Figure 3

Integrated single-cell Aldefluor assay and functional protein assay.

(a) Scheme of the experimental procedure. Aldefluor-stained cells are loaded while the chip is in the stand state. Conversion to the sit state is used to isolate single cells and perform other on-chip steps of the assay. Sandwich immunoassay is completed by adding biotinylated secondary antibodies and fluorophore-labeled streptavidin. (b,c) Cells are randomly distributed in microchambers before lysis (b) and after lysis (c) by diffusion of concentrated lysis buffer from channels to chambers. (d) Examples of detected proteins in red (Cy5 channel) and the reference signal in green (Cy3 channel) for single cells in each row. (e) Calibration curve for immunoassays performed on-chip using recombinant uPA, p-ERK1, p-S6K, VEGF and IL-8. Error bars denote standard deviation from the mean. (f ) Fluorescence readouts obtained for different concentrations of recombinant protein used in calibration. (g) Scatter dot plot showing fluorescence data for secreted (uPA, VEGF, IL-8) and intracellular phosphorylated proteins (p-ERK1, p-S6K) from 1-cell experiments in the microchip compared to background levels from 0-cell experiments with P < 0.001 (***). All data are from the same chip to keep consistency. P values less than 0.05 (*) were considered statistically significant. All scale bars are 500 μm.