Fig. 4

Enhanced fluorescence immunoassays on plasmonic AgNIS for highly sensitive detection of PSA. A Schematic of the sandwich immunoassay format implemented with biotin-BSA. CF647 fluorescence label with optimal enhancement on AgNIS was selected for signal readout. B ATR-FTIR spectra of native BSA and BSA on AgNIS. Dotted lines indicate typical amide I and II bands. C Conformational changes of BSA in the secondary structure after immobilization on AgNIS. Kinetic curves for dynamic binding processes of D BSA and AgNIS, E cAb and antigen, F antigen and dAb using CF647-BSA, CF647-dAb-antigen, and CF647-dAb probes, respectively. Calibration curves of the fluorescence signal intensity as a function of PSA concentrations for G AgNIS and H PS. Inset shows the corresponding fluorescence images. I Calibration curve of sandwich-type conventional ELISA for PSA with HRP-labeled dAb. Absorbance was measured at 450 nm with TMB substrate. J Comparison of signal intensities for 30 nM PSA spiked in PBS, serum, plasma, and whole blood on the AgNIS platform. Data were normalized by the mean fluorescence intensity (MFI) of PBS. One-way analysis of variance (ANOVA) Tukey's HSD post hoc test was used for statistical analysis. Evaluation for K intra-assay and L inter-assay coefficients of variability (%CV) at low (0.3 nM), medium (3 nM), and high concentrations (30 nM) of PSA