开发用于早期检测乳房炎的电化学芯片——体细胞计数和免疫功能评价

Somatic cell count (SCC) is widely used to monitor bovine mastitis. Dairy farmers in Japan must send bovine milk to a laboratory for this inspection because the SCC device is large and expensive. In recent years, focusing on the increase in neutrophils in the early stage of mastitis, the method that can test for an early stage of mastitis by evaluation of the immune function of somatic cells in milk have been studied. In the current study, we previously evaluated cellular respiratory activity1. And we evaluated somatic cell count by elevation of respiration activity of somatic cells in milk and elevation of immune function by phorbol 12-myristate 13-acetate(PMA)-stimulated respiratory burst measurement using scanning electrochemical microscopy (SECM)2. In addition, we developed an electrochemical chip that enables counting of somatic cells in milk and evaluation of immune function. In SECM, we applied the oxygen reduction potential using a Platinum (Pt) microelectrode as a probe, scanned the vicinity of cells and between bulk and calculated the oxygen consumption of cells from the difference in current value between the two points to evaluate respiration activity. Fig.1A shows the arrangement of the electrodes. We made electrochemical chips by photolithography with multiple Pt electrodes and used SU-8 to form an insulating film to determine the electrode size. Fig. 1B is a schematic representation of a well prepared with Polydimethylsiloxane (PDMS). This well can hold 100 µL of sample/solution. Fig. 1C shows the method of respiration activity evaluation and respiratory burst evaluation using the electrochemical chip. There is only the measurement solution (11.4 mM glucose-containing phosphate buffer) in Ch1 well and milk sample in ch2 well while ch3 well contains milk sample containing PMA. We evaluated the respiration activity of somatic cells in milk from the difference in oxygen reduction current value between Ch1 and Ch2 (ΔI1) and estimated the number of somatic cells. We compared the difference in oxygen reduction current value (ΔI2) of Ch2 and ch3 with ΔI1 and evaluated the immune function by evaluation of respiratory burst. Our study will report the results on the number of somatic cells and the real-time monitoring of the respiratory burst.References(1) H. Kikuchi, A. Prasad, R. Matsuoka, S. Aoyagi, T. Matsue, S. Kasai: Frontiers in Physiology 7, 25, 1-6 (2016)(2) R. Kumagai, A.Prasad, S. Kasai, PACIFIC RIM MEETING on electrochemical and solid state science 2020, 11.4-9. 2020 Figure 1