Subclinical mastitis is a common infectious disease of dairy cows. Even without obvious clinical symptoms, this disease results in a significant economic damage in food industry. To develop rapid, highly sensitive, and portable diagnostic devices for subclinical mastitis, we used the fact that the number of neutrophils increases in raw milk of mastitic cows. Neutrophils secrete superoxide anion (O₂^‒). Therefore, O₂^‒ can be an indicator for the number of neutrophils and mastitis.

　To detect O₂^‒ electrochemically, we used a gold working electrode with immobilized superoxide dismutase (SOD) via a self-assembled monolayer (SAM) of L-cysteine. When an appropriate positive potential is applied to the working electrode, direct electron transfer occurs between O₂^‒ and the electrode via SOD (Fig. 1C). Therefore, the concentration of O₂^‒ is measured as an increase in current. Separation and concentration are other critical functions. In the flow channel structure shown in Fig. 1B, outlet 2 was first closed, and raw milk was flowed from the inlet to outlet 1. While passing through the microchannel, neutrophils tended to accumulate in the lower part and adhered to the bottom of the microchannel modified with P-selectin. Then, a phosphate buffer solution (PBS) was flowed to outlet 1 continuously. During this step, neutrophils remained at the bottom, whereas fat globules were eliminated (Fig. 2A). Furthermore, with a glass bottom modified with P-selectin, the cell density was approximately 2.1 times higher than the case with a blocked glass bottom (Fig. 2B). Finally, outlet 1 was closed and outlet 2 was opened. PBS was similarly drained and neutrophils rolled on the bottom surface toward the micropillars and were trapped there (Fig. 2C, D). We then carried out the detection of O₂^‒ secreted from neutrophils in real mastitic milk samples. The output current increased with the increase in the density of neutrophils and the seriousness of mastitis. Furthermore, current observed with real mastitic milk containing 5 × 10⁵ cells/mL could be clearly distinguished from that of normal milk.