On-chip immune cell activation and subsequent magnetic bead-based cytokine detection Patthara Kongsuphol*, Yunxiao Liu, Qasem Ramadan Institute of Microelectronics, Agency for Science Technology and Research (A*STAR), 11 Science Park Rd, Singapore Science Park II, Singapore, 117685 *Corresponding author: P. Kongsuphol, email: kongsupholp@ime.a-star.edu.sg Abstract Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of a panel of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of double-layered microfluidic chambers was fabricated to enable simultaneous cell culture and detection of the induced cytokines at multiple time-points. Each culture chamber comprises two fluidic compartments which are dedicated for cell culture and immune assay, respectively. The two compartments are separated by a porous membrane, which allows the diffusion of cytokines molecules from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The LPS-induced cytokines were quantified in situ after differential stimulation of U937 monocytes and differentiated macrophages. This study provides a proof-of-concept for future automated system development which will be integrated into a complex cellular system for disease modeling. 1. Introduction Monocytes and macrophages play an essential role in the immune system and maintaining tissue homeostasis 1 . Upon activation by either endogenous or exogenous factors, monocytes and macrophages release different types of cytokine to regulate and trigger proper immune responses in which these responses can be either defense, inflammation, or tissue remodeling 2,3 . Cytokines mediate many physiological events that contribute to disease development. Due to the importance of cytokines role as pathogenesis biomarker, it is essential to quantify cytokines in an accurate and timely manner even in a minute amount of biological samples. The quantitative detection of cytokine profile and subsequent immunophenotyping holds great promises for a better understanding of many disease mechanisms, personalized diagnostic and immune therapy. As a result of growing knowledge in cytokine, the release of cytokines from immune cells is being used as a parameter for many disease diagnosis and prognosis especially in infectious and autoimmune diseases 4–7 . In cancer research, considerable attention is paid to the possible role of cytokines-based in cancer therapy 8,9 . Currently, enzyme-linked immunosorbent assay (ELISA) is the most accepted method of cytokine detection which enables high sensitivity multiplexed detection however conventional ELISA is a time- consuming process that requires multiple incubations and several washing steps. On-chip cytokine detection offers a much shorter operational time as compare to ELISA 10–14 . Furthermore, On-chip systems allow analysis of minute quantities of patient-derived samples, which is often difficult to analyze using conventional cell culture and detection systems 15 . Therefore, miniaturization of in vitro analytical systems has, thus, attracted significant attention. Zhu et al. 16,17 described a technique that combines cell culture, cell activation, and cytokine detection processes on a single chip offering a miniaturized and efficient