Hemin-DNA Switch-Assisted Microfluidic Chemiluminescence Immunoimaging for Rapid and Quantitative Screening of Specific Hybridomas

Highly sensitive detection of cell secretions at the single-cell level is critical for efficient cell screening and monoclonal antibody (mAb) production. Herein, we present a highly sensitive microfluidic chemiluminescence immunoimaging (μCLII) method for rapid and quantitative screening of high-yield specific hybridoma cells (HSHCs) by detecting single-cell secreted antibodies on a rationally designed lantern-shaped microchamber array chip. This method can effectively capture and isolate single hybridoma cells to <i>in situ</i> detect their secreted antibody by visual CL imaging with a proximity recognition amplification strategy. The proposed strategy can be performed by antibody-triggered proximity hybridization to release a DNA sequence, which initiates a rolling circle amplification reaction to turn on abundant hemin-DNA enzymes for generating strong CL emission of the imidazole-enhanced luminol-H₂O₂ system. This method shows a detection limit of ∼60 antibodies per chamber, and thus enables a precise sorting of HSHCs within 70 min. Using antiproprotein convertase subtilisin/kexin type 9 mAb (PCSK9-mAb) as a model, all of the cells selected by the μCLII method show high-producing hybridomas with PCSK9-mAb yield more than 80 fg during the screening process, indicating a promising potential of the proposed method for single-cell analysis and mAbs screening.