Double lateral flow immunosensing of undeclared pork and chicken components of meat products.

Adulteration of meat products is a serious problem in the modern society. Consumption of falsified meat products can be hazardous to health and/or lead to violating religious dietary principles. To identify such products, rapid and simple test systems for point-of-need detection are in demand along with complex laboratory methods. This study presents the first double lateral flow (immunochromatographic) test system, which allows simultaneous revealing two prevalent types of falsifications-undeclared addition of pork and chicken components to meat products. In the proposed test system, porcine myoglobin (MG) and chicken immunoglobulin Y (IgY) were used as specific biomarkers recognizable by antibodies. Within the optimization of the analysis, the concentrations of the immune reagents and regimes of their application on the working membrane were selected, which provided minimal limits of detection (LODs) for both analytes. The developed test system enables the detection of MG and IgY with the LODs of 10 and 12 ng/mL, respectively, which accords to addition of 0.1% of the undeclared meat compounds. The applicability of the test system to control the composition of raw meat mixtures and cooked food products was confirmed. The developed approach can be considered as a promising tool for monitoring composition of meat products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05944-y.

Comparison of competitive and sandwich immunochromatographic analysis in the authentication of chicken in meat products.

Cheap chicken meat is often used as an undeclared substitute in meat products. In this study, two formats of the immunochromatographic assay (ICA) of immunoglobulins of class Y (IgY) as a biomarker for chicken authentication were developed. In both competitive ICA (cICA) and sandwich ICA (sICA), gold nanoparticles (GNP) were conjugated with anti-species antibodies. A simple procedure of sample preparation, which took only 30 min, was proposed. Test systems demonstrated high sensitivity and rapidity: visual limits of detection of IgY and assay durations were 12/14 ng/mL and 10/15 min for cICA and sICA, respectively. The absence of cross-reactivity with the mammalian species confirmed the high specificity of the test systems. Good applicability of the assays was confirmed for the detection of chicken in raw meat mixtures: as low as 3% and 0.2% (w/w) of chicken could be revealed in beef and pork by cICA and sICA, respectively. The influence of heat processing of meat-based products on immune recognition and, consequently, the analytical performance of the test systems was revealed. It was shown that sICA is preferable for the detection of IgY even in thermally processed meat. The proposed ICAs can be recommended for rapid on-site control of meat products' composition.

Sensitive immunoenzyme assay for the detection of antibiotic flumequine in honey.

Fluoroquinolone antibiotics are used to cure and protect bees and apiaries from infections. Consequently, they may contaminate honey and other products of beekeeping. In this study, a highly sensitive immunoenzyme assay (EIA) was for the first time developed for the determination of a fluoroquinolone flumequine (FLU) in honey. The EIA was carried out in an indirect competitive format with colorimetric detection. The analysis was characterized by a low limit of detection of 30 pg mL-1. The polyclonal antibodies used showed no cross-reactivity with 24 other (fluoro)quinolones; the assay was highly specific only toward FLU. Different coating FLU-protein conjugates were tested to achieve the most sensitive competitive immunodetection. A highly simplified and rapid (3-5 min) sample preparation was proposed based on the 100-300 times dilution of honey by a buffer. The developed EIA has been tested to detect FLU in honey of different origins, namely acacia, flower, buckwheat, chestnut, and linden honey. It has been demonstrated that 76.2-115.9% of FLU could be determined by the assay. The versatility, simplicity, and rapidity of the EIA enable us to propose this method as an effective tool to control the contamination of honey.

Sensitive Immunochromatographic Determination of Salmonella typhimurium in Food Products Using Au@Pt Nanozyme.

In this study, we developed a sensitive immunochromatographic analysis (ICA) of the Salmonella typhimurium bacterial pathogen contaminating food products and causing foodborne illness. The ICA of S. typhimurium was performed using Au@Pt nanozyme as a label ensuring both colorimetric detection and catalytic amplification of the analytical signal due to nanozyme peroxidase-mimic properties. The enhanced ICA enabled the detection of S. typhimurium cells with the visual limit of detection (LOD) of 2 × 102 CFU/mL, which outperformed the LOD in the ICA with traditional gold nanoparticles by two orders of magnitude. The assay duration was 15 min. The specificity of the developed assay was tested using cells from various Salmonella species as well as other foodborne pathogens; it was shown that the test system detected only S. typhimurium. The applicability of ICA for the determination of Salmonella in food was confirmed in several samples of milk with different fat content, as well as chicken meat. For these real samples, simple pretreatment procedures were proposed. Recoveries of Salmonella in foodstuffs were from 74.8 to 94.5%. Due to rapidity and sensitivity, the proposed test system is a promising tool for the point-of-care control of the Salmonella contamination of different food products on the whole farm-to-table chain.

A Sensitive Fluorescence Polarization Immunoassay for the Rapid Detection of Okadaic Acid in Environmental Waters.

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.

Highly Sensitive Immunochromatographic Detection of Porcine Myoglobin as Biomarker for Meat Authentication Using Prussian Blue Nanozyme.

This study was aimed at the sensitive immunodetection of porcine myoglobin (MG) as a species-specific biomarker in meat products. The enhanced lateral flow immunoassay (LFIA) was created in the sandwich format using monoclonal antibodies (Mab) with specificity to porcine MG and labeled by Prussian blue nanoparticles (PBNPs) as peroxidase-mimicking nanozymes. Signal amplification was provided by the colored product of oxidation catalyzed by the PBNPs. Several Mab-PBNP conjugates with different antibody loads were synthesized; the one that provided the best analytical characteristics of the LFIA was selected. Advanced optimization of the test system was carried out. As a result, the visual limit of detection (LOD) of MG was 1.5 ng/mL. Involvement of the catalytic nanozyme properties allowed the LOD to be decreased by ~9 times in comparison to the LFIA based on gold nanomarkers, and by ~27 times compared to the LFIA based on PBNP coloration. The assay time was 30 min, including catalytic enhancement. A simple technique of meat sample pre-treatment aimed at effective MG extraction and matrix disposal was proposed. The specificity of the LFIA towards the pork meat was demonstrated. The applicability of the created test system was shown by testing extracts obtained from finished meat products.

Application of Au@Pt Nanozyme as Enhancing Label for the Sensitive Lateral Flow Immunoassay of Okadaic Acid.

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 fork¼ chains.

Lateral Flow Test System to Control Total Content of Muscle Tissues in Raw Meat Products.

Assessment of the composition of meat-containing products is the task in demand due to their frequent deviations from declared recipes. The paper presents the developed test system for immunochromatographic determination of total meat content. The assay is based on the simultaneous use of monoclonal antibodies, which specifically interacts with mammalian skeletal troponin I, and polyclonal antibodies, which specifically detect bird immunoglobulin Y. To integrate the detection of both types of meat by the same test strip, the antibodies are mixed in the analytical zone of the test strip and in complex with a gold nanoparticle label. The chosen ratios of the antibodies for both mixtures provide the same contribution of different types of mammalian and bird raw materials of muscle tissues to the label binding. The test system demonstrates suitability for products containing beef, pork, rabbit, lamb, chicken, and turkey meat. The minimal detectable content of meat in samples is 0.1%. The samples for the testing are diluted 100 times, thus eliminating matrix effects, and providing high reproducibility of the color intensity for extracts of different compositions. The obtained results allow the recommendation of the developed test system for rapid on-site control of meat products.

Triple immunochromatographic test system for detection of priority aquatic toxins in water and fish.

Aquatic toxins are a group of toxic compounds produced by several types of freshwater and marine algae and cyanobacteria and transported through the food chains of water bodies. Potential contamination of aquaculture products (raw and processed fish and seafood) with aquatic toxins requires the use of efficient screening methods for their control. In this study, a multiplex immunochromatographic test system for the simultaneous detection of three aquatic toxins-phycotoxins domoic acid (DA) and okadaic acid (OA), and cyanotoxin microcystin-LR (MC-LR)-is for the first time developed. For this, a competitive indirect immunochromatographic analysis (ICA) based on gold-labeled secondary antibodies was carried out. The LODs/cutoffs/working ranges of the ICA were 0.05/0.3/0.07-0.29, 1.3/100/3.2-58.2, and 0.1/2.0/0.2-1.1 ng/mL for MC-LR, DA, and OA, respectively. The assay duration was 18 min. The developed test system was used to analyze water samples from natural sources (salt and fresh water) and fish samples. For sample preparation of water, simple dilution with a buffer was proposed; for fish samples, methanol-water extraction was utilized. It was demonstrated that the triple LFIA specifically detected target aquatic toxins with recoveries of 85.0-121.5%. The developed multiplex LFIA can be considered a promising analytical solution for the rapid, easy, and sensitive control of water and food safety.

Double Immunochromatographic Test System for Sensitive Detection of Phycotoxins Domoic Acid and Okadaic Acid in Seawater and Seafood.

In this investigation, a double immunochromatographic analysis (ICA) of two relevant phycotoxins, domoic acid (DA) and okadaic acid (OA), was developed for the first time. The ICA was performed in the indirect competitive format using gold nanoparticles conjugated with anti-species antibodies. Under optimal conditions, the instrumental detection limits/cutoffs for simultaneous detection of DA and OA were 1.2/100 and 0.1/2.5 ng/mL, respectively. The time of the assay was 18 min. The ICA was applied to test seawater and a large panel of seafood, including mussels, tiger shrimps, octopuses, whelks, crabs, and scallops. The proposed simple sample preparation method for seafood takes only 20 min. For seawater, a dilution by buffer was implemented. The assay recoveries varied from 80.8% to 124.5%. The competitive potential of the proposed technique as a tool to control natural water and seafood samples is determined by its simplicity, rapidity, and sensitivity.

Cascade-Enhanced Lateral Flow Immunoassay for Sensitive Detection of Okadaic Acid in Seawater, Fish, and Seafood.

In this investigation, a new approach for developing a sensitive lateral flow immunoassay (LFIA) was proposed for the detection of the hazardous marine toxin okadaic acid (OA). It is based on the indirect format with anti-species antibodies labeled by gold nanoparticles (AuNPs) and cascade signal amplification. The latter is performed by first passing a mixture of anti-OA antibodies and a tested sample along the immunochromatographic test strip and then performing several cycles of the interaction of anti-species antibodies conjugated with AuNPs with free antibodies, which bind to anti-species antibodies but are not specific to the target analyte. As a result, branched aggregates are formed, due to which the colorimetric signal intensification occurs. The developed test system enabled the detection of OA with an instrumental detection limit of 30 pg/mL and a cutoff of 1 ng/mL, which exceeds these characteristics in the LFIA without amplification by 7 and 2 times, respectively. The OA recoveries from seawater, fish, and seafood varied from 76.9% to 126%. The test system may be required for point-of-care monitoring of samples for phycotoxin contamination; the developed principle of signal amplification can be used in cases where highly sensitive detection of trace amounts of a contaminant is required.

Rapid detection of phycotoxin domoic acid in seawater and seafood based on the developed lateral flow immunoassay.

A lateral flow immunoassay (LFIA) of phycotoxin domoic acid (DA) contaminating seawater and marine organisms was developed in this investigation. Nine clones of monoclonal antibodies against DA were produced and characterized. The test system was implemented in the indirect competitive format, where gold nanoparticles as a marker were conjugated with secondary antibodies. The developed test system allows for the detection of DA with a cutoff of 60 ng mL-1 and an instrumental detection limit of 1.4 ng mL-1 within 15 min. The LFIA was applied to detect DA in seawater, mussels, shrimps, and octopuses. A simple method of seafood sample preparation was proposed. The entire analytical cycle, from obtaining a sample to the estimation of final results, takes only 30 min. The assay recoveries ranged from 88.5% to 124%. The developed analytical method is a promising solution for rapid on-site monitoring of marine toxicants in water and food throughout the farm-to-fork chain.

Sensitive lateral flow immunoassay for the detection of pork additives in raw and cooked meat products.

In this study, we developed a lateral flow immunoassay (LFIA) for the detection of pork additives in meat products. LFIA of porcine immunoglobulins (IgG) as a molecular biomarker was carried out in a sandwich format for species identification. Gold nanoparticles as a nano-dispersed label were conjugated to secondary antibodies specific to anti-porcine IgG. The test system was characterized by high specificity, which was confirmed by the absence of cross-reactivity with any other species tested. A short technique of sample preparation was proposed aimed at the effective extraction of IgG from meat samples. The developed LFIA enabled the detection of a pork ingredient at a level as low as 0.063% (w/w) in raw meat within 35 min including sample preparation. A large panel of real meat samples was analyzed by the LFIA. The results showed that porcine IgG can be reliably recognized both in raw meat products and processed meat foodstuffs.

Sensitive lateral flow immunoassay of an antibiotic neomycin in foodstuffs.

Aminoglycosides belong to a class of antibiotics now widely used in agriculture and veterinary medicine and expected to contaminate food products. In this study, a sensitive lateral flow immunoassay (LFIA) of an aminoglycoside neomycin (NEO) was developed. Two methods of immunochromatographic detection based on various techniques of gold nanoparticles (AuNPs) introduction as a label were compared. It was demonstrated that the indirect labeling (a conjugation of anti-species antibodies with a marker) allowed for an increase in assay sensitivity by 80 times. The test system was characterized by an instrumental limit of detection of 0.1 ng/mL and the cutoff level of 10 ng/mL; the assay duration was 15 min. Specificity only toward NEO was demonstrated. The developed LFIA has been tested to detect NEO in different foodstuffs. It has been demonstrated that 70-119% of NEO (coefficients of variations < 10%) can be determined in milk, turkey meat, honey, and eggs using simple procedures of preliminary sample preparation. Testing the samples showed the coincidence of the results for the developed lateral flow assay and for commercial ELISA kit.

Lateral flow immunoassay for sensitive detection of undeclared chicken meat in meat products.

This study presents the development of an immunochromatographic test system aimed at the detection of chicken additives in meat products. It is based on sandwich-format lateral flow immunoassay (LFIA) of immunoglobulins as a biomarker for species identification. The LFIA based on gold nanoparticles as a label for anti-species antibodies was used to determine chicken immunoglobulins and, accordingly, chicken meat in food products. Absence of cross-reactivity with mammalian species tested in the study confirmed high specificity of the determination. The test system showed good sensitivity and rapidity, allowing for the detection of as low as 0.063% (w/w) chicken meat in raw meat mixtures within 20 min. As a result of the testing of raw and cooked meat products, it was shown that the test system can reliably recognize specific immunoglobulins even after heat processing. The proposed assay can be recommended for rapid on-site screening control of the composition and quality of meat products.

Fluorescence Polarization-Based Bioassays: New Horizons.

Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.

A Comparative Study of Approaches to Improve the Sensitivity of Lateral Flow Immunoassay of the Antibiotic Lincomycin.

This study provides a comparative assessment of the various nanodispersed markers and related detection techniques used in the immunochromatographic detection of an antibiotic lincomycin (LIN). Improving the sensitivity of the competitive lateral flow immunoassay is important, given the increasing demands for the monitoring of chemical contaminants in food. Gold nanoparticles (AuNPs) and CdSe/ZnS quantum dots (QDs) were used for the development and comparison of three approaches for the lateral flow immunoassay (LFIA) of LIN, namely, colorimetric, fluorescence, and surface-enhanced Raman spectroscopy (SERS)-based LFIAs. It was demonstrated that, for colorimetric and fluorescence analysis, the detection limits were comparable at 0.4 and 0.2 ng/mL, respectively. A SERS-based method allowed achieving the gain of five orders of magnitude in the assay sensitivity (1.4 fg/mL) compared to conventional LFIAs. Therefore, an integration of a SERS reporter into the LFIA is a promising tool for extremely sensitive quantitative detection of target analytes. However, implementation of this time-consuming technique requires expensive equipment and skilled personnel. In contrast, conventional AuNP- and QD-based LFIAs can provide simple, rapid, and inexpensive point-of-care testing for practical use.

Lateral Flow Immunoassay to Detect the Addition of Beef, Pork, Lamb, and Horse Muscles in Raw Meat Mixtures and Finished Meat Products.

A lateral flow immunoassay for sensitive detection of skeletal troponin I (TnI) as a specific, thermostable marker of muscle tissue was developed. Due to the antibodies' choice, the assay specifically detects mammalian TnI (in beef, pork, lamb, and horse) but does not detect bird TnI (in chicken or turkey), thus enabling differentiation of these types of raw meat materials. The assay is based on a sandwich format of the analysis using gold nanoparticles as labels. The time of the assay is 15 min, and the TnI detection limit is 25 ng/mL. A buffer solution is proposed for efficient extraction of TnI from muscle tissues and from finished meat products that have undergone technological processing (smoking-cooking-smoking, cooking and smoking). The possibility of detecting beef addition in minced chicken down to 1% was demonstrated.

Design of Multiplex Lateral Flow Tests: A Case Study for Simultaneous Detection of Three Antibiotics.

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.

Development of a double immunochromatographic test system for simultaneous determination of lincomycin and tylosin antibiotics in foodstuffs.

This study is devoted to the development of a sensitive immunochromatographic analysis (ICA) for simultaneous determination of tylosin (TYL) and lincomycin (LIN) as antibiotics of the macrolide and lincosamide classes, widely used in animal husbandry and implicated in the contamination of foodstuffs. The ICA was implemented in an indirect competitive format, using antispecies antibodies conjugated with gold nanoparticles (GNPs) as a label. After the multistep optimization, the developed double ICA allowed for antibiotics detection with instrumental limits of detection/cutoff levels of 0.09/2 ng/mL and 0.008/0.8 ng/mL for TYL and LIN, respectively, within 10 min. The cross-reactivity was 40% to lincosamide clindamycin and negligible to other antibiotics tested. The test system allowed for the detection of TYL and LIN in milk, honey, and eggs. The recoveries of antibiotics from foodstuffs were 87.5-112.5%. The results demonstrate that the developed double ICA is an effective approach for the detection of other food contaminants.