Thermal-stabilized nanoflower luciferase-based detection of SCC and TPC in raw milk

Simultaneous determination of Somatic Cell Count (SCC) and Total Plate Count (TPC) in raw milk reflects milk cleanliness and cow health. The luciferase was thermally unstable and required storage at -20 °C, with repeated freeze-thaw cycles reducing activity. To overcome this limitation, an inorganic hybrid nanoflower luciferase was synthesized by mixing genetically engineered free luciferase with PBS and zinc acetate, followed by incubation, centrifugation, washing, and resuspension. Transmission electron microscopy confirmed successful assembly of luciferase into nanoflower structures. The nanoflower luciferase remained stable over 75 % of its initial activity after 21 days at 4 °C, whereas free enzyme required -20 °C.Compared with genetically engineered free luciferase, the nanoflower form effectively and simultaneously detected SCC and TPC in raw milk even after 2-fold dilution. The limits of quantification were 1 × 10⁴ cells/mL for SCC and 1 × 10⁵ CFU/mL for TPC.