Digital Counting of Quantum Dots Nanobeads on Lateral Flow Test Strips Enables Ultrasensitive Point-of-Care Diagnostics

Lateral flow assay (LFA) is a versatile platform for the rapid detection of various pathogens, pollutants, and toxins at point of care (POC). However, a traditional lateral flow assay strip is still not sensitive enough for early detection of disease, and previous works to enhance the sensitivity of LFA systems have suffered from additional signal amplification chemistry, tedious operation procedure, or expensive signal amplification instruments. In this study, we introduced a digital counting strategy for signal measurement to improve the detection sensitivity of quantum dot nanobead-based lateral flow immunoassays, offering an alternative to traditional fluorescence intensity-based quantification. The ultrasensitive single-molecule POC detection system is composed of an ultrabright fluorescent quantum dot nanobead-based lateral flow assay strip and a portable fluorescent microscopy reader for numerating single immunocomplexes on a test line. As a proof-of-concept, we performed digital LFA to detect interleukin-6 in 1% BSA with LoD as low as 0.02 pg/mL, which is ∼100 times more sensitive than analog LFA. Moreover, an amplification-free assay for HIV RNA was also demonstrated by combining CRISPR-Cas13 reaction and digital LFA with a sensitivity of as low as 89 copies/mL. A prototype portable and affordable fluorescent microscopy reader was designed and demonstrated its ability to be used for on-site testing. All in all, the developed digital LFA system not only has single-molecule level sensitivity but also satisfies the rapidity, easy operation, portability, and low-cost requirements for POC testing.