Integrated nanoparticle size with membrane porosity for improved analytical performance in sandwich immunochromatographic assay

The use of luminescent nanobeads to improve the sensitivity of sandwich immunochromatographic assay (ICA) has obtained increasing concern. Illustrating the relationship among luminescent intensity, nanobead size, nitrocellulose membrane aperture, and ICA sensitivity is important for achieving the optimal target detection. Thus, we synthesized six differently sized quantum dot beads (QBs) (95, 140, 180, 235, 325, and 405 nm) as ICA labels and applied them in three aperture membranes (10, 15, and 25 μm). Results indicate that increasing the QB size to less than an appropriate size of 235 nm is beneficial for ICA sensitivity because of the increased fluorescence. However, oversized QBs result in reduced sensitivity due to the decreased diffusion or settlement of the QB on the membrane that causes obvious background signal. The small aperture membrane perfectly matching the QB size contributes to ICA sensitivity by decreasing the migration velocity of the QB probe for increased binding of the QB@analyte complex at the T zone. Consequently, the best detection of hepatitis B surface antigen with a sensitivity of 0.156 ng/mL is achieved using 235 nm QBs in 15 μm membrane. Further performance evaluation of our QB235-ICACN95 demonstrates excellent accuracy, selectivity, and practicability. This work provides a new idea to manipulate the sensitivity of sandwich ICA by tuning the QB size and the membrane aperture, and a theoretical guidance for selecting the probe and membrane to achieve the best detection of target analytes.