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This paper describes the design, fabrication, and validation of a paper-based diagnostic device for the rapid diagnosis of hypocalcemia in dairy cattle at the point-of-care (POC). The device incorporates a 3D printed calcium ion-selective membrane (ISM) as the sensing element for free—unbound—calcium in real bovine whole blood samples. With a linear response range of 100 mM to 97.7 μM, the sensor covers the clinically relevant concentrations of Ca 2+ associated with both healthy cattle as well as those suffering from hypocalcemia. The components of the Ca 2+ ion-selective electrodes were successfully translated to a paper-based device to provide a sensing platform that is simple to use, disposable, and low-cost, and is therefore well-situated for applications at the POC. The paper-based calcium sensor showed a Nernstian response between 10 mM and 100 μM and required only 12 μl of sample to perform …