Team:ESBS-Strasbourg/Notebook/Microfluidics

• Camille Bernard
• Fabian Stiefel
• Georgio Kourjian
• Jens-Sebastian Kalchschmidt
• Morgane Griesbeck
• Pierre Dillard
• Raphaël Doineau
• Renaud Renault
• Sebastien Pigeot
• Thea Ziegler
• Yohann Lacotte
• Yves Gendrault

Microfluidic is an emerging technique using the laws of fluid mechanics combined with the principal of emulsion. For this technique an emulsion with aqueous micro droplets in an oil solution is created. This water-in-oil emulsion is pumped through extremely small channels, offering with each droplet a new compartmentalization and so a new reaction chamber. Biological processes are usually restrained to the natural compartmentalization system which are the organelles and the cells itself with all the included biomembranes. This emerging technique offers opportunities to easily study biological processes such as enzymatic reactions, either in vitro or in vivo. This is one of the multiple possibilities provided by such a powerful tool. Indeed the droplets made by the water in oil emulsion have about the same size as the cells but are much simpler than microorganisms. So there is less interference with the cell metabolism.

The main advantage of this technique is the possibility to study the reaction of each compound separately. If the emulsion is correctly calibrated it is possible to create droplet short enough to contained just one compound. This high-throughput screening method in such a short volume of solution offers the possibility to realize selection (e.g. peptide binding screening) faster and cheaper than any conventional screening. Moreover, the manipulation allows also an increased efficiency in time and precision control, a more precise control analysis on short time and a possible automation of the process. Microfluidics is also a modular technique. The design of the micro canal system depends on the needed circumstances and can perfectly harmonize a lot of different techniques due to a wide range of different modules. Here is a table representing the advantages of microfluidic and their importance:

The complete potential of this technology is not yet understood, so the following list can just give a glimpse of possible applications:

• High through put drug screening in individual cell.
• Enzymatic assays, half life time and thermodynamically favorable conditions measurements…
• Posttranslational modifications (eg. chromophore).
• Semi-closed-loop system (eg. Cellular culture and medium change without stress)
• Chemistry in droplet-based microfluidic system.
• FADS (Fluorescent Activated Droplets Sorting).

There are a lot of different possibilities with this method and so a lot of different ways to design an experience. The first step is to design the micro canal system wanted depending on the needs required for the experiment. After an impression, a mask of the system is realized and a mold created from this mask. Next the chip is casted by using a polymer. For the microfluidic chips used in biology there are two mains basis polymers PMMA (PolyMethyl MethAcrylate) and PDMS (PolyDiMethyl Siloxane). For chemical reactions or enzymatic reactions, droplets are usually water in an oil fluid. For the test on whole cells, the perfluorocarbon are most commonly used (Perfluorocarbons can dissolve more than 20 times the amount of O2, and three times the amount of CO2, than water. A mouse can survive one hour undamaged immerged in oxygenated perfluorocarbon)

Figure 1: A basic microfluidic chip with three different inputs and one output.

Figure 2: common features for microfluidic manipulation

Load	to obtain formation of the droplet thanks to a bottleneck
Formulate	to obtain a droplet containing different compounds not mixed using laminar flow
Combine	to fuse two or more droplets
Sequential add	to obtain a progressive fusion of different droplets sequentially
Mix	to mix to get an homogenous droplet
Timing	to delay the droplets for an incubation time
Split	to split equally the droplets
Heat/Cool	to obtain a variation of temperature along the canal (eg. microfluidic thermocycler)
Detect	to detect the advancement of the reaction (eg. Spectrometry)
Direct	to sort the droplets, thanks to a magnet, following the fluorescence for example
Store	to store the droplets before or after a reaction
Re-loading	to re-load droplets contained in a store module

Unfortunately the time frame of the project and process in the summer did not allow us to use this technique; most of the preparation and experimental planning were ready. However from the beginning, we had the idea to use microfluidic as a tool to characterize our system and different promoters. We wanted to use the advantage of the system to separate the cells in single droplets where they could grow. Cells are separated at the beginning during the “load phase”. One unique cell is present which gives a very clear signal.

In our scenario the tagged protein is GFP. After the cells would have been separated we could measure the GFP signal for each cell which would have given us an estimation of the protein concentration of GFP before the activation of the system. Then we would have activated the protease with a light impulse and then measure the time the GFP needs to vanish. The test could have been conducted in thousands of cells, allowing the collection of a huge amount of data and therefore obtain a very precise characterization of the system in vivo. It could have helped to determine, the pulse time and intensity needed for activation/deactivation, the delay necessary for degradation of all tagged proteins, the quantity of proteins through degradation regulation after a succession of activating and deactivating pulses.

As a second application of microfluidics in our project, we wanted to use this tool to characterize the GFP expression level with different inducible promoter send with the biobrick registry pack. For this matter it is important to know the half-life and the maturation time of the GFP used. We would have used a well known promoter as referential (may be BBC_J23100)

Figure 3: Scheme representing the microfluidic channel that would have been used for the protease and promoter characterizations

The different measurements cells by cells and at different times permit to have a precise measure of the decrease of the fluorescence upon the time.

We are all disappointed that the conducting of these experimentations was not possible. Microfluidics seems to be an excellent technique and it would have been much rewarding for us to handle this technology. For the next generation of iGEM teams the microfluidic is a very attractive technique for the characterization of biobrick parts as promoters as this technique can, after the set up, give thousands measured points for many different parts in a very short time.

All the knowledge and the data shown are from the professor Andrew Griffiths at the “Laboratoire de Biologie Chimique – ISIS (Institut de Science et d'Ingénierie Supramoléculaires) – CNRS UMR 7006”.