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 us...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-guided+assembly+of+heterotypic+three-dimensional+living+cell+microarrays.">Laser-guided assembly of heterotypic three-dimensional living cell microarrays.</a> Biophysical Journal. 91 (9), 3465-3473 (2006).</li><li>Cerf, A., Cau, J. C., Vieu, C., Dague, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+properties+of+dead+or+alive+single-patterned+bacteria.">Nanomechanical properties of dead or alive single-patterned bacteria.</a> Langmuir. 25 (10), 5731-5736 (2009).</li><li>Cerf, A., Cau, J. C., Vieu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+assembly+of+bacteria+on+chemical+patterns+using+soft+lithography.">Controlled assembly of bacteria on chemical patterns using soft lithography.</a> Colloids and Surfaces: B Biointerfaces. 65 (2), 285-291 (2008).</li><li>Rozhok, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+motile+bacterial+cells+to+prealigned+holed+microarrays.">Attachment of motile bacterial cells to prealigned holed microarrays.</a> Langmuir. 22 (26), 11251-11254 (2006).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+living+bacteria+arrays+with+cellular+resolution.">Microcontact printing of living bacteria arrays with cellular resolution.</a> Nano Letters. 7 (7), 2068-2072 (2007).</li><li>Ingham, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+micro-Petri+dish,+a+million-well+growth+chip+for+the+culture+and+high-throughput+screening+of+microorganisms.">The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (46), 18217-18222 (2007).</li><li>Moffitt, J. R., Lee, J. B., Cluzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-cell+chemostat:+an+agarose-based,+microfluidic+device+for+high-throughput,+single-cell+studies+of+bacteria+and+bacterial+communities.">The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities.</a> Lab on a Chip. 12 (8), 1487-1494 (2012).</li><li>Ni, S., Isa, L., Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+assembly+as+a+tool+for+the+heterogeneous+integration+of+micro-+and+nanoscale+objects.">Capillary assembly as a tool for the heterogeneous integration of micro- and nanoscale objects.</a> Soft Matter. 14 (16), 2978-2995 (2018).</li><li>Ni, S., Leemann, J., Buttinoni, I., Isa, L., Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+colloidal+molecules+from+sequential+capillarity-assisted+particle+assembly.">Programmable colloidal molecules from sequential capillarity-assisted particle assembly.</a> Science Advances. 2 (4), 1501779 (2016).</li><li>Ni, S., Leemann, J., Wolf, H., Isa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+Insights+into+mechanisms+of+capillary+assembly.">Nanoparticle Insights into mechanisms of capillary assembly.</a> Faraday Discussions. 181, 225-242 (2014).</li><li>Pioli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+capillarity-assisted+particle+assembly+in+a+microfluidic+channel.">Sequential capillarity-assisted particle assembly in a microfluidic channel.</a> Lab on a Chip. 21 (5), 888-895 (2021).</li><li>Geissler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+metal+nanowires+using+microcontact+printing.">Fabrication of metal nanowires using microcontact printing.</a> Langmuir. 19 (15), 6301-6311 (2003).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+prevention+of+bubble+formation+and+accumulation+for+long-term+culture+of+pancreatic+islet+cells+in+microfluidic+device.">Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device.</a> Biomedical Microdevices. 14 (2), 419-426 (2012).</li><li>Crowley, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+cell+death+by+propidium+iodide+uptake+and+flow+cytometry.">Measuring cell death by propidium iodide uptake and flow cytometry.</a> Cold Spring Harbor Protocols. 7, 647-651 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;m (red)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoRed-Fi267</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 1.08 &#181;m (green)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoGreen-Fi182</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 2.07 &#181;m (green)</td>
			<td>microParticles GmbH</td>
			<td>PS-FluoGreen-Fi183</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent PS particles of diameter 2.08 &#181;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&#39;&#39; ID, 0.06&#39;&#39; 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 &#60;100&#62; 1-10 &#937; cm</td>
		</tr>
		<tr>
			<td>S&#252;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, ...

This method allows producing user-defined patterns of microorganisms within a microfluidic channel. Once patterned, the microorganisms can be monitored to evaluate their long-term physiology and interaction with high throughput. The use of capillary forces enables a nonspecific route for patterning, which is applicable across different classes of materials.For example, colloidal particles and bacteria. Moreover, it allows to produce large arrays with exquisite control of the spatial arrangement of the material of interest. To date, we tested and applied the method on colloidal particles and microbial cells.As a result, it can find applications in material engineering and fields requiring quantitative single-cell analysis like drug screening. To begin, prepare a PDMS mixture by mixing the elastomer with its cross-linking agent. Then stir the mixture vigorously to blend the two components uniformly until air bubbles are formed and the PDMS mixture looks opaque.Now, degas the mixture in a vacuum desiccator until all air bubbles are removed and the mixture looks transparent again. To obtain a 400 micrometer thick template floor of the microfluidic chip, pour three grams of the mixture on the silicon master and place the silicon master on a spin coater to spin coat at 21 times G for five seconds and 54 times G for 10 seconds. Degas again for removing the trapped air bubbles as described earlier.Pour 20 grams of the PDMS mixture into the 3D printed mold to make the microchannel which will serve as the roof of the microfluidic chip and degas it for 30 minutes as described earlier. Bake the silicon wafer and the 3D printed mold at 70 degrees Celsius for at least two hours, then cut the PDMS along the outline of the 3D printed mold and peel it off. Cut the PDMS with a blade around the microchannels and punch the holes that will serve as inlet and outlet of the microfluidic channel.Now cut the PDMS and peel it 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. Gently rub the templates and microchannels using a 1%detergent solution for five minutes and then rinse with deionized water. Next, rinse the templates and microchannels with isopropanol, before rinsing them with deionized water.Now dry the templates and microchannels at room temperature for one minute with compressed air at one bar. Place the templates and the microchannels in a plasma cleaner with the bonding surfaces facing up. After turning on the plasma cleaner, plasma treat the templates and microchannels for 40 seconds, then take them out from the plasma cleaner and immediately bond the microchannels on top of the templates.Store the microfluidic chips in an oven at 70 degrees Celsius for five days to ensure PDMS hydrophobic recovery. On the day of the experiment, set the box incubator at 37 degrees Celsius several hours before the experiment, then set up the syringe pump and the heated glass plate on the microscope stage, setting the same temperature as that of the box incubator. 90 minutes before the experiment, put the microfluidic chip in a vessel filled with 100%ethanol and flush the channel with 100%ethanol for at least 10 minutes, then place the microfluidic chip in a vacuum desiccator and degas for at least 30 minutes.Now exchange the ethanol with distilled water and vacuum treat the chip for at least 30 minutes. Put the microfluidic chip in the oven at 70 degrees Celsius for 10 minutes to remove any traces of liquid left in the channel. Pipette one milliliter of MOPS medium into a centrifuge vial and add 10 microliters of 0.132 molar potassium phosphate.Aliquot 100 microliters of the overnight culture into the centrifuge vial and centrifuge the culture at 2, 300 times G for two minutes. Gently discard the supernatant to resuspend the pellet in one milliliter of fresh MOPS medium with 0.015%of Tween 20 and 0.01%of potassium phosphate and load the bacterial suspension in a one milliliter syringe. To secure the syringe and the tubing connection, directly insert a needle into the tubing.Now 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. Set the syringe pump at a flow rate of 0.07 to 0.2 microliters per minute to withdraw the bacterial suspension and monitor the patterning process via microscope software. Once the template has been patterned with cells, 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 minutes and prewarmed at 30 degrees Celsius.Now set the syringe pump at a flow rate of two microliters per minute to gently flush the channel. Once the channel has been filled, again increase the flow rate. Acquire images of growing bacteria at the desired magnification and time interval.Pipette 900 microliters of a 0.015%Tween 20 aqueous solution into a centrifuge vial and then pipette 100 microliters of the original colloidal suspension into it. Centrifuge the suspension at 13, 500 times G for one minute and gently replace the supernatant with the aqueous Tween 20 solution. Load the colloidal suspension in a one milliliter 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. Withdraw the colloidal suspension at a flow rate of 0.07 to 0.2 microliters per minute and image the patterning process via microscope software. Increase the flow rate once the meniscus reaches the end of the template quickly.The straight channel geometry was exploited to pattern stationary phased cells of a fluorescent Escherichia coli strain depositing 83%of the 5, 000 analyzed traps. Patterned bacteria resumed growth at different times within 1.5 hours from when the channel was filled with fresh LB.Once growth was resumed, single bacterial cells started forming individual colonies which expanded until a surface layer was formed and the single cell resolution was lost. Analysis conducted over 55, 000 traps showed that green-red dimers made by two and one micrometer diameter particles were formed in 93%and 89%of analyzed traps respectively.And green-red-green trimmers were formed in 52%of the traps. The distance between patterned particles can be precisely controlled by performing two depositions in opposite directions, thus trapping such particles at the opposite ends of each trap. It is important to ensure uniform temperature across the template to prevent condensation during the patterning process.To this end, we place a heated glass plate underneath the template and set it at the same temperature as the box incubator. This method can be used in a variety of biological studies involving quantitative single-cell analysis. A unique advantage is that its high throughput nature which allows patterning of thousands of cells providing large statistics within the same experimental arena.

patterning-microorganisms-microparticles-through-sequential

Research

JoVE Journal

Engineering

3.0K visualizações.  ETH Zurich. Este método permite produzir padrões definidos pelo usuário de microrganismos dentro de um canal microfluido. Uma vez padronizados, os microrganismos podem ser monitorados para avaliar sua fisiologia de longo prazo e interação com alto rendimento. O uso de forças capilares permite uma rota inespecífica para a padronização, que é aplicável em diferentes classes de materiais.Por exemplo, partículas coloidais e bactérias. Além disso, permite produzir grandes matrizes com con...