Electronic Supporting Information Enzyme-responsive fluorescent nanoemulsion based on lipophilic dye liquid Ryoutarou Oishi, a Kaho Maki, a Tatsumi Mizuta, a Kenji Sueyoshi, a,b Tatsuro Endo, a Hideaki Hisamoto a a. Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University 1-1 Gakuencho, Nakaku, Sakai, Osaka 599-8531, Japan E-mail: hisamoto@chem.osakafu-u.ac.jp b. Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 5-3 Yonban-cho, Chiyoda, Tokyo 102-8666, Japan Experimental 1. Material The lipophilic fluorescent alkaline phosphatase (ALP) substrate, 4-[2-(dodecylamino)-2-oxoethyl]-2-oxo- 2H-chromen-7-yl phosphate (12-PC), and ALP-responsive lipophilic dye liquid, [P 66614 ] 2 [12-PC], were prepared according to the methods described in our previous report. 1 Pluronic F-127 (F-127), poly(vinyl chloride) (PVC), and ALP were purchased from Sigma Aldrich (St. Louis, MO, USA), while sodium chloride was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Tetrahydrofuran (THF) and bis(2-ethylhexyl)sebacate (DOS) were obtained from Kanto Chemical (Tokyo, Japan). Tetrahexylammonium hydroxide (N 6666 OH) was purchased from Tokyo Chemical Industry (Tokyo, Japan), and 2-[4-(2- hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was purchased from Dojindo (Kumamoto, Japan). The poly(ethylene terephthalate) (PET) film was obtained from Teijin Limited (Tokyo, Japan). 2. Preparation and characterization of PC-NE [P 66614 ] 2 [12-PC] (26.7 mg) and F-127 (33.4 mg) were dissolved in 5.0 mL of THF. Three milliliters of the mixture was added to 27 mL of ultrapure water at a flow rate of 2 mL/min. After the evaporation of THF, a dispersion of PC-NE was obtained. The particle size and zeta potential were measured using nanoparticle analyzer (NanoPartica SZ-100V2, HORIBA, Kyoto, Japan). The dispersion was dropped onto a copper grid Electronic Supplementary Material (ESI) for Analyst. This journal is © The Royal Society of Chemistry 2021