Team:EPFL/Description/Aptamers
Microfluidics
Microfluidics is a powerful tool in synthetic biology with a wide range of applications such as transcription factors - DNA interactions and screening aptamers. Microfluidics chips consist of micron-sized channels where volumes on the scale of micro-liters flow can interact.
The use of microfluidics has many advantages including high sensitivity, reducing costs and increasing throughput.
These microfluidics chips are made of polymeric organosilicion compounds such as PDMS (polydimethylsiloxane) and contain integrated mechanical systems (sandwiches, necks, buttons, …) allowing a great modularity in experiments.
Since we are working with proteins and DNA aptamers, it was necessary to use small reagent volumes in order to minimize the cost of our experiments but also to measure with high precision the affinity between the protein and the aptamers. Initially, our plan was to go with surface plasmon resonance (SPR) to test the aptamers but we couldn't afford our local Biacore SPR machine. Furthermore, microfluidics could also be a viable implementation for our diagnostic test.1
How does it work?
A MITOMI (Mechanically Induced Trapping of Molecular Interactions) 768 chip is a simple double layer microfluidic device composed of inter-connected channels and chambers2. The device is composed of two layers, referred to as the control and the flow, which are separated by a thin layer of PDMS. The so-called control layer allows the opening or closing of the flow channels by pressurizing the control channels using an incompressible fluid such as water. Under the control layer, the so-called flow layer encapsulates the reagents. Both layers are finally aligned and fixed on an epoxy-coated glass slide as shown in Figure 1 (A). In Figure 1 (B), a magnified view shows two unit cells and the fluid within these cells can be controlled by three separate mechanical valves: the sandwiches, the neck and the button.
Once the chip is ready to be used, reagents are held within small pieces of Tygon tubing that are plugged into the inlets of the flow layer with connecting metal pins. Tubes filled with deionised water are plugged into the control layer in order to open or close mechanical systems. The tubings that are plugged into the flow inlets are used to flow all the chemicals (NeutrAvidin, protein, aptamers, …).
Before running an experiment, the glass surface of the channel is patterned with NeutrAvidin at the button area, while all other parts of the channel are passivated with biotinylated BSA as shown in Figure 2, enabling the specific binding of biotinylated proteins or DNA oligos to the button area.
Since the first reactant will need to bind to NeutrAvidin, the first compound, which is being flown in the various channels, must be biotinylated. At least one compound of the experiment must integrate a fluorophore since the chip is being analyzed under a fluorescent microscope.
To summarize, the general workflow starting from mold fabrication to data analysis of a MITOMI experiment is shown in Figure 3.
References
1. Maerkl, Sebastian J., and Stephen R. Quake. "A systems approach to measuring the binding energy landscapes of transcription factors." Science 315.5809 (2007): 233-237.
2. Piraino, Francesco, et al. "A digital–analog microfluidic platform for patient-centric multiplexed biomarker diagnostics of ultralow volume samples." ACS nano 10.1 (2016): 1699-1710.
