To evaluate cAMP-specific PDE4B inhibitor activity, C, M, Y, and K cartridges were refilled with reaction sample solutions. To optimize cAMP activity using a conventional inkjet printer, cAMP (Y cartridge), Eu-cAMP tracer (M cartridge), and ULight anti-cAMP mAb (C cartridge) were sequentially printed on parchment paper, and incubated for 50 min at room temperature (25 °C and a relative air humidity-RH, of ∼50%) inside of an airtight container. To evaluate the optimal PDE4B enzyme concentration using a conventional inkjet printer, cAMP (Y cartridge) and PDE4B (M cartridge) were consequently printed on parchment paper, and incubated for 30 min at room temperature (25 °C and a relative air humidity-RH, of ∼50%) inside of an airtight container. Then, Eu-cAMP tracer (Y cartridge) and ULight anti-cAMP mAb (M cartridge) were sequentially printed on the same spot and then incubated at room temperature. PDE4B-cAMP interaction and their respective FRET signals were detected based on concentration and in a time-dependent manner. Similarly, inhibition of PDE4B enzyme was investigated using the inkjet-printing system. To verify this, cAMP (Y cartridge), and PDE4B (M cartridge), and roliparm/roflumilast (C cartridge) were successively printed on parchment paper, and incubated for 30 min at room temperature inside of an airtight container. Then, Eu-cAMP tracer (Y cartridge) and ULight anti-cAMP mAb (M cartridge) were serially printed on the same spot and then incubated for 50 min at room temperature. Our previous report discussed the impact of measuring solution density and volume printed per spot. Hence, the number of mole printed was evaluated using solution density, and volume printed on the surface, as described by Lee et al.18. Briefly, solution density was measured by pycnometer, and is expressed as mass per unit volume. The density was calculated using the following formula: ds = (Ws − Wb)/(Ww − Wb), where Ws is solution weight, Wb is bottle weight, and Ww is water weight (supplementary Table S1). Therefore, printed volume was evaluated using cartridge weight loss before and after printing, and solution densities (volume printed = (cartridge weight loss/solution density) *area). The effective number of mole of substance printed on the surface was estimated using printed volume and initial solution stock concentration by means of a standard equation: number of mole = molarity * volume.