A subscription to JoVE is required to view this content. Sign in or start your free trial.
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, 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.
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....
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.
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.......
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.......
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.
....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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2025 MyJoVE Corporation. All rights reserved
We use cookies to enhance your experience on our website.
By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies.