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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 antagoni....

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 us.......

Representative 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.......

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.......

Acknowledgements

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 Zürich, Switzerland), Cameron Boggon (ETH Zürich, Switzerland) and Dr. Fabio Grillo for the insightful discussions.

....

Materials

NameCompanyCatalog NumberComments
Alcatel AMS 200SE I-SpeederAlcatel Micro Machining Systemdeep reactive ion exchange system
Alconoxdetergent
AZ400K developerMicroChemicalsAZ400K
BD 10 mL Syringe (Luer-Lock)BD300912used to flush fresh Lysogeny broth into the microfluidic channel
Box IncubatorLife Imaging Servicesused to ensure a uniform and constant temperature in the channel
CentrifugeEppendorf5424Rused to replace the overnight media with fresh minimal media
Centrifuge vialEppendorf301200861.5 mL
CETONI Base 120CETONI GmbHsyringe pump
Fluorescent PS particles of diameter 0.98 µm (red)microParticles GmbHPS-FluoRed-Fi267
Fluorescent PS particles of diameter 1.08 µm (green)microParticles GmbHPS-FluoGreen-Fi182
Fluorescent PS particles of diameter 2.07 µm (green)microParticles GmbHPS-FluoGreen-Fi183
Fluorescent PS particles of diameter 2.08 µm (red)microParticles GmbHPS-FluoRed-Fi180
Gigabatch 310 MPVA TePlaused to plasma treat a 10 cm silicon wafer
H401-T-CONTROLLEROkolabcontroller of the heated glass plate
H601-NIKON-TS2R-GLASSOkolabheated glass plate
Heidelberg DWL 2000Heidelberg InstrumentsUV direct laser writer
Insulin syringes, U 100, with luerCodan Medical ApSCODA6216401 mL syringe used to withdraw the liquid suspension during the patterning process
KlayoutOpensourceused to design the features on the silicon master
LB Broth, Miller (Luria-Bertani)Fisher Scientific244610Lysogeny broth flushed into the microfluidic channel
Masterflex transfer tubingMasterflexHV-06419-050.020'' ID, 0.06'' OD
MOPS (10x)TeknovaM2101diluted tenfold with milliQ water and used to replace the overnight medium
Nikon Eclipse Ti2Nikon Instrumentsmicroscope
openSCADOpensourceused to design the mold
OPTIspin SB20ATM group51-0002-01-00spin developer
Plasma chamber ZeptoDiener ElectronicZEPTO-1used to plasma treat the template and microchannel to bond them
Positive photoresist AZ1505MicroChemicalsAZ1505
Potassium phosphate dibasicSigma AldrichP3786added to MOPS 1x
Prusa curing and Washing machine CW1SPrusaused to ensure all polymer is cured and uncured polymer is removed from the mold
Prusa Resin - ToughPrusa Research a.s.UV photosensitive 405nm liquid resin for 3D printing
Prusa SL1 3d printerPrusaused to print the mold
ScaleVWR-CH611-2605used to weight PDMS mixture
Silicon wafer (10 cm)Silicon Materials Inc.N/Phos <100> 1-10 Ω cm
Süss MA6 Mask alignerSUSS MicroTec Groupused to align the chrome-glass mask and the substrate, and expose the substrate
Sylgard 184Dow Corningsilicone elastomer kit; curing agent
Techni Etch Cr01Technicchromium etchant
Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silaneSigma Aldrich448931used to silianize the 3D printed mold
TWEEN 20Sigma AldrichP1379used to ensure an optimal receding contact angle during the patterning process
Veeco Dektak 6 MVeecoprofilometer
VTC-100 Vacuum Spin CoaterMTI corporationvacuum spin coater

References

  1. Choi, C. H., et al. Preparation of bacteria microarray using selective patterning of polyelectrolyte multilayer and poly(ethylene glycol)-poly(lactide) deblock copolymer. Macromolecular Research. 18 (3), 254-259 (2010).
  2. Smriga, S., Fernandez, V. I., Mitchell, J. G., Stocker, R.

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