(Invited) Electrochemical Luminescent Imaging of Activated Neutrophils Using Multi-Microwell Array Electrode

Abstract

Vasculitis is an autoimmune disease leads to various sicknesses (e.g. arteriosclerosis). Mechanisms of vasculitis have not been accurately understood, and early detection and diagnosis of vasculitis remain constant challenges. One of the causes of vasculitis is neutrophil-derived reactive oxygen species (ROS) that attack blood vessels. Therefore, detection of ROS derived from activated neutrophils could be important for early diagnosis of vasculitis. Herein, we developed a device for detection of ROS from activated neutrophil at single-cell resolution using electrochemiluminescence (ECL) method. ECL is it’s a highly sensitive method, which is suitable for detection of ROS from a single activated neutrophil. Results & discussion We constructed a multi-microwell array electrode device that enables to detect the generation of ROS from activated neutrophils. Microwell array was fabricated using PDMS and put on an electrode. By sealing neutrophils into the microwells, we could prevent diffusion of ROS, and keep its concentration high. Luminescence was generated by the reaction between luminol and ROS, and its intensity was quantified using EM-CCD camera. Neutrophils were isolated from healthy human blood, and were activated by adding PMA. The activated neutrophils are recognized as a model cells for vasculitis researches. The activated cells and non-activated cells were trapped in microwells and their luminescence was quantified by the EM-CCD camera. As a result, we succeeded in detecting ROS from activated neutrophils. This device may have potential to diagnose vasculitis by comparing the number and condition of activated neutrophils between healthy people and vasculitis patients.