Abstract
Lateral flow immunoassays (LFIAs) have wide application in point-of-care testing, particularly in resource-poor settings. To achieve signal amplification in a gold nanoparticle-based lateral flow assay without an additional procedure or the need for complex fabrication, a new and simple method was developed for using a “stacking pad” configuration that adds an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format. This design helps to accumulate the antibody and antigen on the stacking pad, hence extending the antigen/antibody binding interactions to enhance the test’s detection sensitivity. With the enhanced lateral flow assay, as low as 1 ng/mL of Protein A and 15.5 ng/mL of C-reactive protein can be visualized with the naked eye. We also successfully applied the stacking pad system in the analysis of C-reactive protein in human serum and synovial fluid samples. These results suggest that this stacking pad LFIA can provide sensitive and on-site prognosis for detection in synovial fluid and serum samples in resource-limited settings.
Highlights
A lateral flow immunoassay (LFIA) is a simple diagnostic device based on the chromatography-like migration of a labeled analyte through multiple membranes, including a sample pad, conjugation pad, detection pad, and absorbance pad, ending in the visible result of an immobilized captured reagent
Compared to the conventional LFIA device (Fig. 1(a)), the proposed sLFIA shown in Fig. 1(b) integrates an additional stacking pad between a detection pad that dry-stores the capture and control probes, and a conjugate pad that dry-stores the AuNP-anti-Protein A or AuNP-anti-C-reactive protein (CRP) conjugates using 30 nm AuNPs
Compare to the similar thickness of SP1 (0.5 mm), SC1 (0.5 mm), and SG1 (0.6 mm), the results demonstrate that the SC1 cellulose substrate displayed the best performance, in which the intensity increased by almost 2-fold compared to the LFIA test without a stacking pad
Summary
A lateral flow immunoassay (LFIA) is a simple diagnostic device based on the chromatography-like migration of a labeled analyte through multiple membranes, including a sample pad, conjugation pad, detection pad, and absorbance pad, ending in the visible result of an immobilized captured reagent. We introduce an innovative “stacking pad” configuration by adding an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format (sLFIA), which can accumulate the antibody and antigen on the stacking pad, extending the antigen/antibody binding interactions to enhance the test’s detection sensitivity. This concept was adapted based on the function of the stacking gel in polyacrylamide gel electrophoresis (PAGE), which allows for proteins to be packed in a concentrated area, enabling increased antibody/antigen interaction time[24,25,26]. Various membrane materials, including polyester, cellulose, and glass fiber, were examined as the stacking pad to further increase the detection limit of the colorimetric signal
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