Fig. 2. D4 sandwich immunoassay for quantification of HER2 expression level and its in vitro assessment.

a–c, Schematic and operation of D4 immunoassay. a Spots of immobile cAb and an excess of “soluble” fluorescently labeled dAb are printed directly onto POEGMA-coated glass. b Dispensing sample fluid onto chip surface leads to dissolution of soluble dAb spots, followed by diffusion-driven mixing and antibody “sandwich” formation if HER2 is present. HER2 binding is detected by fluorescence imaging. c Representative fluorescence image of D4 assay after exposure to HER2-spiked RIPA buffer. Scale bar, 1 mm. d Representative dose-response curve generated from D4 chips for RIPA buffer spiked with recombinant HER2. Error bars: mean ± s.d. of duplicate assays. e, Assessment of cultured breast cancer cell lines (BT474, BT20, MDA-MB-231, MDA-MB-468) by D4 assay and comparison with western blotting. Inset: Western blot against HER2 for each cell line, with representative D4 cAb spots underneath. Main: D4 signal intensity (mean ± 95% CI) of ≥6 replicates. Significant difference by one-way ANOVA (F(4, 31) = 179.4, p < 0.0001). Bars with different letters indicate different groups (Tukey post hoc test, p ≤ 0.05). f Concordance analysis of 8 different patient-derived tumor cell lines in culture for HER2 expression by D4 assay vs. ELISA performed by clinical lab; specimens indexed “a” through “h”. D4 results are mean ± s.d. of duplicate assays. Pearson’s r = 0.975 (p < 0.0001, 95% CI: 0.845–0.996).