Rapid Diagnostic Device for Subclinical Mastitis Based on Electrochemical Detection of Superoxide Produced from Neutrophils in Fresh Milk

Abstract

Electrochemical microdevices were fabricated to identify mastitic cows based on the increased number of neutrophils in raw milk. Because neutrophils produce superoxide (O₂^·-), the amount of O₂^·- can be used as an early indicator for subclinical mastitis. In the microdevices, O₂^·- was detected on a gold electrode using superoxide dismutase immobilized via a self-assembled monolayer of cysteine. In a preliminary test using xanthine oxidase to produce O₂^·-, one of the devices detected the production and rapid extinction of O₂^·-. When neutrophils obtained from a mastitic cow were concentrated by centrifugation and introduced into the device, a current increase distinctly different from the background was observed. Furthermore, a micropillar structure was fabricated on the gold electrode to trap and collect neutrophils, thereby facilitating the concentration of these cells around the electrode. The measured current clearly depended on the number of neutrophils in raw milk samples, demonstrating the applicability of the device for rapid diagnosis of subclinical mastitis.