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Yi (Brian) Liu, Eric Reed Swapan Jain, Christopher LaFratta (mentors)
Bard College, Annandale-on-Hudson, NY
October 8, 2011 Americas Regional Jamboree: Indianapolis
Directional Quorum Sensing in a Microfluidic Device
Microfluidics Chemistry Lab-on-a-Chip Microanalysis
Biochemical Microdevices
Quake et al. Science (2005), 310, 1793
DiCarlo et al. Lab Chip (2006), 6, 1445
Lab Chip, 2005, 5, 1024-1032
Language of Bacteria: Quorum Sensing
N-Acyl Homoserine Lactone (AHL)
Bacteria communicate with each other using messenger molecules
http://bacterianeurons.blogspot.com/2008/07/bacterially-speaking-bonnie-bassler.html
luxI luxR + GFP
luxAB luxCDE
Directional Quorum Sensing
A Zener diode allows flow in one direction, but also in the other above a certain threshold I
V
A diodes only allows the flow of current in one direction
We want to use quorum sensing molecules in a directional way
I
V
What is a Zener Diode?
CCD
X
Y
Yellow Filter White Illumination
X-Y stage
405 nm Laser Diode
Direct Laser Writing (DLW)
pinhole
PBS λ/2 405 nm laser diode
TV PC
XY stage
Direct Laser Writing Setup
create microfluidic master by DLW
Develop master
Cast PDMS over master
Plumb holes in PDMS
Bond PDMS to glass
Inject hydrogel containing eColi and trap by DLW
Making Microfluidics for E. coli Capture
5 um
Optical micrographs of hydrogel micropatches containing E.coli within a microfluidic channel.
Crooks et al. Anal. Chem. 2003, 75, 22-26
Other Groups That Capture Bacterial by DLW
Acrylate Monomers
Photoinitiators
Hydrogel
PDMS
Polyethylene glycol diacrylate (PEG-DA)
C
O
PO
O
Lucirin TPO-L 2-Hydroxy-2-methylpropiophenone
SR-499 ethoxylated (6) trimethylolpropane acrylate
Materials Used in Fabrication of Device
H2C
C
H2C
H2C
H2CH3C
OH2C
H2C O C C
HCH2
O
2
OH2C
H2C O C C
HCH2
2
O
OH2C
H2C O C C
HCH2
2
O
500 μm
Direct Laser Writing: Preparing a Master
Acrylate Master on Glass Molded PDMS Microfluidic
Structures made by DLW
luxI luxR + GFP
Parts Used
luxAB: encodes luciferase, which produces light in the presence of luciferin.
luxR+GFP: generates GFP in the presence of AHL.
luxI: synthesizes AHL
luxCDE: encodes luciferin, which produces light in the presence of luciferase.
luxAB luxCDE
Plasmid Prep and Restriction Digestion
luxR
C L 3 kb 1 kb
Agarose gel illustrating the circular (C) and linear (L) form of luxR gene.
E. Coli Fluorescence with AHL
0
500
1000
1500
2000
2500
3000
475 525 575 625
Inte
nsity
Wavelength (nm)
No AHL1 nM AHL10 nM AHL50 nM AHL100 nM AHL
0
1000
2000
3000
lux R lux I + lux R
Fluo
resc
ence
Inte
nsity
Enhanced Fluorescence with AHL Producer
receiver
sender
Fluorescent E. coli in a 60μm tube
Fluorescent E. coli being entrapped in hydrogel
luxI luxR + GFP
Polymerized E. coli Patches in a Microfluidic Channel
60 μm
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.
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
Acknowledgements •Prof. Brooke Jude, Microbiologist, Bard College.
Funding provided by: •Bard Summer Research Institute.
•Science, Math, and Computing Division
•Walvax Biotechnology Co. Ltd (China)