PEdELISA assay platform for monitoring CAR T-therapy associated CRS. (A) Concept of the instantaneous single-molecule binary counting of pre-equilibrium protein-binding events. The combination of pre-equilibrium reaction quenching with single-molecule counting can theoretically achieve an assay with a near-0 incubation time without losing linearity. (B) Schematic and photo image of the PEdELISA system, which comprises a disposable microfluidic chip (inset), an automated fluidic dispensing and mixing module (left), and a 2-dimensional inverted fluorescence scanning module (right). (C) Two-step ultrafast, multiplex PEdELISA process for the pre-equilibrated assay system, including 5-minute magnetic bead incubation for the formation of antibody-antigen-antibody immune-complexes (step 1), buffer exchange, 1-minute avidin-HRP labeling (step 2), and 5-minute continuous washing using the automated fluidic dispensing module. The PEdELISA chip has 8 circular biosensor patterns formed by a cluster of 66 724 arrayed microwells. Fluorescence color-encoded magnetic beads coated with different capture antibodies (nonfluorescent and Alexa Fluor 488 [AF 488]) are pre-deposited into each physically separated biosensor pattern. This arrangement can permit multiplex analyte detection with 2 colors × 8 patterns = 16 plexes. Each chip can quantify up to 16 samples simultaneously per batch run. (D) Digital readout process, which involves loading of HRP fluorescence substrate (QuantaRed), sealing of beads with fluorocarbon oil, and signal reading based on automated fluorescence scanning to count fluorescently activated “on”-state microwells. Data analysis is then performed by a convolutional neural network–guided image-processing algorithm for high throughput and accurate single-molecule counting.