Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

The overall purpose of this device is to provide a new microfluidic platform technology that realizes automated cascade reactions in small aqueous droplets. This method could help answer key questions to the field of synthetic biology by reproducing reaction sequences of microorganisms performing complex reaction sequences with enzymes and screening new catalysts. The main advantage of our lab-in-a-drop approach is that it is very adaptable.

As opposed to channel-based microfluidic chips, the only prerequisite is a hydrophilic-reaction environment. Though this method can provide insight into enzymatic reactions, it can also be applied to other systems, such as inorganic catalysts or stringing of ligands. I first had the idea for this method when I tried to design a microfluidic device with integrated product separation but without a complex system of valves and separators.

By moving the reaction solution as a droplet, reaction control becomes very simple. First, design in 3D print the coil bodies. Then, use a winding machine to wrap the bodies with a 08-millimeter copper wire 45 hundred times each.

Place a Peltier element on an electric board. Screw the first layer of coils onto the Peltier element to form a square. Then, secure the second layer of coils on top with the 3D-printed plastic frame or other non-conductive material.

Connect the electric board to the magnet control with a ribbon cable. Insert neodymium magnets into the coils. Place a one-millimeter thick quartz glass or plastic plate on top of the coil matrix.

Secure the cover in place with screws to finish assembling the actuation platform. To begin the nanoparticle synthesis under an inert atmosphere, first suspend 85 grams Iron(III)chloride hexahydrate and 3 grams Iron(II)chloride tetrahydrate in 200 milliliters of a four-to-one water-to-ethanol solution. Then, add 0.2 milliliters PFOTES and stir the mixture at 500 RPM with a magnetic stir bar.

Add 1.5 molar ammonium hydroxide to the mixture to achieve a pH of 8.0. And then continue stirring the solution for 24 hours to obtain the hydrophobic magnetic nanoparticles. Affix a bar magnet with an adhesive force of 25 kilograms to the bottom of the reaction flask.

Pour off the solution and wash the particles three times with a four-to-one water-to-ethanol solution. Remove the magnet and dry the particles at 60 degree Celsius for 24 hours. Characterize the particles with scanning electron microscopy.

Prepare a solution of 0.1 micrograms per milliliter horseradish peroxidase in 0.1 molar pH 6.5 potassium phosphate buffer with 10 millimolar hydrogen peroxide. Then, prepare a 20-micromolar solution of the fluorescent probe 10-Acetyl-3, 7-dihydroxyphenoxazine in potassium phosphate buffer. Then, gently grind the dry nanoparticles using a glass mortar and pestle.

Transfer the particles to a polystyrene weighing pan and add 10 microliters of the peroxidase solution. Gently swirl the pan for about 10 seconds to achieve nanoparticle self assembly around the peroxidase solution. Transfer this microreactor to the actuation platform.

Repeat this process with 10 microliters of the fluorescent probe solution and place the second microreactor on the platform. Store the remaining particles at room temperature. Use the Peltier element to keep the reaction solutions in the microreactors at 25 degree Celsius.

Mount a fluorescence microscope about 10 millimeters above the microreactors and connect the microscope to a computer. Using the magnet control activate the magnets and turn to position the microreactor of fluorescent probe solution under the microscope. Turn on the excitation light.

Raise the coil magnet to open the microreactor and begin recording the fluorescence microscopy image. After two-to-five seconds, lower the magnet to close the microreactor. Then, use the magnet control to move the peroxidase microreactor adjacent to the fluorescence microreactor.

Open the fluorescent probe microreactor. The peroxidase microreactor is pulled into the probe microreactor and opened, initiating the reaction. Monitor the reaction by fluorescence microscopy opening and closing the merged microreactor as needed.

Microreactors were created by coding droplets of solution with hydrophobic iron nanoparticles. These microreactors can be moved, opened, closed, and merged by manipulation of neodymium magnets under an actuation platform. The actuation platform can also be constructed with iron cores in the coil bodies.

This provides enough magnetic force to move a microreactor by more than 10 millimeters but is insufficent to open the microreactor. Thus, a neodymium magnet must be used to perform reactions in small solution volumes. A representative enzymatic reaction between a peroxidase and a fluorescent label was performed and monitored by fluorescent microscopy.

The Michaelis-mentioned kinetics observed during the reaction agreed well with literature values, indicating the the microreactor setup does not influence the reaction progress. After watching this video, you should have a good understanding of how to synthesize hydrophobic microparticles and to generate aqueous microdroplets with them. The platform technology can be used for automated reaction control by droplet merging.

Additionally, single microreactors filled with substrate can moved on top of small surface zones with mobilized enzymes. Using multiple immobilzation zones with different enzymes enables a sequential reaction cascade with transportable and immediate products. This prototype development is just the beginning.

Currently, we are implementing an automated dispenser system above the reaction platform to reposit immobilized enzymes at any position. The coil matrix will be increased to 10 times 10 positions. This technique can pave the way for computer-controlled planning and execution of biochemical reaction sequences.