Rapid diagnostic device for mastitis based on electrochemical detection of superoxide produced from neutrophils in fresh milk

To identify dairy cows suffering from subclinical mastitis, we used the fact that the number of neutrophils increases in raw milk. The neutrophils, a type of white blood cells, produce superoxide (O₂). Therefore, the amount of O₂ produced in raw milk can be used as an indicator for mastitis. To this end, we fabricated a device consisting of thin-film threee-lectrode systems. A self-assembled monolayer (SAM) of cysteine was formed on a gold electrode and superoxide dismutase (SOD) was immobilized there. Compartments were formed with polydimethylsiloxane (PDMS) by replica molding. A preliminary test that uses xanthine oxidase (XOD) to produce O₂ showed characteristic peaks originating from the production and rapid extinction of O₂. When a raw milk sample obtained from a cow suffering from subclinical mastitis was injected into the device and opsonized zymosan was added to activate neutrophils, a current increase distinctly different from the background was observed, demonstrating the applicability of this device for rapid diagnosis of subclinical mastitis.