A Microcavity Chemiluminescent Immunoimaging Protocol for Fast in Situ Detection of Antibody Secreted from Single Hybridoma Cells

Abstract Chemiluminescent immunoassay is the most widely used protein detection technique in clinical diagnosis but still faces substantial challenges in antibody‐related single‐cell analysis due to the lack of a homogeneous immunoassay with strong and stable chemiluminescence signal. Herein, a single‐cell microfluidic platform for efficient in situ detection of antibody secreted from single hybridoma cells through a microcavity chemiluminescent immunoimaging (MCCLII) protocol is reported, which consist of an imidazole‐enhanced chemiluminescence system, a proximity‐triggered DNA nanomachine and a hybrid‐regulated hemin‐DNA switch. In MCCLII, antibody secreted from single hybridoma cells can be visualized through a homogeneous proximity CL assay, which converses the target antibody to DNA for triggering the DNA nanomachine and then activates the catalytic activity of hemin‐DNA switch to produce strong and persistent chemiluminescence for micro‐imaging. The MCCLII realizes immunoimaging detection down to 66 antibody molecules in 0.79‐nL microchamber and demonstrates the possibility of specific hybridoma cell screening within 30 min, which provides a simple and fast antibody screening platform to promote antibody‐drug production.