PDE4B is a member of the phosphodiesterase family of proteins, which plays a critical role in regulating intracellular levels of cyclic AMP (cAMP)28. cAMP-specific 3′,5′-cyclic phosphodiesterase 4B is a therapeutic target for the treatment of several inflammatory disorders29. Here, we present the development of a simple, cost-effective, less time-consuming, and highly specific PDE4B assay on a non-fabricated parchment paper surface using inkjet-printing technology. Mainly, this assay is based on the competition between the europium (Eu) chelate-labeled cAMP tracer and sample cAMP for binding sites on cAMP-specific monoclonal antibodies (mAb) labeled with the ULight dye on parchment paper. PDE4B enzyme and their respective inhibitors (roliparm and roflumilast)30,31 were utilized to study FRET-based enzyme-inhibitor interactions on paper. In connection with this, Fig. 1 represents the scheme of the present study. When ULight-monoclonal antibody is bound to the Eu-labeled cAMP tracer, a light pulse at 320/340 nm excites the Eu chelate molecule of the cAMP tracer. The energy emitted by the excited Eu chelate is transferred by FRET to ULight molecules on the antibodies, which in turn emit fluorescence at 665 nm. Residual energy from the Eu chelate will produce fluorescence at 615 nm. After printing the reaction components on parchment paper, fluorescence energy transfer between Eu-cAMP and ULight-anti-cAMP provides a specific binding signal in the absence of free cAMP (Fig. 1A), while energy transfer does not occur in the presence of free cAMP (Fig. 1B). FRET between Eu-cAMP and ULight-anti-cAMP provides a particular signal, that is utilized here as an optical method to quantify cAMP-specific PDE4B and their respective inhibitor interactions on paper. In the presence of PDE4B, the cAMP is degraded into AMP, which is not recognized by the ULight-mAb. This leads to an increase in FRET signal, which is proportional to the concentration of degraded cAMP (Fig. 1C). Similarly, in the presence of PDE4B inhibitor, cAMP remains intact and competes with the Eu-cAMP tracer for binding to the Ulight-mAb. This results in a decrease in FRET signal, which is proportional to increased inhibitor concentration (Fig. 1D).Figure 1Schematic diagram. The schematic diagram shows that the reaction components were printed on parchment paper, and the fluorescence energy transfer between Eu-cAMP and ULight-anti-cAMP was determined under four different conditions, including: (A) absence of free cAMP; (B) presence of free cAMP; (C) presence of cAMP and PDE4B enzyme; and (D) presence of cAMP, PDE4B enzyme, and PDE4B inhibitor.