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

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

New and Improved Nanomaterials and Approaches for Optical Bio- and Immunosensors

The current state in the development of biosensors is largely associated with the search for new approaches to simplify measurements and lower detection limits [...

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

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

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

Peroxidase-Mimicking Nanozyme with Surface-Dispersed Pt Atoms as the Label for the Lateral Flow Immunoassay of C-Reactive Protein

We report a new approach to synthesize nanozyme with low consumption of precious Pt-precursor and high peroxidase-mimicking activity. The synthesis includes the formation of gold nanoparticles (Au NPs), the overgrowth of a silver layer over Au NPs (Au@Ag NPs), and the galvanic replacement of Ag with PtCl62- leading to the formation of Au@Ag–Pt NPs with uniformly deposited catalytic Pt sites. The reported approach facilitates up to 20-times lower consumption of Pt precursor. The use of Au@Ag-Pt NPs as the catalytic label in lateral flow immunoassay results in a 65-fold lower limit of detection (15 pg/mL in serum)

Triple Enhancement for Sensitive Immunochromatographic Assay: A Case Study for Human Fatty Acid-Binding Protein Detection

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

QSAR analysis of immune recognition for triazine herbicides based on immunoassay data for polyclonal and monoclonal antibodies.

A common task in the immunodetection of structurally close compounds is to analyze the selectivity of immune recognition; it is required to understand the regularities of immune recognition and to elucidate the basic structural elements which provide it. Triazines are compounds of particular interest for such research due to their high variability and the necessity of their monitoring to provide safety for agricultural products and foodstuffs. We evaluated the binding of 20 triazines with polyclonal (pAb) and monoclonal (mAb) antibodies obtained using atrazine as the immunogenic hapten. A total of over 3000 descriptors were used in the quantitative structure-activity relationship (QSAR) analysis of binding activities (pIC50). A comparison of the two enzyme immunoassay systems showed that the system with pAb is much easier to describe using 2D QSAR methodology, while the system with mAb can be described using the 3D QSAR CoMFA. Thus, for the 3D QSAR model of the polyclonal antibodies, the main statistical parameter q2 ('leave-many-out') is equal to 0.498, and for monoclonal antibodies, q2 is equal to 0.566. Obviously, in the case of pAb, we deal with several targets, while in the case of mAb the target is one, and therefore it is easier to describe it using specific fields of molecular interactions distributed in space