Name: patterning of microorganisms and microparticles through sequential capillarity-assisted assembly
Uploaded: 2021-11-04T13:00:00.000+00:00
Duration: 10 min 17 s
Description: Watch this Scientific Journal Video about Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly at JoVE.com

The authors acknowledge support from SNSF PRIMA grant 179834 (to E.S.), an ETH Research Grant ETH-15 17-1 (R. S.), and a Gordon and Betty Moore Foundation Investigator Award on Aquatic Microbial Symbiosis (grant GBMF9197) (R. S.). The authors thank Dr. Miguel Angel Fernandez-Rodriguez (University of Granada, Spain) for the SEM imaging of bacteria and for the insightful discussions. The authors thank Dr. Jen Nguyen (University of British Columbia, Canada), Dr. Laura Alvarez (ETH Z&#252;rich, Switzerland), Cameron Boggon (ETH Z&#252;rich, Switzerland) and Dr. Fabio Grillo for the insightful discussions.

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.
<ol>
	<li>Design the features using computer-aided design (CAD) software.</li>
	<li>Prepare the chrome-glass mask with a layer of positive photoresist by exposing the designed features with a UV direct laser writer.
	<ol>
		<li>Develop the chrome-glass mask with a spin developer using a developer at 1:4 photoresist to water ratio for 15 s.</li>
		<li>Immerse the mask in chromium etchant for 50 s.</li>
		<li>Plasma-treat a 10 cm silicon wafer for 3 min at 600 W.</li>
		<li>Vapor-deposit a layer of hexamethyldisilizane and bake at 110 &#176;C to improve the adhesion toward the photoresist.</li>
		<li>Deposit a layer of photoresist at 4,500 &#215; g for 2 min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>To complete the fabrication of the silicon master, etch the wafer via deep reactive ion exchange, adjusting the etching time (&#60;2 min) to achieve the desired depth (measured with a profilometer).</li>
</ol>
2. Microchannel mold preparation
<ol>
	<li>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.</li>
	<li>Print the channel with a 3D printer for ~1 h.</li>
	<li>Wash and postcure the mold in a dedicated curing and washing machine.
	<ol>
		<li>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.</li>
		<li>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 &#176;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&#39;s proprietary slicer software.</li>
	</ol>
	</li>
	<li>Place the print in a UV oven for 12 h and place it in an oven at 80 &#176;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.</li>
	<li>Silanize the mold through vapor deposition of Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane.
	<ol>
		<li>Place the 3D mold in a vacuum desiccator along with 20 &#181;L of 100% concentrated Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane pipetted on an aluminum foil placed close to the mold.</li>
		<li>Create a vacuum in the desiccator to generate vapor and leave for 40 min.</li>
		<li>Remove the mold from the desiccator and rinse it with pure ethanol before pouring PDMS on it.</li>
	</ol>
	</li>
</ol>
3. Fabrication of the microfluidic chip
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Pour 3 g of the mixture on the silicon master to produce the template, which will serve as the &#34;floor&#34; 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.
	<ol>
		<li>To obtain a 400 &#181;m-thick template, pour 3 g of PDMS on the silicon master, place the silicon master on a spin coater, and spin coat at 21 &#215; g for 5 s and 54 &#215; g for 10 s, and then degas again as described in step 3.2 to remove trapped air bubbles.</li>
	</ol>
	</li>
	<li>Pour 20 g of the PDMS mixture on the 3D-printed mold to make the microchannel, which will serve as the &#34;roof&#34; of the microfluidic chip, and degas it as described in step 3.2 for 30 min.</li>
	<li>Bake both the silicon wafer and the 3D printed mold at 70 &#176;C for at least 2 h.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store the microfluidic chips in an oven at 70 &#176;C for five days to ensure PDMS hydrophobic recovery and have a receding contact angle within the optimal range, between 30 and 60&#176;10,11.</li>
</ol>
4. Bacterial patterning
<ol>
	<li>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 &#176;C. Add 50 &#181;g/mL of kanamycin for the cells to retain the prpsM-GFP plasmid.</li>
	<li>On the day of the experiment, set the box incubator (Figure 2A) at 37 &#176;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.</li>
	<li>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 &#176;C for 10 min to remove any traces of liquid left in the channel. 
	NOTE:&#160;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.</li>
	<li>Pipette 1 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) medium (1x) into a centrifuge vial and add 10 &#181;L of 0.132 M potassium phosphate (K2HPO4). Take the overnight culture out of the 37 &#176;C incubator and aliquot 100 &#181;L into the centrifuge vial and centrifuge the culture at 2,300 &#215; 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&#176;.</li>
	<li>Load the bacterial suspension in a 1 mL syringe and connect the syringe to the chip through microfluidic tubing.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Set the syringe pump to withdraw the bacterial suspension (Figure 3A-I) at a flow rate of 0.07-0.2 &#181;L/min, which in the described geometry corresponds to a meniscus receding speed of 80-100 &#181;m/min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C.</li>
	<li>Set the syringe pump at a flow rate of 2 &#181;L/min to gently flush the channel. Once the channel has been filled, increase the flow rate (15 &#181;L/min) according to the specific experimental needs.</li>
	<li>Acquire images of growing bacteria at the desired magnification and time interval.</li>
</ol>
5. Colloidal patterning
<ol>
	<li>Pipette 900 &#181;L of a 0.015% v/v Tween 20 aqueous solution into a centrifuge vial and pipette 100 &#181;L of the original colloidal suspension into it. Centrifuge the suspension at 13,500 &#215; 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&#39;s solvent from the stock suspension.</li>
	<li>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.</li>
	<li>Withdraw the colloidal suspension at a flow rate of 0.07-0.2 &#181;L/min, which corresponds to a meniscus receding speed of 1-2 &#181;m/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&#39; 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&#39; sequence. The composition of the resulting colloidal arrays is solely given by the sequence of colloidal suspensions used to fill the traps (Figure 3B).</li>
</ol>

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The authors have no conflicts of interest to disclose.

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...

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.&#160;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 methods7,8,9,10 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 sCAPA13,14,15 (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 in13,14,15. In the original sCAPA technology13,14,15, 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&#39; 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.

<table><tbody><tr><td>Alcatel AMS 200SE I-Speeder</td><td>Alcatel Micro Machining System</td><td></td><td>deep reactive ion exchange system</td></tr><tr><td>Alconox</td><td></td><td></td><td>detergent</td></tr><tr><td>AZ400K developer</td><td>MicroChemicals</td><td>AZ400K</td><td></td></tr><tr><td>BD 10 mL Syringe (Luer-Lock)</td><td>BD</td><td>300912</td><td>used to flush fresh Lysogeny broth into the microfluidic channel</td></tr><tr><td>Box Incubator</td><td>Life Imaging Services</td><td></td><td>used to ensure a uniform and constant temperature in the channel</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424R</td><td>used to replace the overnight media with fresh minimal media</td></tr><tr><td>Centrifuge vial</td><td>Eppendorf</td><td>30120086</td><td>1.5 mL</td></tr><tr><td>CETONI Base 120</td><td>CETONI GmbH</td><td></td><td>syringe pump</td></tr><tr><td>Fluorescent PS particles of diameter 0.98 &amp;micro;m (red)</td><td>microParticles GmbH</td><td>PS-FluoRed-Fi267</td><td></td></tr><tr><td>Fluorescent PS particles of diameter 1.08 &amp;micro;m (green)</td><td>microParticles GmbH</td><td>PS-FluoGreen-Fi182</td><td></td></tr><tr><td>Fluorescent PS particles of diameter 2.07 &amp;micro;m (green)</td><td>microParticles GmbH</td><td>PS-FluoGreen-Fi183</td><td></td></tr><tr><td>Fluorescent PS particles of diameter 2.08 &amp;micro;m (red)</td><td>microParticles GmbH</td><td>PS-FluoRed-Fi180</td><td></td></tr><tr><td>Gigabatch 310 M</td><td>PVA TePla</td><td></td><td>used to plasma treat a 10 cm silicon wafer</td></tr><tr><td>H401-T-CONTROLLER</td><td>Okolab</td><td></td><td>controller of the heated glass plate</td></tr><tr><td>H601-NIKON-TS2R-GLASS</td><td>Okolab</td><td></td><td>heated glass plate</td></tr><tr><td>Heidelberg DWL 2000</td><td>Heidelberg Instruments</td><td></td><td>UV direct laser writer</td></tr><tr><td>Insulin syringes, U 100, with luer</td><td>Codan Medical ApS</td><td>CODA621640</td><td>1 mL syringe used to withdraw the liquid suspension during the patterning process</td></tr><tr><td>Klayout</td><td>Opensource</td><td></td><td>used to design the features on the silicon master</td></tr><tr><td>LB Broth, Miller (Luria-Bertani)</td><td>Fisher Scientific</td><td>244610</td><td>Lysogeny broth flushed into the microfluidic channel</td></tr><tr><td>Masterflex transfer tubing</td><td>Masterflex</td><td>HV-06419-05</td><td>0.020&#039;&#039; ID, 0.06&#039;&#039; OD</td></tr><tr><td>MOPS (10x)</td><td>Teknova</td><td>M2101</td><td>diluted tenfold with milliQ water and used to replace the overnight medium</td></tr><tr><td>Nikon Eclipse Ti2</td><td>Nikon Instruments</td><td></td><td>microscope</td></tr><tr><td>openSCAD</td><td>Opensource</td><td></td><td>used to design the mold</td></tr><tr><td>OPTIspin SB20</td><td>ATM group</td><td>51-0002-01-00</td><td>spin developer</td></tr><tr><td>Plasma chamber Zepto</td><td>Diener Electronic</td><td>ZEPTO-1</td><td>used to plasma treat the template and microchannel to bond them</td></tr><tr><td>Positive photoresist AZ1505</td><td>MicroChemicals</td><td>AZ1505</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786</td><td>added to MOPS 1x</td></tr><tr><td>Prusa curing and Washing machine CW1S</td><td>Prusa</td><td></td><td>used to ensure all polymer is cured and uncured polymer is removed from the mold</td></tr><tr><td>Prusa Resin - Tough</td><td>Prusa Research a.s.</td><td></td><td>UV photosensitive 405nm liquid resin for 3D printing</td></tr><tr><td>Prusa SL1 3d printer</td><td>Prusa</td><td></td><td>used to print the mold</td></tr><tr><td>Scale</td><td>VWR-CH</td><td>611-2605</td><td>used to weight PDMS mixture</td></tr><tr><td>Silicon wafer (10 cm)</td><td>Silicon Materials Inc.</td><td></td><td>N/Phos &amp;lt;100&gt; 1-10 &amp;Omega; cm</td></tr><tr><td>S&amp;uuml;ss MA6 Mask aligner</td><td>SUSS MicroTec Group</td><td></td><td>used to align the chrome-glass mask and the substrate, and expose the substrate</td></tr><tr><td>Sylgard 184</td><td>Dow Corning</td><td></td><td>silicone elastomer kit; curing agent</td></tr><tr><td>Techni Etch Cr01</td><td>Technic</td><td></td><td>chromium etchant</td></tr><tr><td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td><td>Sigma Aldrich</td><td>448931</td><td>used to silianize the 3D printed mold</td></tr><tr><td>TWEEN 20</td><td>Sigma Aldrich</td><td>P1379</td><td>used to ensure an optimal receding contact angle during the patterning process</td></tr><tr><td>Veeco Dektak 6 M</td><td>Veeco</td><td></td><td>profilometer</td></tr><tr><td>VTC-100 Vacuum Spin Coater</td><td>MTI corporation</td><td></td><td>vacuum spin coater</td></tr></tbody></table>

patterning of microorganisms and microparticles through sequential capillarity-assisted assembly

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.

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...

Watch this Scientific Journal Video about Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly at JoVE.com

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

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.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

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Research

JoVE Journal

Engineering

3.1K Views.  ETH Zurich. 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.

Video: Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Chemistry

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Bioengineering

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

A Novel Ink-Free, Label-Free Magnetic-Archimedes Cell Patterning Method

Cell Patterning Using Magnetic-Archimedes Strategy

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell patterning strategy based on the Magnetic-Archimedes (Mag-Arch) effect is introduced. This approach enables precise control of cell distribution without the use of ink materials or labeling particles. By introducing a paramagnetic reagent to enhance the magnetic susceptibility of the cell culture medium, cells are repelled by magnets and arrange themselves into a pattern complementary to the magnet sets positioned beneath the microfluidic substrate.
In this article, detailed procedures for cell patterning using the Mag-Arch-based strategy are provided. Methods for patterning single-cell types as well as multiple cell types for co-culture experiments are offered. Additionally, comprehensive instructions for fabricating microfluidic devices containing channels for cell patterning are provided. Achieving this feature using parallel methods is challenging but can be done in a simplified and cost-effective manner. Employing Mag-Arch-based cell patterning equips researchers with a powerful tool for in vitro research.

Author Spotlight: Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications 

This protocol describes an ink-free, label-free, substrate-independent, high-throughput cell patterning method based on the Magnetic Archimedes effect.

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Micropatterning and Assembly of 3D Microvessels

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Simple, Affordable, and Modular Patterning of Cells using DNA

Pattern Generation for Micropattern Traction Microscopy

Cell Patterning Using Magnetic-Archimedes Strategy

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Micropatterning and Assembly of 3D Microvessels

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Simple, Affordable, and Modular Patterning of Cells using DNA

Pattern Generation for Micropattern Traction Microscopy

Cell Patterning Using Magnetic-Archimedes Strategy

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates