Although significant FRET signals were acquired with W and W/O cAMP, it is important to assess the amount of cAMP involved in the reaction. In our previous work, we developed a novel method using an inkjet printer to evaluate the number of mole printed on the surface based on volume printed per spot and their respective solution density32. This approach enhanced the application of the inkjet-printing technique to study enzyme inhibitory assay. The described method32 was applied in the present study to evaluate the number of mole of cAMP printed on paper. Initially, the reaction sample solution densities were evaluated using a pycnometer and expressed in g/mL (Supplementary Table S1). The printed volume was evaluated using solution densities, and cartridge weight loss before and after printing. Thus, the printed volume was calculated using the formula: printed volume = (cartridge weight loss/solution density) * area. Supplementary Table S2 shows that 27.8 nL of cAMP was printed/spot area to react with 30 nL of Eu-cAMP tracer and 30 nL of ULight anti-cAMP mAb, which were printed on the same spot area. The effective mole printed on the surface was estimated using estimated printed volume and initial solution stock concentration using a standard equation: number of mole = molarity * volume. It was determined that 2.79 × 10−16 mole of cAMP was printed per spot area.