4Institute for Analytical Instrumentation of RAS, 31 Ivana Chernykh, 198095, St. Petersburg, Russia peter.belobrov@gmail
Microfluidic bioassay based on bacterial luciferase and NADH:FMN-oxidoreductase PI Belobrov1,2, IA Denisov1, КA Lukyanenko1, AS Yakimov1, EN Esimbekova1,2, KI Belousov3, AS Bukatin1, IV Kukhtevich1, VV Sorokin1, AA Evstrapov3,4 1Siberian
Federal University, 79 Svobodny Pr., 660041, Krasnoyarsk, Russia of Biophysics SB RAS, 50/50 Aсademgorodok St., 660036, Krasnoyarsk, Russia 3ITMO University, 49 Kronverksky Pr., 197101, St. Petersburg, Russia 4Institute for Analytical Instrumentation of RAS, 31 Ivana Chernykh, 198095, St. Petersburg, Russia
[email protected] 2Institute
Abstract The disposable microfluidic chips [1] for measurement of water pollution can be made with biomodule based on enzymes of luminous bacteria: luciferase and NADH:FMN-oxidoreductase [2, 3]. Interaction of organic and inorganic pollutants with these enzymes from the bacterial luminescence system leads to quenching of light emission and changing of shapes of measured kinetic curves. Here we show the automation of this biomodule for ecological bioassay with microfluidic techniques. Luminescence chemical reactions in biomodule are described by the equations: NADH:FMN-oxidoreductase NADH + FMN + H⁺ ——————————→ FMNH₂ + NAD⁺ luciferase FMNH₂ + RCHO + O₂ ————→ FMN + RCOOH + H₂O + hν The components of the reaction: luciferase (EC 1.14.14.3) from Photobacterium leiognathi, NADH:FMNoxidoreductase (EC 1.6.99.3) from Vibrio fischeri, NADH and tetradecanal were immobilized in gel made from potato starch and placed in reactor chamber. FMN for reaction activation was deposited separately in chip by the process of droplet drying. Channelized surface of poly(methyl methacrylate) microfluidic chips was formed by direct cutting with the milling machine MDX-20 (Roland, Japan). Sealing of microfluidic chips was carried out by spraying of acetone or dichloroethane on flat half of chip followed by pressing of channelized half with pressure ~ 4 N/mm². The influence of acetone and 1,2dichloroethane on enzymes was estimated. The kinetics of light emission during bioluminescent reaction was recorded with the single tube luminometer GloMax 20/20 (Promega, USA). The bioluminescent reaction starts when FMN mixed with components around gel film solving in reaction chamber. As FMN activates the reaction of light emission it is important to have a uniform concentration of FMN in all parts of the reaction chamber. To improve mixing and achieve reproducible results different topologies of microfluidic chip (Fig. 1) were studied. Dynamics of FMN dissolution and mixing was studied using numerical simulations. It was shown that FMN dissolution takes 0.7–0.8 s depending on the flow rate but the passive mixing efficiency is not enough to achieve uniform concentration profiles in the reaction chamber. Because of that we suppose that active mixing strategies should be implemented in the microfluidic chip for increasing results reproducibility. In topology (c) sample proceeded through the input channel into a serpentine mixer with dried FMN, where it dissolved FMN, and then stirred with it. Dissolved FMN with a sample then entered the reaction chamber. In (b) and (c) passive mixing was used and in (d) and (e) - active mixing. In topology (d) FMN was dried next to the immobilized enzyme system in reaction chamber. When the flow got to the chamber it stopped at the output hole, which was located in the end of the reaction chamber. Then the active mixing started for a few seconds followed by the measurement of the kinetics. Topology (e) differs from (d) in a way that the flow came to the reaction chamber from two different directions and stopped at the “stop-line”. Active mixing join two droplets through this “stop-line“ and mixing them, then the measurement of kinetics is starting. The influence of surface modification of microfluidic chip to the properties of immobilized luminescent system components was studied in topology (a) with 4 different surfaces (Fig. 2). The experiments were carried out with clean water without any pollutant. The immobilization with gelatin (3) was shown to be the best option. We assume, that gelatine protects enzymes during drying process,
so they provide better repeatability of measurements and the highest intensity. Observed decreasing of luminescence in sealed chips is due to the lack of O₂ in the reaction chamber. Active mixing provides more uniform concentration of FMN in reaction chamber and therefore is more preferable. Dean vortices significantly influence the speed of FMN dissolution. Starch gel film on top of gelatin film provides maximum reproducibility of the results. Acknowledgement The research was supported by the grant of the Russian Science Foundation (project no.15-19-10041). References [1] Sackmann, E., Fulton, A., Beebe, D., Nature, 507 (2014) 181-189. [2] Esimbekova E., Kondik A., Kratasyuk V., Environmental monitoring and assessment, 7 (2013) 59095916. [3] Esimbekova E., Kratasyuk V., Shimomura O., Adv Biochem Eng Biotechnol, 144 (2014) 67-109. Figures
Fig. 1. Evaluated microfluidic chip topologies. Inlets at the bottom. Dried gel droplets are shown by nets. Dried FMN droplets are shown by dots.
Fig. 2. Maximal intensity of bioluminescent reaction for unsealed (black) and sealed (grey) microfluidic chips with different types of surface treatment before compounds immobilization: (1) the droplet with reactants was dried in a reactor chamber (2) the PMMA was treated with 1M NaOH solution for 18 hours, then washed and a droplet of gel was dried in a reaction chamber (3) surface of reaction chamber was covered with gelatin, then the droplet of gel with reactants was dried on top of it (4) the surface of reaction chamber was modified with amylose and amylopectin molecules covalently bonded by ethylenediamine dihydrochloride to it. Percent demonstrates the decrease in luminescence intensity in sealed chip compared to unsealed chips.
