Firstly, optimal cAMP activity was measured to evaluate the cAMP-specific PDE4B enzyme inhibitory assay. The spot size 0.3 cm with an area of 0.071 cm2/spot was used for printing. The cAMP (Y cartridge), Eu-cAMP tracer (M cartridge), ULight anti-cAMP mAb (C-cartridge), and Buffer (K cartridge) were sequentially printed on parchment paper based on a layer-by-layer process using a conventional inkjet printer (Fig. 2A). Two different sets of experiments (W cAMP and W/O cAMP) were performed to verify cAMP activity in the presence of Eu-cAMP tracer and ULight anti-cAMP mAb. The reaction components were printed on parchment paper in reference to Fig. 2A. Hence, two different spot areas were printed, W and W/O cAMP. Immediately, Eu-cAMP tracer and ULight anti-cAMP mAb were printed on the cAMP pre-printed spot surface and incubated for 30 min (Fig. 2B,C). Competitive fluorescent energy transfer occurred in the presence and absence of cAMP measured at the 665-emission range using a fluorescent microscope, and the FRET images were analyzed with MetaMorph software. Figure 2B(1 and 2) and 2C(1 and 2) represent the monochrome and fluorescent dye color image of the FRET signal acquired with W and W/O cAMP, respectively. Three different regions (R1, R2, and R3) were selected to confirm the uniform FRET signal distributed on parchment paper, and their representative individual intensity profiles and percentage intensity profiles are shown in Fig. 2B(3 and 4) and 2C(3 and 4), respectively. Individual and average FRET signals corresponding to R1, R2, and R3 that acquired W and W/O cAMP are graphically represented as Fig. 2D.Figure 2Optimization of cAMP activity using the inkjet-printing method. (A) Represents printing order. (B) FRET signals were acquired with cAMP, and their respective images were illustrated as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (C) FRET signals were acquired without cAMP, and their respective images were shown as: 1. monochrome image; 2. fluorescent dye color image; 3. intensity profile of three different regions; and 4. percentage intensity profile of three regions. (D) Fluorescence signal generated on a single spot was measured. The graph illustrates the individual fluorescence signal produced with and without cAMP by three different regions/spot areas.