Fig. 2: Establishment of LIT immunoassay.

a Representative bright-field images of self-assembled GRASPs in LIT before and after rapid aspiration with different buffer solutions (PBST and serum). Scale bar = 50 µm. b The recovery rate after different cycles of washing. Median fluorescent intensity, background signal, and signal-to-noise ratios measured in LIT immunoassays at varying concentrations of detection antibodies (c) and SAPE (d). The standard curves for IL-8 measured by LIT (e) and suspension GRASPs assay (f), with representative bright-field and fluorescent images of GRASPs from both assays (g, h). Scale bar = 10 µm. i The cross-binding percentages of six cytokine analytes at 1 ng/ml were measured in a six-plex LIT assay. j Representative bright field and fluorescent images of cross-binding assay. Scale bar = 20 µm. k Standard curves plotted for a six-plex LIT assay (IL-1β, IL-4, IL-5, IL-6, IL-8, and GM-CSF). Median fluorescent intensities of IL-5 (l) and IL-8 (m) at concentrations of 1000 pg/ml, 200 pg/ml, and 40 pg/ml measured in two-plex LITs over 180 days of storage. n Stability of IL-5 and IL-8 over 6 months, as evidenced by the estimated error of MFI at the concentration of 1 ng/ml. All images shown are representative images from three independent experiments. Data shown are means ± standard deviation (S.D.) for three independent experiments. General data were analyzed with Origin 2021. The five-parameter logistic regression model was used to fit the standard curves. LOD and LOQ were defined as the concentrations corresponding to signal values of mean(blank) + 3σ(blank) and mean(blank) + 10σ(blank), respectively, where mean(blank) is the average signal and σ(blank) is the standard deviation of the blank measurements.