Microfluidic PDMS Device: “The Mother Machine”

The use of microfluidics in biological research has gained much popularity in recent years1. Subfields that have been impacted by this technology range from tissue engineering2, cancer stem cell research3, gene expression of embryonic stem cells4, protein interactions5, diagnostic medicine as well as microbial physiology and behaviour6-7, to name a few. A specific contribution to the field of microbiology is the ability to observe and manipulate single cells in a controlled manner.

Schematic overview of the microfluidic device bound to the cover glass.
Figure 1. Schematic overview of the PDMS microfluidic device bound to the cover glass.

We fabricated a versatile device with growth channels, capable of being used to study different bacterial species in a high-thoughput manner (Figure 1). In its design, cells are confined in the growth channels oriented perpendicularly to a trench through which growth medium is flown. This polydimethilsiloxane (PDMS) device makes it possible to study a large number of cells over multiple generations. Cells are immobilized, in the absence of chemical fixation, at the far end of such a growth channel (ca. 25 µm in length). The length of the growth channels is chosen so as to ensure sufficient supply of nutrients to the bacteria by diffusion. Such an immobilization scheme allows one to simultaneously study numerous different cells for extensive periods of time.

Schematic overview of the three principal steps in creating the PDMS device.
Figure 2. Schematic overview of the three principal steps in creating the PDMS device.

The channels in the microfluidic device are formed out of PDMS, which has a number of attractive features that makes it an excellent material. For example, sub-micron sized structures down to ca. 100 nm are possible in PDMS, and its porosity enables the immobilized organism to receive oxygen. The fabrication of the device contains three principal steps (Figure 2). Firstly, the pattern of the device is etched into a 4″ silicon (Si) wafer (Figure 2, step 1), accomplished by employing Electron Beam Lithography (EBL) and specific dry etching protocols. Secondly, we use PDMS to create a negative mold of the structure that was fabricated in the Si wafer (Figure 2, step 2). Finally, we use this PDMS mold to fabricate the final device in PDMS (Figure 2, step 3). To optimize fabrication yield, we employ a wafer much larger than the size of a single device, allowing use to fabricate multiple individual devices (current protocol allows 24).

Figure 4. a) Image of the final Si wafer folling the fabrication.
Figure 3. a) Image of the final Si wafer following fabrication. One can observe the 24 structures etched into the Si wafer. The inlet outlet ports and the main trench can be seen. b) SEM image of fabricated sub-micron channels in the silicon wafer. Here is a side view of a part of the main trench and the small growth channels.

After the final PDMS device is created from the mold, the bacteria can be loaded into the channels by means of centrifugation. Keeping a constant flow of nutrients, one is now able to start the high-throughput live-cell imaging experiment. Typically a measurement with the PDMS device lasts for ~14h, tracking ~70 cell cycles per channel, which equates to ~840 cell cycles per experiment (maximum yield).

Currently we use the device to study genetically engineered strains of E. coli to study dynamics of proteins that are part of the DNA replication process. The proteins of interest are fluorescently labelled and tracked during the stages of the cell cycle. Our setup allows to track three protein populations, labelled by blue, red and yellow/green fluorescent proteins. Here is an example of an E. coli strain containing a fluorescently labelled (CFP) chromosomal locus.

Figure 4. a) Brightfield image of a loaded PDMS device with genetically engineered E. coli, having a fluorescently labelled (CFP) chromosomal locus. b) The corresponding fluorescence image after illuminating the sample with a 457 nm laser.

The growth of this E. coli strain and chromosomal dynamics can be observed in the following movies: Brightfield Movie, CFP Fluorescence Movie.

More information on the fabrication protocol can be found in the relevant publication: Moolman et al. (2013)

This PDMS microfluidic device is used to study the sliding clamp (ß2) protein, part of the DNA replication machinery in Escherichia coli. To learn more about how we prepare the microfluidic device in studying bacteria: Moolman et al. (2014)

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