Fig. 2.

Representative microchip-based methods for single-cell proteomic analysis. a) Microengraving: individual cells are enclosed into microwells and generate microarrays of proteins from each microwells. The protein microarrays are identified by an antibodies-coated glass slide. The slide is replaced at various time points for dynamic analysis. Reproduced from ref. [53] with permission from the Proceedings of the National Academy of Sciences. b) Single-cell western blotting: single cells are captured into polyacrylamide gel microwells and go through in situ cell lysis, electrophoresis for protein separation, photo-induced protein immobilization. Through cycles of antibody stripping and protein reprobing, multiple proteins from the same cell can be detected. Reproduced from ref. [64] with permission from Nature Publishing Group. c) Droplet microfluidics: single T cells are encapsulated in agarose-gel droplets together with functionalized capture beads, and then secrete cytokines that bind to the beads confined within the droplets. The droplets are gelled and washed to break the emulsion, incubated with fluorescently labeled detection antibodies, and quantified by flow cytometry. Reproduced from ref. [71] with permission from the Royal Society of Chemistry. d) Single-cell barcode microchip: single cells are loaded and isolated into high-throughput microchambers. The secreted proteins from cells are captured by multiplex antibody barcode arrays coated on a glass substrate, and then verified by sandwich-type immunoassay. Reproduced from ref. [75] with permission from Nature Publishing Group. e) Microbeads array: each microbeads are functionalized by a specific ssDNA sequence and then converted into antibody microbeads array by cDNA-antibody conjugates for secreted proteins detection. Successive hybridization (labeling) and denaturation (quenching) are continuously performed for decoding process, exhibiting ordered fluorescent colors on every microbead. The ordered colors are corresponding to different types of antibodies carried on the microbeads. Reproduced from ref. [93] with permission from Wiley.