On the basis of the sensitivity and specificity studies, the practical application of the inkjet printing-based system for determining PDE4B inhibitory activity was investigated. For this purpose, the printing-output setting was fixed as 100, to print cAMP, PDE4B, Eu-cAMP tracer, ULight anti-cAMP mAb, and buffer. In addition, the printing-output setting (C value) was altered by a two-order difference per spot (from 0 to 88) to vary the amount of roliparm or roflumilat to be printed per spot area. Figure 4A(1)–A(4) and Fig. 5A(1)–A(4) show the response order of printing cAMP, PDE4B, and roliparm/roflumilast. Finally, the buffer was printed to avoid drying. Reaction samples printed-paper was incubated for 30 min inside of an airtight container. Immediately, Eu-cAMP tracer and ULight anti-cAMP mAb (Fig. 4A(6) and (7) and Fig. 5A(6) and (7)) were printed from different cartridges on the same spot area, and incubated for 50 min. FRET signals were detected at 665 nm using a fluorescent microscope.Figure 4Inkjet-printing based determination of PDE4B inhibition by roliparm. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roliparm, and the secondary Y-axis represents the ejection volume of roliparm. Error bars represent the standard deviation of three independent measurements.Figure 5Inkjet-printing based determination of PDE4B inhibition by roflumilast. (A) Represents printing order; (B) fluorescence signal generated on parchment paper as a function of C value; (C) graph represents the average fluorescence intensity of the individual images (4A) as a function of C value; and (D) the X-axis represents the number of mole of roliparm printed per spot, the primary Y-axis represents the FRET signals produced in the presence of cAMP, PDE4B and roflumilast, and the secondary Y-axis represents the ejection volume of roflumilast. Error bars represent the standard deviation of three independent measurements.