Development of a rapid calcium testing strip with metal-enhanced fluorescence for early detection of hypocalcemia in...

Hypocalcemia (milk fever) is a severe metabolic disease in dairy cattle, affecting up to 50% of multiparous cows postpartum and leading to significant economic losses and increased risk of secondary disorders. Rapid, low cost, and on-site detection of low blood calcium levels (< 1.1 mM) is needed for timely intervention, yet current diagnostic methods are costly, complex, or require centralized laboratory analysis, delaying treatment. This study addresses the need for a simple, rapid, and cost-effective diagnostic tool by developing a gold nanoparticle-functionalized test strip for the quantification of ionized calcium (Ca²⁺) in bovine plasma and milk. The assay uses the Ca²⁺-sensitive fluorescent dye Fluo-5 N, with gold nanoparticles (AuNPs) integrated into nitrocellulose membrane to amplify the fluorescent signal based on the metal-enhanced fluorescence (MEF) effect. This test is designed for rapid, dip-and-read operation. Optimal assay performance was achieved through a simple, scalable fabrication process costing an estimated $0.14 CAD per strip and yielding a rapid 30-second readout after sample application. MEF optimization demonstrated a threefold increase in fluorescence intensity compared to controls without AuNPs. The test strip established strong linear correlations across clinically relevant dilutions in bovine plasma (R² = 0.981) and skim milk (R² = 0.941), with 95.0% to 108.6% recovery across different milk dilutions and an average recovery of 96.7% in skim milk, confirming its robustness against matrix effects in the target biological fluids. The functional detection range, spanning from a limit of detection of 431µM to 1.1 mM, and with a limit of quantification of 616 μM, effectively covering the critical diagnostic window for subclinical hypocalcemia (< 1.1 mM). Additionally, the test strips demonstrated long-term stability, maintaining performance for at least 120 days when stored at 4 °C. Lastly, the simple dipping-based sample application was validated as statistically equivalent to pipetting, showing potential for adaptation to farm use. This simple, portable technology could significantly improve herd health management with the potential to enable rapid, on-site screening for subclinical hypocalcemia, facilitating early treatment, and reducing the economic burden due to milk fever.