Paper-based fluoroimmunoassay for rapid and sensitive detection of antigen

A paper strip assay is developed for fast, simple and inexpensive detection of IgG protein in a quantitative manner with high sensitivity and selectivity. In the first step, polystyrene (PS) microbeads conjugated with primary anti-goat IgG are immobilized on paper strips. Upon capturing target goat IgG and subsequent incubation with Cy3-labeled secondary anti-goat IgG, a sandwich type immunoassay is formed on paper strips. Detailed study reveals that BSA blocking is effective in preventing non-specific protein adsorption on paper strips and the concentration of the signalling anti-goat IgG has to be controlled to minimize background signal. The paper-based fluoroimmunoassay has shown high specificity for goat IgG detection with a detection limit of 20 ng mL−1 using a fluorescence array scanner under optimized conditions and demonstrated generic applications in practical samples. To test the feasibility of the assay for colorimetric detection, gold nanoparticles are conjugated to the secondary anti-goat IgG to replace Cy3. Small Au nanoparticles (NPs) are retained on the paper strip upon target capturing and sandwich assay formation. The detection is signalled by a visible color change of the PS spot from white to blue with Au development. The Au NP stained colorimetric immunoassay requires no equipment, has simple operating steps, and enables detection of 5 μg mL−1 IgG with the naked eye.