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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

Abstract

We demonstrate a method for the generation of controlled, dynamic chemical pulses―where localized chemoattractant becomes suddenly available at the microscale―to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert ('caged') by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

Introduction

Microbes rely on chemotaxis, the process of detecting chemical gradients and modifying motility in response1, to navigate chemical landscapes, approach nutrient sources and hosts, and escape noxious substances. These microscale processes determine the macroscale kinetics of interactions between microbes and their environment2,3. Recent advances in microfluidics and microfabrication technologies, including soft lithography4, have revolutionized our ability to create controlled microenvironments in which to study the interactions of microbes. For example, past expe....

Protocol

1. Fabrication of the Microfluidic Device for the Single Chemical-pulse Experiment

  1. Design the channel using computer-aided design (CAD) software and print it onto a transparency film to create the photo mask (Figure 1A).
  2. Fabricate the master by soft lithography (under clean-room conditions).
    1. Clean a silicon wafer (4 inches) in quick succession with acetone, methanol and isopropanol, then dry using nitrogen. Bake the wafer in the oven at 130 .......

Representative Results

We used the microfluidic and millifluidic devices (Figure 1) to study bacterial accumulation profiles under dynamic nutrient conditions. Bacterial trajectories were extracted from recorded videos acquired by phase contrast microscopy of the accumulation-dissipation dynamics of a bacterial population following a chemical pulse released by photolysis (Figure 2 and Figure 3). By averaging millions of trajectories, the spatiotemporal dy.......

Discussion

This method allows researchers to study bacterial chemotaxis under controlled, dynamic gradients in micro- and millifluidic devices, enabling reproducible data acquisition. The near-instantaneous creation of chemical pulses at the microscale by photolysis aims to reproduce the types of nutrient pulses that bacteria encounter in the wild from a range of sources, for example, the diffusive spreading of plumes behind sinking marine particles25, or the nutrient spreading from lysed phytoplankton cells.......

Acknowledgements

The authors thank the FIRST microfabrication facility at ETH Zurich. This work was supported by an Australian Research Council Discovery Early Career Researcher Award DE180100911 (to D.R.B.), a Gordon and Betty Moore Marine Microbial Initiative Investigator Award GBMF3783 (to R.S.), and a Swiss National Science Foundation grant 1-002745-000 (to R.S.).

....

Materials

NameCompanyCatalog NumberComments
(3-Aminopropyl) triethoxysilane (APTES)Sigma-AldrichA3648>98% purity, highly toxic
CELLSTAR tubeGreiner Bio-One21026150 ml
CentrifugeEppendorf5424Rto eliminate spent media from the bacterial culture
Digital Incubators Incu-LineVWR-CH390-0384to bake 3D master
DusterVWR-CH16650-22to clean the wafer and microchannels
Hot plateVWR-CH444-0601to bond the microchannels
IsopropanolSigma-AldrichW292907
LightSafe micro centrifuge tubesSigma-AldrichZ6883121.5 ml
MATLABMathworksfor image analysis and bacterial tracking
Microcentrifuge tubeEppendorf301200861.5 ml
Microscope glass slideVWR-CH631-1552
Microscope Nikon Eclipse TiENikon InstrumentsMEA53100with motorized stage
MNI-GlutamateTocris Bioscience1490>98 % purity, photosensitive
Mold printing equipmentStratasysObjet30 3D printer
Mold printing service3D Printing StudiosCustomhttps://www.3dprintingstudios.com/
Nanodrop One UV-Vis SpectrophotometerThermo Fisher ScientificND-ONE-Wto calibrate the uncaging
NIS ElementsNikon InstrumentsMicroscope Imaging Software
Oven Venti-LineVWR-CH466-3516to bake PDMS (with forced convection)
Photoresist SU-8-3050MicroChem Corp.SU8-3050
Plasma chamber ZeptoDiener ElectronicZEPTO-1to functionalize the surfaces before bonding
Polycarbonate membraneSterlitechPCT04471000.4 µm pore size, 19 % open area, 24 µm thickness
Polyethylene microtubingScientific CommoditiesBB31695-PE/2I.D. x O.D.: 0.015" x 0.043" / 0.38mm x 1.09mm
Polystyrene Petri dishVWR-CH25373-100bottom surface (90 mm x 15 mm) to bond the millifluidic device
ScaleVWR-CH611-2605to weight PDMS mixture
sCMOS camera Andor ZylaOxford Instrumentsfor phase contrast and fluorescence microscopy (max 100 fps)
Sea saltInstant OceanProduct No. SS1-160p
SolidWorks 2015Dassault Systemes SolidWorksUsed to design the mold
Spectra X light engineLumencolorfor LED 395 nm
Sylgard 184Dow Corning110-41-155PDMS Si Elastomer Kit; curing agent
Syringe (Luer-Lok)B Braun Omnifix4616308F
Syringe NeedleAganiA228from 10 to 30 ml
Syringe Pump 11 Pico Plus EliteHarvard Apparatus70-4506Terumo Agani 23 gauge 5/8 inch (16mm)
VeroGreyStratasysDual Syringe Pump
Vortex-GenieScientific IndustriesSI-0236Mold Material

References

  1. Armitage, J. P., Lackie, J. M. . The biology of the chemotactic response. , (1991).
  2. Azam, F., Malfatti, F. Microbial structuring of marine ecosystems. Nature Reviews Microbiology. 5 (10), 782-791 (2007).
  3. Buchan, A., LeCleir, G. R., Gulvik, C. A., González,....

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MicrofluidicsChemical GradientsMicrobial BehaviorChemotaxisPhotolysisPDMSSoft Lithography3D PrintingMaster FabricationMicroenvironments

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