Fig. 3

Fluorescence multiplexing and point-of-care capabilities of AgNIS. A Estimation of surface-immobilized BSA by a non-fluorescence enzymatic assay using BSA-HRP complexes. Assay solutions after enzymatic reactions were transferred into clear-bottomed wells for more accurate measurement of OD at 450 nm to circumvent intrinsic absorption interference from AgNIS. B Absorbance at 450 nm from BSA-HRP adsorbed on PS and AgNIS after enzymatic reactions. No statistical significance (P = 0.1683) was found (n = 10). C Multiplexed fluorescence verification of CF488A, CF555, and CF647 on AgNIS. Native BSA was included for concentration compensation. Specific fluorescence can be simultaneously and accurately differentiated in the corresponding channel. D Schematic design and E photographs of a smartphone-based POCT prototype for multiplexed fluorescence imaging. White light LED inverted to DC was used for illumination with appropriate excitation filter sets. F Transmission spectra of the excitation filters overlaid with the emission spectrum of the white-light excitation source (black). G Transmission spectra of the emission filters set in front of the camera for fluorescence detection. H Representative fluorescence images were scanned with the POCT prototype for PS and AgNIS modified with fluorophore-labeled BSA (left panel) or with immunoassay detection of 100 nM PSA (right panel)