Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

1. Silicon master preparation

NOTE: The PDMS templates bearing the microfabricated traps that form the template for colloidal and microbial patterning were fabricated according to the method introduced by Geissler et al.¹⁷. The silicon master was prepared by conventional lithography in a cleanroom. See the following steps for the procedure and the Table of Materials for the equipment.

1. Design the features using computer-aided design (CAD) software.
2. Prepare the chrome-glass mask with a layer of positive photoresist by exposing the designed features with a UV direct laser writer.
   1. Develop the chrome-glass mask with a spin developer using a developer at 1:4 photoresist to water ratio for 15 s.
   2. Immerse the mask in chromium etchant for 50 s.
   3. Plasma-treat a 10 cm silicon wafer for 3 min at 600 W.
   4. Vapor-deposit a layer of hexamethyldisilizane and bake at 110 °C to improve the adhesion toward the photoresist.
   5. Deposit a layer of photoresist at 4,500 × g for 2 min.
   6. Expose the feature to UV through the chrome-glass mask with a mask aligner for 2 s and develop with a developer as for the mask.
3. To complete the fabrication of the silicon master, etch the wafer via deep reactive ion exchange, adjusting the etching time (<2 min) to achieve the desired depth (measured with a profilometer).

2. Microchannel mold preparation

1. Design the channel with CAD software and slice it with a slicer software to convert the designed model into instructions for the 3D printer, setting the slicing distance at 0.05 mm.
2. Print the channel with a 3D printer for ~1 h.
3. Wash and postcure the mold in a dedicated curing and washing machine.
   1. Put the mold in a container filled with pure isopropyl alcohol (IPA) and vortex the liquid (wash for 20 min). Take the container filled with IPA out of the machine.
   2. Once the washed mold is removed from the IPA container, put it back into the washing and curing machine and postcure it for 15 min at 35 °C.
      NOTE: The Table of Materials reports the 3D printer and the curing and washing machine used in the mold preparation protocol. The 3D model was sliced with the 3D printer's proprietary slicer software.
4. Place the print in a UV oven for 12 h and place it in an oven at 80 °C for 12 h.
   NOTE: This step ensures that all the polymer is cured and all uncured polymer is removed from the print as it would prevent PDMS from curing.
5. Silanize the mold through vapor deposition of Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane.
   1. Place the 3D mold in a vacuum desiccator along with 20 µL of 100% concentrated Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane pipetted on an aluminum foil placed close to the mold.
   2. Create a vacuum in the desiccator to generate vapor and leave for 40 min.
   3. Remove the mold from the desiccator and rinse it with pure ethanol before pouring PDMS on it.

3. Fabrication of the microfluidic chip

1. Prepare a PDMS mixture by mixing the elastomer with its crosslinking agent (Table of Materials). Stir the mixture vigorously to blend the two components uniformly until air bubbles are formed, and the PDMS mixture looks opaque.
   NOTE: The amount of cross-linker should be 10% by weight of the amount of elastomer.
2. Degas the mixture of elastomer and crosslinking agent in a vacuum desiccator to remove the trapped air bubbles. Transfer the mixture in the desiccator to the container used for mixing (step 3.1). Continue the degassing process until all the bubbles have been removed and the mixture looks transparent again.
   NOTE: The time typically required for desiccation is 30 min.
3. Pour 3 g of the mixture on the silicon master to produce the template, which will serve as the "floor" of the microfluidic chip.
   NOTE: The thickness of the PDMS layer can be tuned by changing the amount of mixture poured on the silicon master.
   1. To obtain a 400 µm-thick template, pour 3 g of PDMS on the silicon master, place the silicon master on a spin coater, and spin coat at 21 × g for 5 s and 54 × g for 10 s, and then degas again as described in step 3.2 to remove trapped air bubbles.
4. Pour 20 g of the PDMS mixture on the 3D-printed mold to make the microchannel, which will serve as the "roof" of the microfluidic chip, and degas it as described in step 3.2 for 30 min.
5. Bake both the silicon wafer and the 3D printed mold at 70 °C for at least 2 h.
6. Peel the PDMS layer off the 3D printed mold, cut the PDMS with a blade around the microchannels, and punch the holes that will serve as inlet and outlet of the microfluidic channel.
7. Peel the PDMS off the silicon master and cut the PDMS layer into smaller pieces with the same dimensions of the microfluidic channels that will be bonded on top of the templates.
8. Gently rub the templates and microchannels using a 1% detergent solution (see the Table of Materials) for 5 min and then rinse with deionized water. Next, rinse the templates and microchannels with isopropanol and rinse them with deionized water. Dry the templates and microchannels at room temperature for 1 min with compressed air at 1 bar.
9. Place the templates and the microchannels in a plasma cleaner with the surfaces to be bonded facing up. Turn on the plasma cleaner, and plasma treat the templates and the microchannels for 40 s. Take them out from the plasma cleaner and immediately bond the microchannels on top of the templates by putting them in contact with one another.
10. Store the microfluidic chips in an oven at 70 °C for five days to ensure PDMS hydrophobic recovery and have a receding contact angle within the optimal range, between 30 and 60°^10,11.

4. Bacterial patterning

1. One day prior to the experiment, grow a population of Escherichia coli (strain MG1655 prpsM-GFP). Inoculate the culture directly from the frozen stock and grow overnight for 20 h in lysogeny broth (LB) medium in a shaker incubator at 37 °C. Add 50 µg/mL of kanamycin for the cells to retain the prpsM-GFP plasmid.
2. On the day of the experiment, set the box incubator (Figure 2A) at 37 °C several hours before the experiment to have a uniform and stable temperature before starting the experiment. Set up the syringe pumpand the heated glass plate on the microscope stage (Figure 2B,C), setting the temperature at the same temperature as the box incubator.
   NOTE: The box incubator in which the entire system, including the microscope (Figure 2E), is enclosed ensures that a uniform and constant temperature is maintained throughout the entire experiment when the channel is flushed with medium.
3. Ninety minutes before the experiment, put the microfluidic chip in a vessel filled with 100% ethanol (EtOH) and flush the channel with 100% EtOH for at least 10 min. Place the microfluidic chip in a vacuum desiccator and degas for at least 30 min. Exchange the EtOH with distilled water and vacuum-treat the chip for at least 30 min. Put the microfluidic chip in the oven at 70 °C for 10 min to remove any traces of liquid left in the channel.
   NOTE: This step is necessary to prevent bubble formation in the channel when flushed with culture medium. This bubble prevention protocol was adapted from Wang et al.¹⁸.
4. Pipette 1 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) medium (1x) into a centrifuge vial and add 10 µL of 0.132 M potassium phosphate (K₂HPO₄). Take the overnight culture out of the 37 °C incubator and aliquot 100 µL into the centrifuge vial and centrifuge the culture at 2,300 × g for 2 min. Gently pipette the supernatant out of the centrifuge vials, and resuspend the pellet in 1 mL of fresh MOPS medium with 0.015% v/v of Tween 20 and 0.01% v/v of potassium phosphate.
   NOTE: The typical concentration of the bacterial suspension is in the range of 0.015-0.15 vol%, which ensures the formation of an extended and mobile accumulation zone and prevents the accumulation of particles on the substrate. Replacing the overnight medium with fresh medium minimizes the risks of releasing films of bacteria on the template. The lack of carbon source in the fresh medium prevents cells from growing in the suspension during template patterning. A concentration of 0.015% v/v of Tween 20 in the suspension is necessary for the contact angle of the receding liquid on the PDMS template to fall within the optimal range, between 30 and 60°.
5. Load the bacterial suspension in a 1 mL syringe and connect the syringe to the chip through microfluidic tubing.
   1. To secure the connection between the syringe and the tubing, directly insert a needle with an outer diameter of 0.6 mm into the tubing. To avoid scattering any suspension remaining in the inlet vicinity across the channel, flush fresh medium through the hole used as an outlet during the patterning process (Figure 3A-III), rather than the hole used as the inlet.
   2. Mount the syringe on the syringe pump and inject the suspension into the microfluidic chip through the inlet located at the upstream part of the channel until the suspension covers the template region with traps.
      NOTE: During the liquid injection process, air can escape through an outlet located at the downstream end of the channel.
   3. Set the syringe pump to withdraw the bacterial suspension (Figure 3A-I) at a flow rate of 0.07-0.2 µL/min, which in the described geometry corresponds to a meniscus receding speed of 80-100 µm/min.
   4. Monitor the patterning process via microscope software.
      NOTE: Here, a 10x magnification was used to monitor the receding meniscus on the template, and a 20x magnification was used to monitor the deposition of individual bacteria into the microfabricated traps.
6. Once the template has been patterned with cells (Figure 3A-II), increase the withdrawal flow rate to quickly empty the microfluidic channel and flush it with fresh LB that was previously degassed for at least 30 min and prewarmed at 30 °C.
7. Set the syringe pump at a flow rate of 2 µL/min to gently flush the channel. Once the channel has been filled, increase the flow rate (15 µL/min) according to the specific experimental needs.
8. Acquire images of growing bacteria at the desired magnification and time interval.

5. Colloidal patterning

1. Pipette 900 µL of a 0.015% v/v Tween 20 aqueous solution into a centrifuge vial and pipette 100 µL of the original colloidal suspension into it. Centrifuge the suspension at 13,500 × g for 1 min. Gently remove the supernatant and replace it with the aqueous Tween 20 (0.015% v/v) solution. Repeat this process three times to ensure complete replacement of the supplier's solvent from the stock suspension.
2. Load the colloidal suspension in a 1 mL syringe and connect the syringe to the chip through microfluidic tubing. Inject the suspension into the microfluidic chip through the inlet located within the central section, at the upstream part of the channel, and gradually push the suspension until the template is covered.
3. Withdraw the colloidal suspension at a flow rate of 0.07-0.2 µL/min, which corresponds to a meniscus receding speed of 1-2 µm/min, and image the patterning process via microscope software. Use 10x magnification to monitor the receding meniscus on the template and 20x magnification to observe the deposition of individual particles into the microfabricated traps. Increase the flow rate once the meniscus reaches the end of the template to empty the channel quickly.
   NOTE: Colloidal arrays composed of several particles can be produced by running the patterning process from steps 5.1 to 5.3 in series. The channel is loaded with a new colloidal suspension at each iteration according to the desired colloidal arrays' composition. Each patterning step adds one particle to the colloidal array and can be run sequentially until the traps have been filled with the desired particles' sequence. The composition of the resulting colloidal arrays is solely given by the sequence of colloidal suspensions used to fill the traps (Figure 3B).