Inkjet bioprinting was performed and PDE4B enzyme reaction curve was obtained at three different concentrations (stock concentrations: high −10 µM, medium −5 µM, and low −0.5 µM) using 2.79 × 10−16 mole of cAMP as a substrate on parchment paper. cAMP, PDE4B, Eu-cAMP tracer, and ULight anti-cAMP mAb were sequentially printed on parchment paper based on a layer-by-layer process using a conventional inkjet printer. Figure 3 illustrates the optimization condition under which the FRET signal correlates linearly with time. As shown in Fig. 3A,B, the signal increased significantly as a function of time up to 50 min with a higher concentration of PDE4B; whereas, the signal reached a plateau after only 30 min when medium and low concentration of enzyme were used. Figure 3A represents the FRET images acquired using a fluorescent microscope, and Fig. 3B corresponds to their respective fluorescent intensity determined using Metamorph software. Figure 3C presents the intensity profile of fluorescent signals produced on parchment paper, which confirms the linear correlation between the fluorescent signal and time point. The intensity profile image clearly supports that the FRET signal increased linearly as a function of time up to 50 min with a higher concentration of PDE4B. Similarly, non-linear signals were generated on paper in the presence of medium and low concentration of PDE4B (Fig. 3C). As previously mentioned, the volume printed and number of mole of PDE4B printed per spot area were evaluated. The number of mole of PDE4B printed per spot area corresponds to 2.81 × 10−13 mole, 1.40 10−13 mole and 1.40 10−14 mole, which was ejected from the cartridges filled with high –10 µM, medium −5 µM, and low −0.5 µM concentration of PDE4B, respectively (supplementary Table S2). In Fig. 3D, the graphical plot presents the FRET signal generated in response to the enzymatic reaction on paper. The optimized condition here verifies that 2.81 × 10−13 mole of PDE4B printed/spot degrading 2.79 × 10−16 mole of cAMP leads to an increase in FRET signal, while a significantly lesser FRET signal was generated in the presence of 1.40 10−13 mole and 1.40 10−14 mole of PDE4B. In order to develop a paper-based sensitive assay and ensure cAMP activity using inkjet-printing technique, 2.81 × 10−13 mole of PDE4B and reaction time of approximately 50 min were selected for subsequent experiments.Figure 3Optimization of PDE4B enzyme activity using the inkjet-printing method. (A) Fluorescence signal generated on parchment paper; (B) graph represents the average fluorescence intensity of the individual images (3A); (C) illustrates the intensity profile of the individual images (3A). Figure data A, B, and C were acquired as a function of dose and time point; and (D) the X-axis represents the number of mole of PDE4B printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP and PDE4B, and the secondary Y-axis represents the ejection volume of PDE4B.