SLIDE 1 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
SLIDE 2 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
SLIDE 3 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
SLIDE 4
luxI luxR + GFP luxAB luxCDE Directional Quorum Sensing
SLIDE 5
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?
SLIDE 6
CCD X Y Yellow Filter White Illumination X-Y stage 405 nm Laser Diode
Direct Laser Writing (DLW)
SLIDE 7 pinhole PBS λ/2 405 nm laser diode
TV PC XY stage
Direct Laser Writing Setup
SLIDE 8 create microfluidic master by DLW Develop master Cast PDMS
Plumb holes in PDMS Bond PDMS to glass Inject hydrogel containing eColi and trap by DLW
Making Microfluidics for E. coli Capture
SLIDE 9
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
SLIDE 10 Acrylate Monomers Photoinitiators Hydrogel PDMS Polyethylene glycol diacrylate (PEG-DA)
C O P O O
Lucirin TPO-L 2-Hydroxy-2-methylpropiophenone SR-499 ethoxylated (6) trimethylolpropane acrylate
Materials Used in Fabrication of Device
H2C C H2C H2 C H2 C H3C O H2 C H2 C O C C H CH2 O
2
O H2 C H2 C O C C H CH2
2
O O H2 C H2 C O C C H CH2
2
O
SLIDE 11
500 μm
Direct Laser Writing: Preparing a Master
SLIDE 12
Acrylate Master on Glass Molded PDMS Microfluidic
Structures made by DLW
SLIDE 13
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
SLIDE 14
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.
SLIDE 15
- E. Coli Fluorescence with AHL
500 1000 1500 2000 2500 3000 475 525 575 625 Intensity Wavelength (nm)
No AHL 1 nM AHL 10 nM AHL 50 nM AHL 100 nM AHL
SLIDE 16 1000 2000 3000 lux R lux I + lux R Fluorescence Intensity
Enhanced Fluorescence with AHL Producer
receiver sender
SLIDE 17
Fluorescent E. coli in a 60μm tube
SLIDE 18
Fluorescent E. coli being entrapped in hydrogel
SLIDE 19
luxI luxR + GFP
Polymerized E. coli Patches in a Microfluidic Channel 60 μm
SLIDE 20 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
SLIDE 21 Acknowledgements
- Prof. Brooke Jude, Microbiologist, Bard College.
Funding provided by:
- Bard Summer Research Institute.
- Science, Math, and Computing Division
- Walvax Biotechnology Co. Ltd (China)
