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.
Keywords: Digital ELISA; Microfluidics; Microwell arrays; Single molecule.
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