Holders were designed in FreeCAD and 3D printed on a stereolithography 3D printer (SL1S, Prusa) with transparent resin (Clear Tough Resin, 3DM) using the manufacturer standard parameters. All shapes of paper and nitrocellulose were cut to size with the laser cutter. The design included a buffer reservoir with 1 mL capacity, which is contacted to the electrofluidic layer through a 8.3 mm diameter buffer pad (CF6, Cytiva). After flowing through the electrodes, the buffer was absorbed by two absorbent pads with 450 μL capacity each (CF6, Cytiva). The assay was performed as follows. The sample (10 μL) was mixed with 10 μL of standard. The standard comprised alkaline phosphatase (P7923, Sigma) with a pre-calibrated activity. Next, 2 μL of 100 mM p-aminophenyl phosphate (≥97%, Adipogen) in 1 M Tris•HCl pH 9.8 with 200 mM MgCl2 were added to the mixture followed by incubation for 20 minutes at 37 °C. The samples were put on ice before injection (5 μL injection volume) into the device. The following sera were analyzed: calf serum C8056 (Sigma), human serum H4522 (Sigma) and goat serum G9023 (Sigma). All sera samples were centrifuged at 2 krcf for 10 minutes before use. The electrochemical measurements were performed in chronoamperometry mode (PGSTAT 204, Metrohm) with an applied voltage of 0.25 V vs RE, and sampling resolution of 10 ms. The signals were computed by integrating the current peaks with subtraction of a linear background.