Ways to Reach Lower Detection Limits of Lateral Flow Immunoassays

This chapter considers factors influencing sensitivity of lateral flow immunoassay and modern developments that are focused on reaching lower detection limits. The existing variety of proposed approaches is classified in accordance with the “big five rules” for these assays, including proper sample, receptor, interaction, response, and output. The solutions for rapid extraction of target analytes and preventing negative influence of extractants are considered. Role to antibodies affinity and specificity is characterized. Potential of alternate bioreceptor molecules is discussed. Immunoreactants’ compositions, concentrations, and locations on the test strip are characterized as factors determining assay parameters. The existing variety of labels is compared in terms of their optical and alternate registration. Tools to modulate a sequence of analytical reactions and to form aggregates of the detected labels are considered. The discussed approaches are illustrated through developments of test strips for detection of mycotoxins, veterinary drugs, and other analytes

The Role of White-rot Fungi in Herbicide Transformation

Understanding herbicide transformation is necessary for pesticide development for their safe and efficient use, as well as for developing pesticide bioremediation strategies for contaminated soil and water. Recent studies persuasively demonstrated the key role of soil white-rot fungi in biotransformation of various anthropogenic environmental contaminants. However, often this common knowledge is not associated with specific metabolic processes of fungi and therefore cannot be transformed into specific recommendations for agricultural practice. The given review offers a systematic collection and analysis of the current knowledge about herbicide transformation by white-rot fungi at the cellular and molecular levels. Special attention is given to the role of oxidative enzymes such as laccases, lignin peroxidases, and manganese peroxidases in the biotransformation processes

METHODS OF IDENTIFICATION OF MUSCLE TISSUE IN MEAT PRODUCTS. PREREQUISITES FOR CREATING A MULTI–LEVEL CONTROL SYSTEM

Unfair production and products that do not comply with the declared labeling are currently an acute problem in the field of technical regulation, including with regard to food safety and quality. Given the high added value and multicomponent composition, finished meat products are among the most susceptible to adulteration. Despite the best efforts of regulatory agencies to counteract these inconsistencies, the hidden substitution of cheaper or lower-grade meats is still widespread. One of the main tasks facing research laboratories and testing centers today is the detection of falsification of food products, as well as standardization and certification of techniques necessary to solve such problems. The manufacturer, aware of the current control methods, can go to the deception, using vegetable protein, new unregistered feed additives. To determine the complex changes that occur in products, it is necessary to use methodological approaches in which it is possible to reliably determine these changes. The paper presents an overview of the most commonly used methodologies for assessing the component composition of meat products. Quality assessment of meat products includes control of components of finished products. The most difficult task is to determine the proportion of muscle protein in multicomponent meat products that have undergone heat treatment.Unfair production and products that do not comply with the declared labeling are currently an acute problem in the field of technical regulation, including with regard to food safety and quality. Given the high added value and multicomponent composition, finished meat products are among the most susceptible to adulteration. Despite the best efforts of regulatory agencies to counteract these inconsistencies, the hidden substitution of cheaper or lower-grade meats is still widespread. One of the main tasks facing research laboratories and testing centers today is the detection of falsification of food products, as well as standardization and certification of techniques necessary to solve such problems. The manufacturer, aware of the current control methods, can go to the deception, using vegetable protein, new unregistered feed additives. To determine the complex changes that occur in products, it is necessary to use methodological approaches in which it is possible to reliably determine these changes. The paper presents an overview of the most commonly used methodologies for assessing the component composition of meat products. Quality assessment of meat products includes control of components of finished products. The most difficult task is to determine the proportion of muscle protein in multicomponent meat products that have undergone heat treatment

Сomparison of Au, Au-Pt, and Au-Ag nanoparticles as markers for immunochromatographic determination of nonylphenol

Gold spherical nanoparticles, gold-platinum nanoflowers, and gold-silver nanostars were obtained and compared as labels for immunochromatographic analysis. The nanoparticles were synthesized by chemical reduction from various precursors and then conjugated with staphylococcal protein A to be used in indirect immunochromatographic determination of nonylphenol. The results obtained were evaluated in terms of analytical characteristics and R2 value, as well as the color intensity of the test band. According to the comparison results, it was revealed that the R2 value varied from 0.82 for the gold-silver nanostars to 0.96 for the spherical gold nanoparticles. The working range of determined concentrations was from 2 to 100 μg/mL for unspherical and from 2 to 50 μg/mL – for spherical markers used; the analysis time was 20 min

Theoretical and Experimental Comparison of Different Formats of Immunochromatographic Serodiagnostics

In this study, a comparative theoretical and experimental analysis of two immuno-chromatographic serodiagnostics schemes, which differ in the immobilization of immunoreagents and the order of the formation of immune complexes, is performed. Based on the theoretical models, the assays are characterized to determine which scheme has a higher quantity of the detected complex and thus ensures the sensitivity of the analysis. The results show that for the effective detection of low-affinity antibodies, the scheme involving the immobilization of the antigen on gold nanoparticles and the antibody-binding protein on the test strip was more sensitive than the predominantly used scheme, which inverts the immunoreagents’ locations. The theoretical predictions were confirmed by the experimental testing of sera collected from tuberculosis patients

Post-Assay Chemical Enhancement for Highly Sensitive Lateral Flow Immunoassays: A Critical Review

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

The Steadfast Au@Pt Soldier: Peroxide-Tolerant Nanozyme for Signal Enhancement in Lateral Flow Immunoassay of Peroxidase-Containing Samples

The approach to inhibit endogenous peroxidases by elevated concentrations of hydrogen peroxide while maintaining the high peroxidase-mimicking activity of Au@Pt nanozymes was developed. The approach facilitates selective and highly-sensitive detection of peroxidase-mimicking nanozyme nanozymes in the background of endogenous peroxidases. Au@Pt nanozyme was used as the colorimetric and catalytic label in lateral flow immunoassay of an important plant pathogen – potato virus X. The inhibition of endogenous peroxidases in plant extracts and selective detection of Au@Pt nanozyme provides the lowest limit of detection among immunochemical assays of potato virus X (up to 500 times lower compared to the assay with conventional gold nanoparticles). The proposed approach uses the fundamental principle of enzyme inhibition by the substrate. It is universal and applicable to all matrixes with peroxidase activity. </div

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible