Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

1. Wafer Fabrication

1. Design the microfluidic device containing inlets, outlets, main channels and the PLBRs (Figure 1A) using CAD software.
2. The design presented in this protocol (Figure 2) consists of two seeding inlets, a gradient generator for mixing of two different substrates, one outlet, and six arrays of PLBRs. Each array contains 5 PLBRs, resulting in 30 parallel PLBRs inside one microfluidic device.
3. Create a lithography photomask containing the desired chip layouts (Figure 1B). The photomask was produced in-house by electron beam writing with submicron resolution. The mask used was composed of a chromium layer on a 5 in² glass plate.
   Note: perform all following steps under cleanroom class 100 conditions or better (a process flowchart is shown in Figures 3A and 3B).
4. Clean a 4 in silicon wafer with piranha (10:1 ratio of sulfuric acid and hydrogen peroxide) and hydrofluoric acid for several minutes (Caution: hazardous chemicals). Rinse with deionized (DI) water for approximately 10 sec.
5. Dehydrate wafer for 20 min at 200 °C.
6. Spin coat 1 µm SU-8 2000.5 photoresist onto the wafer (1^st layer) (4 ml resist, spin 10 sec with, v = 500 rpm, and a = 100 rpm/sec, spin 30 sec with v = 1,000 rpm and a = 300 rpm/sec).
7. Place the coated wafer on a hotplate at 95 °C to drive off excess solvent (1.5 min at 65 °C, 1.5 min at 95 °C, and 1 min at 65 °C; ideally use two hotplates).
8. Insert 1^st layer photomask (here the trapping regions of the picoliter reactors) and wafer inside the mask aligner and expose wafer to 350-400 nm (vacuum contact, 64 mJ/cm², t = 3 sec, I = 7 mW/cm²).
9. Perform post exposure bake on a hotplate at 95 °C to initiate the polymerization of SU-8 (1 min at 65 °C, 1 min at 95 °C, and 1 min at 65 °C). Note: after this step the structures in the SU-8 layer can be seen.
10. Place the wafer in a SU-8 developer bath for 1 min and transfer the wafer into a second container with fresh SU-8 developer for few seconds.
11. Rinse the wafer in isopropanol to remove SU-8 developer and dry wafer using nitrogen flow of wafer spinner.
12. Hard bake the wafer for 10 min at 150 °C.
13. Spin coat 9 µm SU-8 2010 photoresist onto the wafer (2^ndlayer) (dispense 4 ml resist, spin 10 sec with v = 500 rpm, a = 100 rpm/sec, and spin 30 s with v = 4,000 rpm, a = 300 rpm/sec).
14. Place the wafer with SU-8 on a hotplate at 95 °C to drive off excess solvent (15 min at 65 °C, 45-60 min at 95 °C, and 10 min at 65 °C). Note: attention has to be paid to wrinkles and bubbles. If the wafer is heated to fast to 95 °C, evaporated solvent may be encapsulated in tiny gas bubbles.
15. Insert photomask with the desired layout (here main channels for nutrient supply) and the wafer into the mask aligner and expose to 350-400 nm (hard contact, 64 mJ/cm², t = 10 sec, I = 7 mW/cm²)
16. Perform post exposure bake on a hotplate at 95 °C to finalize the polymerization of SU-8 (5 min at 65 °C, 3:30 min at 95 °C, 3 min at 65 °C). Note: after this step the structures in the SU-8 layer can be seen.
17. Place the wafer in a SU-8 developer bath for 45 sec, transfer the wafer into a second container with fresh SU-8 developer and develop for 60 sec.
18. Rinse the wafer 20 sec with isopropanol to remove any SU-8 developer residue and dry wafer using pressured nitrogen.
19. Finally hard bake the wafer at 150 °C. As a result the final wafer (Figure 1C) is obtained, which will be used as master mold for PDMS molding.
20. Perform profilometer measurements (Figure 3C) to validate SU-8 structure heights. Note: inaccuracies in structure height may results in inefficient cell trapping or loss of cells during cultivation.

2. Polydimethylsiloxane Chip Fabrication

Note: All following steps should be ideally performed under laminar-flow conditions to prevent dust particles interfering with the fabrication procedure (A process flowchart is shown in Figure 4).

1. Prepare a mixture of polydimethylsiloxane (PDMS) base and curing agent in a 10:1 ratio. Mix carefully until a homogenous solution is achieved which looks opaque. Prepare as much as required for the desired layer height (here 3 mm).
2. Degas the PDMS mixture for approximately 30 min under slight vacuum until all bubbles have disappeared.
3. Prepare molding device (or Petri dish) with appropriate SU-8 wafer and pour the PDMS mixture into it (Figure 1D).
4. Bake the PDMS for 3 hr at 80 °C in the oven.
5. Carefully peel off the PDMS slab from the wafer. Cut the slab into single chips using a clean and sharp scalpel.
6. Wash the chips in a n-pentane bath for 90 min, followed by two acetone washing baths (90 min each). Dry the chips overnight to remove any solvent residue. Caution: perform the PDMS washing under a fume hood. Note: during the n-pentane wash, monomers and dimers are removed from the cured PDMS and the chip size may temporarily double during washing procedure.
7. Store the microfluidic PDMS chips in close containers until the final experiment.
8. Just before the experiment, punch the inlet and outlet holes into the PDMS chip using a needle (or hole-puncher) with a slightly smaller diameter than the connectors that are used to connect tubing with PDMS chip.
9. Clean the microfluidic PDMS chip carefully with isopropanol and use scotch tape to remove any dust particles which might stick on the structured PDMS side. Use the scotch tape several times until no particle can be seen on the chip.
10. Clean a 170 µm thin glass slide with acetone and isopropanol successively. Finally clean with deionized water and dry with pressurized nitrogen.
11. Before plasma-activation, warm up the plasma cleaner and run the plasma for approximately 300 sec. Plasma-oxidize glass slide and PDMS chip (Power 50 W, Time = 25 sec, oxygen flow rate = 20 sccm).
12. Align the PDMS and glass chip before bonding. Finally, place the PDMS chip carefully onto the glass slide (Figure 1E). PDMS and glass will bond within seconds. Note: do not push with tweezers onto the top of the PDMS chip during the bonding process. This may lead to so called roof-collapsing of the channels and small structures.
13. In order to strengthen the bond, bake the final PDMS-glass chip for 10 sec at 80 °C.

3. Preparation of the Bacterial Culture

Note: All cultivations should be prepared in sterile filtered medium to prevent accumulation of undesired particles, which may interfere during cultivation.

1. Use an agar plate containing the desired organisms (here, C. glutamicum ATCC 13032) and inoculate one colony into 20 ml of fresh BHI medium, incubate overnight (≈ 8-14 hr) at 30 °C on a rotary shaker (120 rpm).
2. Transfer 10 µl of the preculture into the desired medium (here CGXII²¹) which will be used during microfluidic cultivation and let the cell grow overnight at 30 °C on a rotary shaker (120 rpm).
3. Transfer the desired amount of cell suspension (between 10-500 µl, depending on the start of the experiment) into the desired medium (here CGXII²¹) which will be used during microfluidic cell cultivation. Note: the best is to use cells from the early logarithmic phase for seeding. For C. glutamicum culture the best optical density (OD₆₀₀) for seeding was between 0.5-2.
4. Transfer 1 ml of the bacterial culture into a sterile 2 ml tube. Note: this should be done right after the microfluidic PDMS chip was assembled to minimize transfer time between shake flask and microfluidic cultivation. Typically the transfer time is around 15 min and should be kept as small as possible to prevent impact on metabolism caused by oxygen limitation and temperature changes.

4. Experimental Setup

Note: All steps are performed with an inverted microscope.

1. Start microscope incubator 2 hrs before the experiment to warm-up the system. Note: the microscopy should be equipped with a full-size incubator to control temperature and if desired gas flow. Additional humidity control is not necessary since the chip system is continuously infused with media.
2. Open incubator system, select the desired objective and if required add immersion oil onto the objective.
3. Mount the chip inside the chip holder. If required fix the glass plate with adhesive tape in order to avoid chip any movement during stage operation.
4. Center the sample on the microscope and focus onto the PLBR arrays.
5. Connect inlets and outlets with appropriate tubing (Figure 1F). Connect tubing to a waste reservoir. A representative chip can be seen in Figure 4D.
6. Insert syringes into pumps and start media flow. Use medium, buffer or if necessary coating solution and rinse the microfluidic channels for approximately 1 hr. Note: coating solution is used to coat channel walls to prevent unspecific cell adhesion.
7. For E. coli, 0.1% solution of BSA is used to coat the channel walls. For C. glutamicum no coating is necessary. After the coating procedure flush the chip with medium prior cell seeding.
8. Before cell seeding and cultivation, check that no leakage occurs and that the temperature is constant.

5. Seeding of Bacterial Cells into the Microfluidic Device

1. Make sure the desired bacteria solutions are available in appropriate syringes connected to tubing.
2. Disconnect buffer or coating solution and connect the cell suspension to the chip. To minimize death volume, undesired air bubbles and to reduce experimental time, change the complete needle as well as tubing, rather than only the syringes.
3. Infuse the cell suspension into the channels at a volumetric flow rate of 200 nl/min until most of the PLBRs are filled with the desired amount of cells (Figure 5A). Note: optimal seeding results depend on the bacterial strain, OD₆₀₀, and growth medium of the preculture. These parameters have to be adapted to increase trapping efficiency and time until a sufficient number of cells are trapped in the reactor structures. For C. glutamicum, typically a cell suspension of OD₆₀₀ 0.5-2 was used; for E. coli the OD₆₀₀ was between 0.5-1.
4. If only a small number of PLBRs are filled, increase the flow rate to 800-1,200 nl/min.
5. Disconnect the cell suspension and connect the growth medium to the chip (Figure 5B). Make sure that no air bubble is introduced during the medium change. Perfuse with fresh growth media at 100 nl/min.

6. Time-lapse Imaging

1. Select specific PLBRs for time-lapse imaging. Typically PLBRs are chosen that contain a single mother cell at the beginning of an experiment. The number of regions of interest that can be investigated in one experiment depends on the desired frame rate and microscopic setup.
2. Select an appropriate frame rate depending of number of PLBRs. Make sure that the microscope can handle the desired amount of ROIs in the time lapse interval.
3. Choose appropriate filter sets (here YFP). Automatically close the shutter during stage movement and after each time-lapse measurement to prevent chromophore bleaching.
4. Configure the time-lapse microscopy sequence and start the experiment.
5. After all PLBRs are overgrown, the experiment can be stopped, the microfluidic PDMS chip can be discarded and the experiment can be evaluated.

7. Analysis

Note: The following steps or parts of the procedure can be performed manually or by image analysis programs such as ImageJ, etc.

1. Determine PLBRs of interest where the cultivation fulfills all desired criteria, e.g. number of mother cells, position of the mother cells, etc.
2. To determine the growth rate of one microcolony count the number of cells in each time frame.
3. Calculate the maximum growth rate by plotting time vs. ln (cell number). The slope of the plot represents the growth rate in [1/hr] (see Figure 6).
4. Fluorescence data analysis strongly depends on the performed experiment. In this report, an example was chosen to illustrate colony-to-colony and cell-to-cell heterogeneity between different isogenic microcolonies (see Figure 7).