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The diagnosis of milk fever or hypocalcemia in lactating cows has a significant economic impact on the dairy industry. It is challenging to identify asymptomatic subclinical hypocalcemia (SCH) in transition dairy cows. Monitoring subclinical hypocalcemia in milk samples can expedite treatment and improve the health, productivity, and welfare of dairy cows. In this study, an attomolar-sensitive sensor is developed using extrusion-based 3D-printed sensing structures to detect the ratio of ionized calcium to phosphate levels in milk samples. The unique geometries of the lateral structure of 3D-printed sensors, along with the wrinkled surfaces, provide a limit of detection down to the attomole (138 a m ) concentration of the target analyte. The calcium-to-phosphate ratio in milk samples not only provides early disease indications but also enables on-site testing. This highly selective test is validated using real milk and blood samples, and the results are compared with those of commercial meters. This fast response (~10 s) low-cost sensor opens a promising tool for the farm-side diagnostic of dairy cows that can promote best practice management of dairy cows. A 3D-printed sensor with surface-wrinkled structures detects subclinical hypocalcemia in dairy cows by measuring the calcium-to-phosphate ratio in milk. With attomolar sensitivity and rapid results, it enables early treatment, improving cow health and productivity.

Biosensors are pivotal in addressing global challenges in healthcare, environmental monitoring, and diagnostics by providing rapid, sensitive, and portable detection solutions. This thesis explores the integration of allosteric transcription factors (aTFs) and Cas12a to create a novel biosensing platform, leveraging the ligand specificity of aTFs and the robust signal amplification of Cas12a’s trans-cleavage activity. The platform focuses on detecting biomarkers such as N-Acetylneuraminic acid (Neu5Ac) and heavy metals like lead, cadmium, and copper, using corresponding aTFs—NanR, CadC, and CsoR. Experimental evaluations address heavy metal interference on Cas12a activity, aTF gating efficiency, and variability among Cas12a orthologs. Our findings showed differences in heavy metal interference on different Cas12a orthologs and the ability for aTFs to gate Cas12a activation. Future development and expansion of the platform could lead to innovative solutions for precise diagnostics and environmental monitoring, addressing challenges in resource-limited and field-deployable settings.