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Lateral flow immunoassay (LFIA) has found a broad application for testing in point-of-care (POC) settings. LFIA is performed using test strips—fully integrated multimembrane assemblies containing all reagents for assay performance. Migration of liquid sample along the test strip initiates the formation of labeled immunocomplexes, which are detected visually or instrumentally. The tradeoff of LFIA's rapidity and user-friendliness is its relatively low sensitivity (high limit of detection), which restricts its applicability for detecting low-abundant targets. An increase in LFIA's sensitivity has attracted many efforts and is often considered one of the primary directions in developing immunochemical POC assays. Post-assay enhancements based on chemical reactions facilitate high sensitivity. In this critical review, we explain the performance of post-assay chemical enhancements, discuss their advantages, limitations, compared limit of detection (LOD) improvements, and required time for the enhancement procedures. We raise concerns about the performance of enhanced LFIA and discuss the bottlenecks in the existing experiments. Finally, we suggest the experimental workflow for step-by-step development and validation of enhanced LFIA. This review summarizes the state-of-art of LFIA with chemical enhancement, offers ways to overcome existing limitations, and discusses future outlooks for highly sensitive testing in POC conditions. [ABSTRACT FROM AUTHOR]

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen–antibodies–labeled immunoglobulin-binding protein (Scheme A); antigen–antibodies–labeled antigen (Scheme B); and immunoglobulin-binding protein–antibodies–labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases. [ABSTRACT FROM AUTHOR]

Finding optimal conditions for competitive lateral flow immunoassay is a controversial task. The content of specific antibodies labeled by nanoparticles should be simultaneously high to reach intense signals and low to register an influence on the signals for minimal concentrations of the target analyte. We propose to use two kinds of complexes of gold nanoparticles in the assay, with antigen–protein conjugates and with specific antibodies. The first complex interacts both with immobilized antibodies in the test zone and with antibodies on the surface of the second complex. In this assay, the coloration is enhanced by the binding of two-colored preparations in the test zone, whereas the antigen in the sample inhibits both the binding of the first conjugate with the immobilized antibodies and with the second conjugate. This approach is realized for the detection of insecticide imidacloprid (IMD), an important toxic contaminant connected with the recent global death of bees. The proposed technique expands the working range of the assay, that is, in accordance with its theoretical analysis. The reliable change of coloration intensity is achieved for a 2.3-times-lower concentration of the analyte. The limit of IMD detection is 0.13 ng/mL for tested solutions and 1.2 µg/kg for initial honey samples. The combination of two conjugates doubles the coloration in the absence of the analyte. The developed lateral flow immunoassay is applicable for five-fold-diluted honey samples without extraction, does not require additional stages (all reagents are pre-applied to the test strip), and is implemented in 10 min. [ABSTRACT FROM AUTHOR]

Changes in the limits of detection (LODs) for a multiplex lateral flow immunoassay (LFIA) caused by different locations of the binding zone on the test strips were studied. Due to the non-equilibrium conditions of the immune reactions in LFIAs, their analytical parameters are susceptible to the binding constants of antigen–antibody reactions and assay duration. Consequently, the integration of several tests into one multiplex assay can cause a significant worsening of the sensitivity. In this study, we propose a simple methodology for the determination of the best arrangement of binding zones, which takes into account the binding constants for immunoreagents. LFIAs of four mycotoxins, namely, aflatoxin B1, deoxynivalenol, T-2 toxin, and ochratoxin A, were integrated into a multiplex test strip. An enzyme-linked immunosorbent assay was applied to determine the equilibrium and kinetic constants of the immunoreactants for each analyte. It was found that the arrangement of binding zones with a descending order of the equilibrium association constants was optimal and provided both lower detection limits and a more uniform coloration. The selected position of the binding zones allowed decreasing the LODs down to 2 and 27 times for ochratoxin A and deoxynivalenol, respectively. The proposed approach can be applied to multiplex LFIAs for different analytes. [ABSTRACT FROM AUTHOR]

The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes. [ABSTRACT FROM AUTHOR]

In this study, a lateral flow immunoassay (LFIA) was developed to detect okadaic acid (OA) belonging to the diarrheic shellfish poisoning group of aquatic toxins. Newly obtained anti-OA monoclonal antibodies and bimetallic core@shell Au@Pt nanoparticles were used in the indirect format of the LFIA. Peroxidase-mimicking nanozyme properties of Au@Pt enabled using them to enhance band coloration on the test strips and, consequently, for increasing the LFIA sensitivity. The instrumental limit of detection (LOD), the working range of detectable concentrations, and the visual cutoff of the assay were 0.5, 0.8–6.8, and 10 ng/mL, respectively. The assay duration was 20 min. The rapid and simple sample preparation procedure was applied for seawater, river water, and fish samples. The total duration of the sample pretreatment and LFIA was 25/40 min for water/fish samples, ensuring testing rapidity. The developed test system provides sensitive control of raw materials and food products and can be used to detect OA at all stages of the food industry «from sea to fork» chains. [ABSTRACT FROM AUTHOR]

The presence of pathogen-specific antibodies in the blood is widely controlled by a serodiagnostic technique based on the lateral flow immunoassay (LFIA). However, its common one-stage format with an antigen immobilized in the binding zone of a test strip and a nanodispersed label conjugated with immunoglobulin-binding proteins is associated with risks of very low analytical signals. In this study, the first stage of the immunochromatographic serodiagnosis was carried out in its traditional format using a conjugate of gold nanoparticles with staphylococcal immunoglobulin-binding protein A and an antigen immobilized on a working membrane. At the second stage, a labeled immunoglobulin-binding protein was added, which enhanced the coloration of the bound immune complexes. The use of two separated steps, binding of specific antibodies, and further coloration of the formed complexes, allowed for a significant reduction of the influence of non-specific immunoglobulins on the assay results. The proposed approach was applied for the serodiagnosis using a recombinant RBD protein of SARS-CoV-2. As a result, an increase in the intensity of test zone coloration by more than two orders of magnitude was demonstrated, which enabled the significant reduction of false-negative results. The diagnostic sensitivity of the LFIA was 62.5% for the common format and 100% for the enhanced format. Moreover, the diagnostic specificity of both variants was 100%. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Food colorants are inalienable part of human life. Since ancient times, they have become firmly established and have undergone a number of changes. During this time, the attitude towards them has changed. The desire to color the food now raises questions about the various side effects of their use. With the development of methods of toxicology and obtaining data on the toxicity of food coloring, restrictions and standards for their content in food have emerged. The development of methods for the analysis of dyes in various food products is becoming increasingly in demand. Development of screening immunoanalytical methods such as enzyme-linked immunoassay and immunochromatographic one suitable for simultaneous analysis of a large number of samples at the same time allows to avoid serious costs when operating the chromatographic equipment. Reduction of the analysis time and simple sample preparation is what unites immunoassay techniques. This review shows the possibilities of modern immunoanalytical methods for analyzing samples and determining food dyes belonging to different classes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] We review recent progress in research on immunochromatographic analyses. [•] We discuss the advantages and the disadvantages of immunochromatographic systems. [•] We discuss strategies to reduce the limits of detection and to increase accuracy. [•] We describe approaches used for simultaneous analysis of multiple compounds. [•] We describe commercially-available tests and proposed directions for future research. [ABSTRACT FROM AUTHOR]

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems. [ABSTRACT FROM AUTHOR]

Many studies have found that gold nanoparticles with branched surfaces (nanoflowers) are markers for immunosensors that provide higher sensitivity gains than the commonly used spherical gold nanoparticles. Although the analytical characteristics of nanoparticle-using systems vary significantly depending on their size and shape, the question of choosing the best gold nanoflowers remains open. This work presents a comparative study of a panel of 33 preparations of gold nanoflowers formed by varying several parameters: the size of spherical nanoparticles-nuclei, the concentrations of nuclei, and tetrachloroauric acid during growth. The sizes of the resulting particles, their sorption capacity under antibody immobilization, mobility along membranes for lateral flow assays, and the effects of these parameters on the limits of detection of lateral flow immunoassay are characterized. The optimality of preparations obtained by growing a 0.2% v/v solution of nuclei with a diameter of 10 or 20 nm with tetrachloroauric acid at a concentration of 0.12 mM was shown. With their use, lateral flow immune tests were developed to determine markers of acute myocardial infarction—fatty acids binding protein and troponins I and T. The use of gold nanoflowers obtained under the proposed protocols led to significant gains in the limits of detection—3 to 10 times under visual detection and over 100 times under instrumental detection—compared to spherical gold nanoparticles. The significant increase under instrumental detection is due to the label's low nonspecific binding. [ABSTRACT FROM AUTHOR]

Three techniques were compared for lowering the limit of detection (LOD) of the lateral flow immunoassay (LFIA) of the receptor-binding domain of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) based on the post-assay in situ enlargement of Au nanoparticles (Au NPs) on a test strip. Silver enhancement (growth of a silver layer over Au NPs—Au@Ag NPs) and gold enhancement (growth of a gold layer over Au NPs) techniques and the novel technique of galvanic replacement of Ag by Au in Au@Ag NPs causing the formation of Au@Ag-Au NPs were performed. All the enhancements were performed on-site after completion of the conventional LFIA and maintained equipment-free assay. The assays demonstrated lowering of LODs in the following rows: 488 pg/mL (conventional LFIA with Au NPs), 61 pg/mL (silver enhancement), 8 pg/mL (galvanic replacement), and 1 pg/mL (gold enhancement). Using gold enhancement as the optimal technique, the maximal dilution of inactivated SARS-CoV-2-containing samples increased 500 times. The developed LFIA provided highly sensitive and rapid (8 min) point-of-need testing. [ABSTRACT FROM AUTHOR]

Determination of the presence in the blood of antibodies specific to the causative agent of a particular disease (serodiagnosis) is an effective approach in medical analytical chemistry. Serodiagnostics performed in the lateral flow immunoassay format (immunochromatography) meet the modern requirements for point-of-care testing and are supported by existing technologies of large-scale diagnostic tests production, thus increasing the amount of attention in a tense epidemiological situation. For traditional lateral flow serodiagnostics formats, a large number of nonspecific immunoglobulins in the sample significantly reduces the degree of detectable binding. To overcome these limitations, an assay based on the formation of immobilized antigen-specific antibody-labeled antigen complexes detection was proposed. However, the requirements for its implementation, providing maximum sensitivity, have not been established. This article describes the mathematical model for the above assay. The influence of the ratio of reagent concentrations on the analysis results is considered. It is noted that the formation of specific antibody complexes with several labeled antigens is the main limiting factor in reducing the detection limit, and methods are proposed to minimize this factor. Recommendations for the choice of the assay conditions, following from the analysis of the model, are confirmed experimentally. [ABSTRACT FROM AUTHOR]

The use of lateral flow immunoassays (LFIAs) for rapid on-site testing is restricted by their relatively high limit of detection (LoD). One possible way to decrease the LoD is to optimize nanoparticle properties that are used as labels. We compare two types of Au nanoparticles: usual quasispherical gold nanoparticles (C-GNPs), obtained by the Turkevich–Frens method, and superspherical gold nanoparticles (S-GNPs), obtained by a progressive overgrowth technique. Average diameters were 18.6–47.5 nm for C-GNPs and 20.2–90.4 nm for S-GNPs. Cardiomarker troponin I was considered as the target analyte. Adsorption and covalent conjugation with antibodies were tested for both GNP types. For C-GNPs, the minimal LoD was obtained with 33.7 nm nanoparticles, reaching 12.7 ng/mL for covalent immobilization and 9.9 ng/mL for adsorption. The average diameter of S-GNPs varied from 20.2 to 64.5 nm, which resulted in a decrease in LoD for an LFIA of troponin I from 3.4 to 1.2 ng/mL for covalent immobilization and from 2.9 to 2.0 ng/mL for adsorption. Thus, we obtained an 8-fold decrease in LoD (9.9 to 1.2 ng/mL) by using S-GNPs. This effect can be related to more effective antibody immobilization and improved S-GNP optical properties. The obtained results can improve LFIAs for various practically significant analytes. [ABSTRACT FROM AUTHOR]

The presented study is focused on the impact of binding zone location on immunochromatographic test strips on the analytical parameters of multiplex lateral flow assays. Due to non-equilibrium conditions for such assays the duration of immune reactions influences significantly the analytical parameters, and the integration of several analytes into one multiplex strip may cause an essential decrease of sensitivity. To choose the best location for binding zones, we have tested reactants for immunochromatographic assays of lincomycin, chloramphenicol, and tetracycline. The influence of the distance to the binding zones on the intensity of coloration and limit of detection (LOD) was rather different. Basing on the data obtained, the best order of binding zones was chosen. In comparison with non-optimal location the LODs were 5–10 fold improved. The final assay provides LODs 0.4, 0.4 and 1.0 ng/mL for lincomycin, chloramphenicol, and tetracycline, respectively. The proposed approach can be applied for multiplexed assays of other analytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Two kinds of gold nanoparticles were applied in one test strip. • Shape (spheres or flowers) of the nanoparticles determines their color (red or blue). • Two-color lateral flow assay was realized for T-2 toxin. • Different coloration of formed lines simplifies interpretation of the assay results. [ABSTRACT FROM AUTHOR]

Hydrochlorothiazide, an antihypertensive and diuretic drug only for clinic purpose, was recently found as an adulterant in functional foods. Due to the potential health risk, it is urgent to develop a simple and rapid assay for its identification. In this work, two hapten structures were rationally designed based on molecular modeling, and a highly sensitive antibody to hydrochlorothiazide was obtained with 50% inhibitive concentration (IC 50) of 6.7 ng/mL for the first time. A lateral flow strip assay was then developed based on time resolved fluorescence, demonstrating a cut-off value of 250 μg/kg to hydrochlorothiazide in functional capsules and tablets, and a negligible cross-reactivity to related compounds. The strip performance for real samples collected from the retail markets was consistent of that of confirmed method (LC-MS/MS). As an ideal tool, the proposed strip could be applied to identify adulterated hydrochlorothiazide in functional foods. [Display omitted] • Two novel hydrochlorothiazide haptens were rationally designed assisted by computer molecular modeling. • A novel antibody against hydrochlorothiazide was produced with high specificity and sensitivity. • A easy-use lateral flow strip was developed for identifying emergent adulterant hydrochlorothiazide in foods. [ABSTRACT FROM AUTHOR]

Immunoreagents with good results in the competitive enzyme-linked immunosorbent assay are often unable to provide the required detection limit in the traditional competitive immunochromatographic assay. The solution may be either the production of new reagents or improving the test strip. In the latter case, the assay is often performed stepwise using additional liquid reagents, but this is a significant drawback for practical use. We introduce a test strip made as a dry chemical device that still provides the two-step immunochemical interactions — formation of a complex of specific antibodies with an antigen and its detection by a conjugate of antispecies antibodies with a nano-sized label. Analysis with this test strip is similar to that with ordinary test strips and requires no additional reagents and manipulation. The use of specific antibodies and marker as two separate components allows to improve the analytical parameters. The new test significantly lowers the limit of detection, making it possible to use antibodies previously ineffective in immunochromatography. The proposed approach was tested by determining zearalenone and aflatoxin B1 mycotoxins. The visual limit of detection for aflatoxin B1 decreased to 0.6 ng/mL compared to 11 ng/mL with an ordinary test strip. For zearalenone, a test strip was created with visual detection limit of 6 ng/mL with reagents inefficient in the traditional test strip (which is not able to detect even 9 μg/mL of zearalenone). Thus, the proposed approach allows obtaining ‘dry’, multi-stage, immunochromatographic test strips, providing a highly sensitive detection method. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×10 2 CFU/mL; 20 min) in comparison with the common analysis (LOD 2×10 3 CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965. [ABSTRACT FROM AUTHOR]