Macromolecular crowding agents enhance the sensitivity of lateral flow immunoassays

Lateral flow immunoassays (LFIA) have a plethora of applications in health, environmental and food sectors for low-cost, simple, and rapid point-of-need testing. Typically, the user only needs to add the sample without any other intervention from sample application to results. A compelling challenge, and a constant pursuit in LFIA is to improve the assay sensitivity without compromising the simplicity and practicality. We report that the addition of water-soluble macromolecular crowding agents leads to an enhancement of the sensitivity, which is attributed to the fact that the exposure of antibodies and micro/nanoparticle conjugates to macromolecularly crowded environment, while migrating through the confining pores of the strip-pads by capillary forces, promotes the interactions that are responsible for analyte recognition and signal generation. The effect was shown by using two of the most widely established LFIA tests worldwide, that is, detection of nucleocapsid protein from SARS-CoV-2 associated with COVID-19 and detection of Strep-A antigen from Streptococcus pyogenes associated with pharyngitis. For immediate demonstration of the sensitivity enhancement, we worked directly on commercially available devices already optimized in terms of reagents and conditions. Of the crowders used, ficoll, Mr 400000, and ficoll, Mr 70000, gave a 5-10-fold improvement of the signal without affecting the background. Because the addition of macromolecular crowding agents is complementary to other strategies of sensitivity enhancement, such as the design of novel labels and the introduction of signal amplification, we anticipate that the proposed modulation will be extended to numerous analytes with a variety of reporters and LFIA configurations.