<p> Supplementary Materials</p><p>FRACTAL-SHAPED MICROCHANNEL DESIGN FOR A KINETIC ANALYSIS OF BIOCHEMICAL REACTIONS IN A DELAY LINE</p><p>Katsuki Hirata1, Tetsuo Ichii2, Hiroaki Suzuki1,2, Tomoaki Matsuura2,3, Kazufumi Hosoda1, Tetsuya Yomo1,2,4 1Graduate School of Information Science and Technology, Osaka University, Japan, 2ERATO, JST, Japan, 3Graduate School of Engineering, Osaka University, Japan, 4Graduate School of Frontier Biosciences, Osaka University, Japan Figure S1. Fluorescence images of emulsion containing hydrolyzed fluorescent substrate (TokyoGreen). (A) Mineral oil containing Span80 and Tween80 is used as the oil phase. TokyoGreen leaked out to the oil phase immediately after emulsification. (B) Fluorocarbon oil (FC-40) containing EA-surfactant is used as the oil phase. TokyoGreen was retained in the droplets. Scale bar: 100 m. Figure S2. Calibration curve of the fluorescence intensity and the hydrolyzed substrate concentration obtained from the images of droplets containing known hydrolyzed substrate concentrations in the channel. This dilution series for calibration was prepared from the solution of TokyoGreen completely hydrolyzed by -glucuronidase enzyme in a test tube, followed by denaturing this enzyme by adding SDS. Figure S3. Actual designs at the droplet formation (flow focusing) site. The area marked with the red square is enlarged. (a) Merging design used to mix enzyme and substrate. (b) Simple straight water-phase inlet used for the gene expression reaction. Supplementary Table 1. Components in the two solutions to complete the enzyme reaction system used in the merging channel design.</p><p>Sol.1 Sol.2 -glucuronidase,1 or 0.2 nM TokyoGreen-GlcU, 5 M Hepes-KOH, 50 mM Hepes-KOH, 50 mM, Mg(OAc)2, 13 mM Mg(OAc)2, 13 mM, potassium glutamate, 100 mM potassium glutamate, 100 mM BSA, 300 mg/ml BSA, 300 mg/ml Transferrin,Alexa Fluor 594, 0.5 M Supplementary Table 2. Components in the gene expression reaction using the PURE system. Components in the lab-made PURE system is listed in our previous report.23</p><p>DNA, 10 nM PURE system Transferrin, Alexa Fluor 594, 1 M T7RNApolymerase, 0.87 M Tokyogreen-GlcU, 50 M Rnaseinhibitor, 0.2 U/mL Figure S4. Lineweaver-Burk plot of the -glucuronidase hydrolysis reaction performed in a test tube. Reaction time courses for various initial substrate concentrations hydrolyzed by 5 pM - glucuronidase was measured using a real-time PCR system (Mx-3005P, Agilent Technologies), from which initial reaction velocities were extracted. Kinetic parameters derived from this plot are -8 -6 3 8 Vmax = 3.310 M/min, Km = 8.710 M, kcat = 6.510 min, and kcat/Km = 7.510 /min M.</p>