Directional Quorum Sensing in a Microfluidic Device Yi (Brian) Liu, Eric Reed Swapan Jain, Christopher LaFratta (mentors) Bard College, Annandale-on-Hudson, NY October 8, 2011 Americas Regional Jamboree: Indianapolis
Biochemical Microdevices Microfluidics Chemistry Lab-on-a-Chip Microanalysis DiCarlo et al . Lab Chip (2006), 6, 1445 Lab Chip , 2005, 5, 1024-1032 Quake et al. Science (2005), 310, 1793
Language of Bacteria: Quorum Sensing Bacteria communicate with each other using messenger molecules N-Acyl Homoserine Lactone (AHL) http://bacterianeurons.blogspot.com/2008/07/bacterially-speaking-bonnie-bassler.html
Directional Quorum Sensing luxI luxR + GFP luxAB luxCDE
What is a Zener Diode? A diodes only allows the flow of current in one direction I V A Zener diode allows flow in one direction, but also in the other above a certain threshold I V We want to use quorum sensing molecules in a directional way
Direct Laser Writing (DLW) CCD 405 nm Laser Diode X Y X-Y stage Yellow Filter White Illumination
Direct Laser Writing Setup PC TV XY stage pinhole 405 nm PBS λ /2 laser diode
Making Microfluidics for E. coli Capture Plumb holes create in PDMS microfluidic master by DLW Bond PDMS to glass Develop master Inject hydrogel Cast PDMS containing eColi over master and trap by DLW
Other Groups That Capture Bacterial by DLW Optical micrographs of hydrogel micropatches containing E.coli within a microfluidic channel. 5 um Crooks et al. Anal. Chem. 2003, 75, 22 -26
Materials Used in Fabrication of Device Acrylate Monomers Hydrogel O H 2 H 2 Polyethylene glycol diacrylate (PEG-DA) H 2 C O C C O C C CH 2 H 2 O H 2 H 2 H 2 H 2 H 3 C C C C O C C O C C CH 2 H 2 O H 2 H 2 H 2 C O C C O C C CH 2 H 2 SR-499 ethoxylated (6) trimethylolpropane acrylate Photoinitiators PDMS O C P O O 2-Hydroxy-2-methylpropiophenone Lucirin TPO-L
Direct Laser Writing: Preparing a Master 500 μm
Structures made by DLW Acrylate Master on Glass Molded PDMS Microfluidic
Parts Used luxI luxR + GFP luxI: synthesizes AHL luxR+GFP: generates GFP in the presence of AHL. luxAB luxCDE luxCDE: encodes luciferin, which produces light in the presence of luciferase. luxAB: encodes luciferase, which produces light in the presence of luciferin.
Plasmid Prep and Restriction Digestion luxR 3 kb C L 1 kb Agarose gel illustrating the circular (C) and linear (L) form of luxR gene.
E. Coli Fluorescence with AHL 3000 2500 2000 Intensity No AHL 1 nM AHL 1500 10 nM AHL 50 nM AHL 1000 100 nM AHL 500 0 475 525 575 625 Wavelength (nm)
Enhanced Fluorescence with AHL Producer 3000 receiver Fluorescence Intensity sender 2000 1000 0 lux R lux I + lux R
Fluorescent E. coli in a 60μm tube
Fluorescent E. coli being entrapped in hydrogel
Polymerized E. coli Patches in a Microfluidic Channel 60 μm luxI luxR + GFP
Goals & Accomplishments • Create a microfluidic device entrapping two strains of E.coli • Create a sending and receiving strains of E.coli • Show directional quorum sensing in a microfluidic device • Combine the two quorum sensing systems to make it bidirectional luxI luxR + GFP luxI + luxR mixture shows significantly higher fluorescence signal vs. luxR alone. Successful entrapment of two strains of E. coli in polymerized hydrogel. Retention of fluorescence capability. Advance our knowledge regarding quorum sensing.
Acknowledgements •Prof. Brooke Jude, Microbiologist, Bard College. Funding provided by: •Bard Summer Research Institute. •Science, Math, and Computing Division •Walvax Biotechnology Co. Ltd (China)
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