Development of quantitative radioactive methodologies on paper to determine important lateral-flow immunoassay parameters

Abstract

The lateral-flow immunoassay (LFA) is a well-established diagnostic technology that has recently seen significant advancements due in part to the rapidly expanding fields of paper diagnostics and paper-fluidics. As LFA-based diagnostics become more complex, it becomes increasingly important to quantitatively determine important parameters during the design and evaluation process. However, current experimental methods for determining these parameters have certain limitations when applied to LFA systems. In this work, we describe our novel methods of combining paper and radioactive measurements to determine nanoprobe molarity, the number of antibodies per nanoprobe, and the forward and reverse rate constants for nanoprobe binding to immobilized target on the LFA test line. Using a model LFA system that detects for the presence of the protein transferrin (Tf), we demonstrate the application of our methods, which involve quantitative experimentation and mathematical modeling. We also compare the results of our rate constant experiments with traditional experiments to demonstrate how our methods more appropriately capture the influence of the LFA environment on the binding interaction. Our novel experimental approaches can therefore more efficiently guide the research process for LFA design, leading to more rapid advancement of the field of paper-based diagnostics.

Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays

“…Indeed, these techniques can help to identify and shortlist the most promising bioreceptors, however, they must be finally tested in a real LFA, in order to choose those that will be used in the final assay since the kinetics can be different for binding in porous media. 69 Capture bioreceptors vs. detection bioreceptors.In order to avoid confusion, in this manuscript we will refer to the bioreceptors conjugated to the nanoparticles as "detection bioreceptors", to the bioreceptors on the membrane as "capture bioreceptors", which are further divided into "test line capture bioreceptors" and "control line capture bioreceptors".…”

Plasmonic molecular assays: Recent advances and applications for mobile health

“…Plasmonic nanomaterials have been utilized to integrate with cellulose paper devices for POC sensing applications [182]. Lateral flow assays (LFAs), also commonly known as "dipstick tests", have been the predominant POC tests in the past decades [183].…”

The Role of Nanoparticle Design in Determining Analytical Performance of Lateral Flow Immunoassays

“…A further calculation of the Damkohler number (Da =k on ’CR/D e ) compares the reaction flux (of a given test site capture antibody concentration C ) to diffusion flux (Figure 1 b). Here, the effective forward reaction rate constant ( k on ’ ) for antibody-labeled GNPs is assumed to be 27 $${\mathrm{normalk}}_{{\mathrm{on}}^{\prime }}=\mathrm{n}·{\mathrm{normalk}}_{\mathrm{on}}$$where k on is the forward rate constant for a single antibody-antigen interaction in the LFA membrane environment, and n is the effective number of antibodies per GNP. With the calculated Pe ≫ 1 (convection dominates diffusion) and Da ≪ 1 (diffusion dominates reaction) shown in Table S2; thus reaction is the rate-limiting step to improve GNP capture (details in Supporting Information section 8).…”

“…Further, directly measuring k on ′ of GNPs within LFA environments using radioactively labeled antibodies will be useful to improve the model and find the ultimate limits of this LFA technology. 27 …”