This invention relates to the field of single-
cell functional analysis and
microfluidic chip technology, and discloses a kit and method for in-situ sequential quantitative detection of secreted and
intracellular proteins in the same single
cell. The kit includes: a microcavity array
chip; a secreted
protein capture antibody barcode chip and an
intracellular protein capture antibody barcode chip; a plastic clamp; a bioaffinity modification
reagent; secreted
protein standards,
intracellular protein standards, secreted
protein detection antibody stock solution, and
intracellular protein detection
antibody stock solution; a fluorescent conjugate,
antibody buffer, antibody blocking solution,
cell lysis buffer, and cell washing buffer. The method of this invention, through a sequential detection process of first capturing and incubating secreted proteins, followed by in-situ
lysis to capture intracellular proteins, achieves high-
throughput, high-sensitivity, and high-specificity in-situ sequential quantitative detection of secreted and intracellular proteins in the same single cell without cell transfer or loss of spatial location information. It can establish a
correlation analysis of "intracellular signaling pathway activation - external functional output" in single cells, providing a powerful tool for
disease mechanism research,
drug screening, and
immunotherapy evaluation.