress in POC testing has largely been provided by advanced manufacturing technology for lateral flow (immunochromatographic) test strips. They are widely used to rapidly and easily control a variety of biomarkers of infectious diseases and metabolic and functional disorders, as well as in consumer protection and environmental monitoring. However, traditional lateral flow tests rely on visual assessment and qualitative conclusion, which limit the objectivity and information output of the assays. Therefore, there is a need for approaches that retain the advantages of lateral flow assays and provide reliable quantitative information about the content of a target compound in a sample mixture. This review describes the main options for detecting, processing, and interpreting immunochromatographic analysis results. The possibilities of modern portable detectors that register colored, fluorescent, magnetic, and conductive labels are discussed. Prospects for further development in this direction are also examined.

The traditional immunochromatographic assay using a conjugate of gold nanoparticles with specific ochratoxin A (OTA) antibodies and a new type of assay with indirect labeling using a combination of free antibodies and a conjugate of gold nanoparticles with anti-species antibodies were compared using the example of OTA detection. In the proposed assay, specific antibodies are included in the sample dilution buffer, which increases the duration of their interaction with the antigen, while a conjugate of anti-species antibodies with the marker is applied to the test strip. The assay was approbated for OTA detection in maize extracts. Transition to indirect labeling was shown to reduce the OTA detection limit by two orders of magnitude up to 0.12 ng/mL. The causes of this improvement are discussed. The high sensitivity of immunochromatography with indirect labeling makes it a promising approach for detection of various antigens with low molecular weight.

Spherical gold nanoparticles are the most commonly used marker in lateral flow assays. However, the widespread practice of using identical coloration for the test and control zones of test strips can lead to erroneous interpretations of the assay's results. We propose an immunochromatographic test strip with lines of different colors. For this purpose, gold nanoparticles of different shapes were used, namely blue nanoflowers in the test zone and red gold nanospheres in the control zone. A detailed synthesis procedure for nanoparticles and their conjugates is considered and design parameters for optimal results are described. For the first time, nanoparticles of different shapes have been combined in the test strip with indirect labeling of specific antibodies (via their interaction with labeled secondary antibodies). Using the T-2 toxin (T2T) as an example, an instrumental detection limit of 30 pg/ml and a working range 0.06-0.9 ng/mL were achieved in an analysis of water-organic corn extracts.

Комплексы магнитных наночастиц с антителами применяются для селективной и высокочувствительной детекции в медицинской диагностике, экологическом мониторинге, контроле качества и безопасности продукции. Магнитные наночастицы используются в качестве управляемого носителя при концентрировании пробы как твердая фаза во время анализа либо детектируемая по магнитным, оптическим или иным свойствам метка. В настоящем обзоре рассмотрены результаты современных исследований, включая способы получения аналитических реагентов, новые методы проведения анализа, варианты детекции магнитных наночастиц.

The format of an immunochromatographic multiassay is first proposed with native antisera and a universal conjugate of antispecies antibodies with gold nanoparticles. This format allows (1) the exclusion of purification and conjugation stages for specific antibodies and (2) significant reduction of the concentration of specific antibodies in the system. The independent use of specific antibodies and a conjugated marker provided a low detection limit and high signal intensity. The proposed format was implemented for the simultaneous detection of two herbicides. The instrumental limits for the detection of atrazine and chlorsulfuron were 0.1 and 0.7 ng/mL, respectively, and the analysis time was 20 min. The suitability of the test system for monitoring these herbicides in nontreated apple and blackcurrant juices is shown. The assay technique is simple, sensitive, and easily transferrable to any other antigen.

A new kind of competitive immunochromatographic assay is presented. It is based on the use of a test strip loaded with (a) labeled specific antibodies, (b) a hapten-protein conjugate at the control zone, and (c) antibodies interacting with the specific antibodies in the analytical zone. In the case where a sample does not contain the target antigen (hapten), all labeled antibodies remain in the control zone because of the selected ratio of reactants. The analytical zone remains colorless because the labeled antibodies do not reach it. If an antigen is present in the sample, it interferes with the binding of the specific antibodies in the control zone and knocks them out. Some of these antibodies pass the control zone to form a colored line in the analytical zone. The intensity of the color is directly proportional to the amount of the target antigen in the sample. The assay has an attractive feature in that an appearance in coloration is more easily detected visually than a decoloration. Moreover, the onset of coloration is detectable at a lower concentration than a decoloration. The new detection scheme was applied to the determination of the mycotoxin deoxynivalenol. The visual limit of detection is 2 ng·mL−1 in corn extracts (35 ng per gram of sample). With the same reagents, this is lower by a factor of 60 than the established test strip. The assay takes only 15 min. This new kind of assay has wide potential applications for numerous low molecular weight analytes.

Magnetic nanoparticles (MNP) are efficient molecular carriers for affine molecules. MNP complexes with antibodies can be used for the selective concentration and highly sensitive detection of various compounds. In this paper, development steps of the enzyme immunoassay using MNP are considered in details. Simple method of MNP synthesis and production of conjugates of antibodies with the aggregated MNP using physical sorption is presented. Simple, yet effective, formats of enzyme immunoassay are given. Using aflatoxin B1 detection as an example, possibility of decreasing detection limit up to 2 pg/mL, with a considerable decrease in the assay time, and performing immune interaction in the media with high organic content is shown.

In this study, we proposed a new amplification scheme to improve the sensitivity of immunochromatographic analysis (ICA) and maintain high-intensity coloration. It was based on the indirect introduction of a label into immune complexes formed via a biotin–streptavidin module. The registered signal (test zone coloration) was amplified by multiplying the label binding. During the assay, biotinylated antibodies interacted with colored conjugates of streptavidin and gold nanoparticles (GNPs), and the addition of a biotinylated protein promoted aggregation of the label. In the developed assay, the detection limit of streptomycin (STR) was 30 ng/mL; the achieved characteristics exceeded those of standard competitive ICA using antibodies linked directly to a label. Because of the universal nature of the proposed amplification scheme, it could be applied to other compounds, including antibiotics from different chemical classes. Such a sensitive ICA amplification scheme could also be considered an efficient screening tool for antibiotics in medical monitoring and food safety.

Complexes of magnetic nanoparticles with antibodies are used for the selective and highly sensitive detection in medical diagnostics, environmental monitoring, and the product quality and safety control. Magnetic nanoparticles are applied as controllable carriers to concentrate samples, as a solid phase in assays, or as labels detected by virtue of their magnetic, optical, or other properties. This review covers the results of recent studies, including methods for the preparation of analytical reagents, new assays, and techniques for the detection of magnetic nanoparticles.

Highly sensitive immunochromatographic test systems were developed for the detection of zearalenone (ZEA) and T-2 toxin (T2T) using magnetite nanoparticles (MNPs) for the labeling. In order to detect an analyte with high sensitivity, the competitive reaction was performed with free specific antibodies, while immune complexes were detected by the reaction with label-conjugated anti-species antibodies. The conditions for the synthesis of magnetite nanoparticles conjugated to anti-species antibodies were optimized. The concentrations of specific reagents that provided the highly sensitive detection of T-2 toxin and zearalenone were found. The instrumental detection limit for the determination of T-2 toxin and zearalenone in baby food samples (oat flakes) was 0.1 and 0.05 ng/mL (2.0 and 1.0 ng/g), respectively. The assay time was 15 min. The results of the present study confirm the possibility of the practical use of magnetite nanoparticles for immunochromatographic assay labeling.

This article proposes a new scheme for increasing the sensitivity of immunochromatographic assays. The scheme based on (a) recognition of dual-component gold nanoparticles (GNPs) conjugated to specific immunoglobulin G (IgG) and biotin, and (b) two additional mono-component conjugates, GNP–biotin and GNP–streptavidin. Antibodies on the surface of the GNP bind to the model antigen used here (the sepsis marker procalcitonin), and additional conjugates amplify the signal creating nanoparticle nets through biotin–streptavidin interaction. This is a one-step multi-interaction assay that requires no additional reagents or manipulations. The assay takes 15 min, and the detection limit is 3 pg mL−1 which is 30 times lower than the conventional test system with the same antibodies. Testing sera of patients with the lowest procalcitonin levels using this method confirmed the efficiency of the new enhancement scheme. In our perception, the approach is universal and can be used in many immunochromatographic assay.

A new scheme of immunochromatographic assay was developed for the highly sensitive detection of low-molecular-weight analytes. This scheme includes the following two steps: the formation of complexes of free specific antibodies with an antigen and their detection by anti-species antibodies conjugated to gold nanoparticles as the label. This scheme was tested with mycotoxin T-2 toxin in maize extracts. The use of specific antibodies and a label as two individual components made it possible to independently vary their concentrations with a simultaneous decrease in the detection limit and an increase in the color intensity. The assay did not require additional reagents and manipulations. The instrumental and visual detection limits of the designed test system were 0.1 and 5.0 ng/mL, respectively (2 and 90 ng per gram of analytes), which are two orders of magnitude lower compared to conventional immunochromatography using the same reagents.

To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts.

The scheme of immunochromatographic competitive analysis with "external" specific antibodies is offered for the first time. The test strip includes all the necessary components for analysis (membranes, protein antigen conjugates, colored nanoparticles), except specific free unconjugated antibodies that are transferred to the buffer used for sample dilution. Thus, the concentrations of the antibodies and markers can be chosen independently, and dilution of the sample with buffer (a routine procedure for any immunochromatographic test) ensures pre-incubation of the antibodies with the sample. Both factors increase the sensitivity of the test. The proposed scheme has been proven to be effective for the determination of deoxynivalenol and T-2 toxin; detection limits of 500 pg/mL and 50 pg/mL, respectively, were achieved. This is an order of magnitude lower than the limits of detection of traditional tests using the same reagents. The analysis was conducted in water-organic extracts (20% methanol); the duration of the analysis is 10 min (it's the same time as for a traditional test). The proposed approach is universal, and it can be used to detect a variety of compounds.

The development of sector of "fast-testing" i.e. test-systems permitting carrying out analysis during home visit of doctor or at primary examination of patient without any additional devices and reagents is predominant tendency in international practice. The immunochromatography is an effective technical solution in out-laboratory diagnostic, which nowadays is actively applied in controlling hundreds of diagnostically significant markers of infectious diseases, metabolic and functional disorders. However, common immunochromatography is focused on qualitative visual evaluation of results of study i.e. conclusion on presence or absence of coloration of particular zones of test-band. Therefore, the technical solutions retaining such merits of immunochromatography as expressness and technical simplicity and at the same time providing objectivity of diagnostic and increasing its informativeness are extremely in demand. The review considers main methodical solutions and tendencies of their practical implementation targeted at device documentation, processing and interpretation of results of immunochromatography analysis. The optical systems of registration in visible area of spectrum dominating in assortment of modern detectors are presented. The new solutions oriented on working with fluorescent, magnetic and electroconductive markers are presented too. The perspectives of further development of this direction are characterized including application as detectors of domestic communication devices and formation of cloud data bases for storage and processing of information concerning results of examinations.

Pseudo-homogeneous enzyme immunoassay (EIA) of aflatoxin B1 (AFB1) was accomplished using anti-AFM1 monoclonal antibodies conjugated with magnetic particles (MPs). The assay includes the concentration of AFB1 from the test sample on the surface of the MP-antibody conjugate, the binding of the AFB1-peroxidase conjugate to free sites of the antibodies, the separation of the complexes that formed from unreacted components by means of magnetic field, and the evaluation of the enzymatic activity of MP-bound peroxidase. A comparative study of antibody conjugates, which were prepared by three different methods, namely, by physical adsorption on native MP and covalent binding to oleic acidor polystyrene-coated MPs, was performed. For these conjugates, the detection limits of EIA for AFB1 are 2.6, 0.4, and 0.6 ng/mL, respectively. The advantages of the pseudo-homogeneous EIA format as a tool for the highly sensitive control of toxic contaminants in food are the shorter time of incubation of immunoassay reagents (5 min; the total assay time is 20 min) and the possibility of concentrating the analyte from the test samples.