Diagnosis and Monitoring of Milk Fever with a Laser-Engraved Calcium Sensor

Milk fever is a metabolic disorder that occurs in dairy animals and its occurrence is more prevalent a few months before and after calving and can lead to a range of clinical symptoms, including loss of appetite, muscle spasms, lateral recumbency, and eventually coma or death if left untreated. The primary cause of these symptoms is a decrease in blood calcium levels, hypocalcemia. Previous studies have categorized cows into three groups based on their serum calcium levels: normocalcemia (≥ 2.0 mmol/L), subclinical hypocalcemia (&lt; 2.0 mmol/L but without any observable clinical signs), and clinical hypocalcemia (with clinical signs). Based on previous studies, the prevalence of subclinical hypocalcemia in the pasture-based system was around %50. Moreover, the productive life of dairy cows with hypocalcemia decreases by 3.4 years, which can have economic impacts on animal husbandry businesses. Since subclinical milk fever does not have symptoms and frequent testing of calcium levels is expensive, subclinical milk fever is rarely diagnosed. Thus, having a Point-of-Use inexpensive sensor for frequent and on-demand calcium measurement is beneficial. For this purpose, we fabricated an inexpensive Laser-induced Graphene based potentiometric electrode to detect calcium levels in the clinically relevant range.<br/>Materials and Methods:<br/>We prepared the calcium selective membrane by mixing poly(vinyl chloride) (PVC) (33%), 2-nitrophenyl octyl ether (66%), calcium Ionophore (IV) and potassium tetrakis(4-chlorophenyl)borate (KTPClB) (1%), with tetrahydrofuran. We fabricated the LIG sensors by directly operating a CO2 laser on a Kapton film. Then, we drop-casted the membranes on the electrodes. We performed all the measurements at room temperature using a 16-channel potentiometer relative to a commercial reference electrode.<br/>Results and Discussion:<br/>We used laser engraving to pattern graphene on a low-cost polyimide film. SEM results indicated a 3D porous structure of LIGs with a thickness of 20 µM. Furthermore, EDX and RAMAN spectroscopy were performed on the sensors to confirm the presence of graphene on the PI films. The results indicate that the sensitivity of our LIG-based sensors is 26.58± 0.28 mV/dec, which is close to the theoretically expected value (29.6 mV/dec). Moreover, the limit of detection (LOD) of the sensor is 1.87 µM, which is ~ 3 orders of magnitude lower than the clinically relevant range of calcium in the body. Another important characteristic of the sensor that ensures its reliability is the selectivity toward the ion of interest. The Selectivity results of the sensor indicates superior selectivity of the sensor over physiologically relevant ions and biologicals. Finally the sensor’s performance in the bovine sample was evaluated. To this purpose, first the sensor where calibrated in two different solutions of artificial serum and then the sensors were exposed to the bovine serum. The results were compared with a commercial Calcium sensor indicating %104.54 recovery of LIG sensors.<br/>Conclusion:<br/>We fabricated a LIG-based potentiometric sensor that shows outstanding sensitivity and selectivity toward the ion of interest. Moreover, the sensor shows %4.54 error compared with a commercial electrode that is suitable and acceptable for in field detection of subclinical milk fever. Moreover, using a laser as an inexpensive, fast, and mass-producible method for fabricating the sensors makes them a suitable choice for in-field frequent use.