Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Summary

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Abstract

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology.

This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence.

The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

Introduction

Spatial patterning of single microorganisms, particularly within experimental arenas that enable full control over environmental conditions, such as microfluidic devices, is highly desirable in a broad range of contexts. For example, arranging microorganisms into regular arrays would permit the accurate imaging of large numbers of individual cells and the study of their growth, physiology, gene expression in response to environmental stimuli, and drug susceptibility. It would also allow studying cell-cell interactions of particular interest in research into cellular communication (e.g., quorum sensing), cross-feeding (e.g., algal-bacterial symbiosis), or antagonism (e.g., allelopathy), with full control over the spatial localization of cells relative to each other. Cell physiology and evolution studies¹, cell-cell interaction studies², phenotypic differentiation screening³, environmental monitoring⁴, and drug screening⁵ are among the fields that can greatly benefit from a technology able to achieve such quantitative single-cell analysis.

Several strategies to isolate and handle single cells have been proposed in recent years, from holographic optical traps⁶ and heterogeneous surface functionalization methods^7,8,9,10 to single-cell chemostats¹¹ and droplet microfluidics¹². These methods are either technically very demanding or affect cell physiology and fail to provide a high-throughput platform to pattern microbes that can be studied over long periods, ensuring single-cell resolution, full optical access, and control over environmental conditions. The goal of this paper is to describe a platform to pattern bacteria with micrometric precision into prescribed spatial arrangements on a PDMS surface through capillarity-assisted assembly. This platform allows precise and flexible spatial patterning of microbes and enables full optical access and control over environmental conditions, thanks to its microfluidic nature.

The technology behind this platform is an assembly technology developed in recent years, named sCAPA^13,14,15 (sequential capillarity-assisted particle assembly) that was integrated into a microfluidic platform¹⁶. The meniscus of an evaporating liquid droplet, while receding over a patterned polydimethylsiloxane (PDMS) substrate inside a microfluidic channel, exerts capillary forces that trap the individual colloidal particles suspended in the liquid into micrometric wells microfabricated on the substrate (Figure 1A). Suspended particles are first transported to the air-liquid interface by convective currents and then placed into the traps by capillarity. Capillary forces exerted by the moving meniscus act on a larger scale compared to forces involved in particle interactions.

Thus, the assembly mechanism is not influenced by the material, dimensions, and surface properties of the particles. Parameters such as particle concentration, the speed of the meniscus, temperature, and surface tension of the suspension are the only parameters that influence the yield of the patterning process. The reader can find a detailed description of the influence of the aforementioned parameters on the patterning process in^13,14,15. In the original sCAPA technology^13,14,15, the colloidal patterning process was carried out in an open system and required a high-precision piezoelectric stage to drive the suspension across the template. This platform exploits a different strategy and allows the patterning to be carried out with standard equipment generally used in microfluidics in a controlled environment, thus minimizing the risks of contaminating the samples.

This microfluidic platform was first optimized on colloidal particles to create regular arrays of inert particles and then successfully applied to bacteria. Both microfluidic platforms are described in this paper (Figure 1B,C). Most of the preparatory steps and the experimental equipment described in the protocol are common for the two applications (Figure 2). We report colloidal patterning to demonstrate that the technique can be used to perform multiple sequential depositions on the same surface to create complex, multimaterial patterns. In particular, one single particle was deposited per trap for each step to form colloidal arrays with a specific geometry and composition, solely dictated by the traps' geometry and filling sequence. As for bacterial patterning, single depositions are described, resulting in one bacterium being deposited per trap. Once cells are patterned on the surface, the microfluidic channel is flushed with medium to promote bacterial growth, the preliminary step of any single-cell study.

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Protocol

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).

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Results

A microfluidic platform that exploits capillarity-assisted assembly to pattern colloidal particles and bacteria into traps microfabricated on a PDMS template was developed. Two different channel geometries have been designed to optimize the patterning of colloids and bacteria through the capillarity-assisted assembly. The first channel geometry (Figure 1B) consists of three 23 mm long parallel sections with no physical barrier between them. The two sections on the sides are 5 mm wide and 1 m...

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Discussion

The microfluidic platform described here allows the patterning of micro-sized objects, such as colloids and bacteria, into prescribed spatial arrangements on a PDMS substrate. The full control over environmental conditions offered by microfluidics and the ability to pattern cells with micrometric precision granted by sCAPA technology makes it a very promising platform for future physiology and ecology studies.

In the experiments presented in this work, the silicon master was realized using the...

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Materials

Name	Company	Catalog Number	Comments
Alcatel AMS 200SE I-Speeder	Alcatel Micro Machining System		deep reactive ion exchange system
Alconox			detergent
AZ400K developer	MicroChemicals	AZ400K
BD 10 mL Syringe (Luer-Lock)	BD	300912	used to flush fresh Lysogeny broth into the microfluidic channel
Box Incubator	Life Imaging Services		used to ensure a uniform and constant temperature in the channel
Centrifuge	Eppendorf	5424R	used to replace the overnight media with fresh minimal media
Centrifuge vial	Eppendorf	30120086	1.5 mL
CETONI Base 120	CETONI GmbH		syringe pump
Fluorescent PS particles of diameter 0.98 µm (red)	microParticles GmbH	PS-FluoRed-Fi267
Fluorescent PS particles of diameter 1.08 µm (green)	microParticles GmbH	PS-FluoGreen-Fi182
Fluorescent PS particles of diameter 2.07 µm (green)	microParticles GmbH	PS-FluoGreen-Fi183
Fluorescent PS particles of diameter 2.08 µm (red)	microParticles GmbH	PS-FluoRed-Fi180
Gigabatch 310 M	PVA TePla		used to plasma treat a 10 cm silicon wafer
H401-T-CONTROLLER	Okolab		controller of the heated glass plate
H601-NIKON-TS2R-GLASS	Okolab		heated glass plate
Heidelberg DWL 2000	Heidelberg Instruments		UV direct laser writer
Insulin syringes, U 100, with luer	Codan Medical ApS	CODA621640	1 mL syringe used to withdraw the liquid suspension during the patterning process
Klayout	Opensource		used to design the features on the silicon master
LB Broth, Miller (Luria-Bertani)	Fisher Scientific	244610	Lysogeny broth flushed into the microfluidic channel
Masterflex transfer tubing	Masterflex	HV-06419-05	0.020'' ID, 0.06'' OD
MOPS (10x)	Teknova	M2101	diluted tenfold with milliQ water and used to replace the overnight medium
Nikon Eclipse Ti2	Nikon Instruments		microscope
openSCAD	Opensource		used to design the mold
OPTIspin SB20	ATM group	51-0002-01-00	spin developer
Plasma chamber Zepto	Diener Electronic	ZEPTO-1	used to plasma treat the template and microchannel to bond them
Positive photoresist AZ1505	MicroChemicals	AZ1505
Potassium phosphate dibasic	Sigma Aldrich	P3786	added to MOPS 1x
Prusa curing and Washing machine CW1S	Prusa		used to ensure all polymer is cured and uncured polymer is removed from the mold
Prusa Resin - Tough	Prusa Research a.s.		UV photosensitive 405nm liquid resin for 3D printing
Prusa SL1 3d printer	Prusa		used to print the mold
Scale	VWR-CH	611-2605	used to weight PDMS mixture
Silicon wafer (10 cm)	Silicon Materials Inc.		N/Phos <100> 1-10 Ω cm
Süss MA6 Mask aligner	SUSS MicroTec Group		used to align the chrome-glass mask and the substrate, and expose the substrate
Sylgard 184	Dow Corning		silicone elastomer kit; curing agent
Techni Etch Cr01	Technic		chromium etchant
Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane	Sigma Aldrich	448931	used to silianize the 3D printed mold
TWEEN 20	Sigma Aldrich	P1379	used to ensure an optimal receding contact angle during the patterning process
Veeco Dektak 6 M	Veeco		profilometer
VTC-100 Vacuum Spin Coater	MTI corporation		vacuum spin coater