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)