Single-molecule immunoassay technology: Recent advances

Detecting diseases at the molecular level aids in early diagnosis and treatment. However, traditional immunological detection techniques, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence, have detection sensitivities between 10−16 and 10−12 mol/L, which are inadequate for early diagnosis. Single-molecule immunoassays can reach detection sensitivities of 10−18 mol/L and can detect biomarkers that are difficult to measure using conventional detection techniques. It can confine molecules to be detected in a small spatial area and provide absolute counting of the detected signal, offering the advantage of high efficiency and accuracy. Herein, we demonstrate the principles and equipment of two single-molecule immunoassay techniques and discuss their applications. It is shown that the detection sensitivity can be improved by 2–3 orders of magnitude compared to common chemiluminescence or ELISA assays. The microarray-based single-molecule immunoassay technique can test 66 samples in 1 h, which is more efficient than conventional immunological detection techniques. In contrast, microdroplet-based single-molecule immunoassay techniques can generate 107 droplets in 10 min, which is more than 100 times faster than a single droplet generator. By comparing the two single-molecule immunoassay techniques, we highlight our personal perspectives on the current limitations of point-of-care applications and future development trends.