INTRODUCTION In the United States, 30-50% of dairy cows experience metabolic disorders due to nutritional deficiencies, leading to economic losses, especially from reduced milk yield due to hypocalcemia. Calcium is crucial for muscle and nerve function, skeletal strength, and immune function. Laboratory screening costs $60 per sample, and on-site testing costs around $1000. With approximately 33% subclinical and 5% clinical hypocalcemia prevalence in US dairy herds, there's a need for smart, cost-effective sensors. Early diagnosis is vital for disease prevention, relying on rapid, farm-side, accurate assessment of calcium (Ca2+) and phosphate (P) ion levels.Herein, an attomole-sensitive sensor was developed using 3D printed structures to detect ionized phosphate to calcium ratios in milk samples, diagnosing milk fever. Unique 3D printed sensor geometries with wrinkled surfaces provide an unprecedented limit-of-detection of 138 aM. Wrinkled surfaces of poly (3-octyl-thiophene) act as ion-to-electron transducers for Ca2+ and P detection. The P/Ca2+ ratio measurement with potentiometry technique not only indicates early disease but also enables on-site testing without any need for mediator or sample preparation. Our sensor validated using cow’s milk, whole milk, and fetal bovine serum, and results were compared with commercial meters. This low-cost, sensitive, and fast response (~10 s) sensor is a promising tool for in-farm diagnostic facilitating management of dairy cows. SENSOR FABRICATION The manufacturing process begins with 3D CAD design of the sensor base (Figure 3c). The sensor is produced using a high-resolution extrusion-based 3D printer, enables the manufacturing of microstructures. Prints undergo washing process, and resin is cured with UV light (405 nm). Figure 2b shows two distinct microstructure types on the sensor base, amplifying surface area. The lateral pattern enhances surface area, aiding 3D microelectrode formation for analyte diffusion. Besides, random wrinkles form unintentionally due to resin instability during UV exposure, serving as microelectrodes and increasing surface area, leading to enhanced sensitivity. Next, a Kapton shadow mask covers the sensor's surface, and an e-beam evaporator applies a gold thin layer to uncovered regions. Ca2+ and P ion sensing electrodes are modified with POT as the ion-to-electron layer, followed by drop-cast Ca2+ and P ion-selective membrane (ISM) on top of POT layer. The reference electrode is constructed by applying Ag/AgCl paste between two working electrodes, covered with a Nafion layer for stability and Cl¯ leaking prevention. To improve repeatability, each sensor section is treated with a high ion concentration solution of Ca2+ and P. RESULTS AND DISCUSSION Surface characterization of the electrode involved electrochemical and surface analyses. FE-SEM images in Figure 2b, d show the lateral pattern and micro-wrinkles of the sensor base. Cyclic voltammetry investigated electrochemical behavior, revealing distinct peaks during ferro/ferricyanide presence (Figure 2a). Gold catalyzed mediator redox reaction, with the POT layer decreasing peak currents due to increased surface resistance. The ISM, primarily PVC, elevated electrical resistance, and selectively allowing Ca2+ and P to pass through rather than mediators, reducing current. Electrochemical Impedance Spectroscopy confirmed findings, with Nyquist plots showing charge transfer resistance (Rct) in Figure 2c. Open circuit potentiometry (OCP) detected Ca2+ and P ions (Figure 1a, 1c). OCP response increasing with higher Ca2+ concentration and decreasing with elevated P ion concentration. Calibration graphs in Figure 1b, 1d demonstrated a linear relationship between ion concentrations and OCP. OCP measurements across various concentrations, sensor washing, and repeated measurements in Figure 1a, 1c indicated sensors repeatability. Finally, sensor tests with fresh milk samples from 15 lactating Holstein multiparous cows revealed notable variation in P levels compared to Ca2+ concentration. Examining the P/Ca2+ ion ratio in milk samples (Figure 6f) assessed milk fever in dairy cows, indicating health status in terms of hypocalcemia, and hypercalcemia. Note: The high quality images will be provided in the poster. Figure 1