Development of a flow-free microfluidic gradient generator

1
OQuinn ,

2
Tullier ,

2
Pojman ,

1
Melvin

Kelly
Michael
John
and Adam
1 Cain Department of Chemical Engineering, 2 Department of Chemistry
Louisiana State University

Abstract
Microfluidic technology has flourished since its emergence over thirty years ago,
allowing for rapid scientific advances in a wide variety of fields. Microfluidics allows for
precise spatial and temporal control of the chemical and physical microenvironment
and has numerous applications ranging from DNA analysis to inkjet printing. In
microfluidics, all fluid flow is laminar, forcing mixing to occur via length-scale diffusion.
In the case of a microfluidic gradient generator, this allows for a chemical gradient to
develop perpendicular to the direction of fluid flow. Applications of microfluidic
gradient generators include quantifying cell migration in response to gradients
chemical cues (chemotaxis) and culturing of cells for extended periods of time.
However, many of the available devices require direct flow through the fluidic
channels and are, thus, incompatible with non-adherent cells like bacteria and algae.
To address this issue, we developed a double-layer microfluidic device consisting of a
top PDMS-layer, imprinted with the fluidic channels, on top of a bottom layer of
agarose, allowing for rapid diffusion of chemical cues. One disadvantage of this
PDMS/agarose system is that the two layers do not readily adhere, requiring an
external chamber to create a tight seal. In an effort to overcome this limitation, we
fabricated devices with a PDMS-alternative polymer made using thiol-ene chemistry.
Both devices were characterized using fluorescent tracers and numerical simulations
to evaluate the mass transfer dynamics. As a proof-of-principle, the device was used
in preliminary studies to culture the model algae Chlamydomonas reinhardtii for
several days by continuous nutrient perfusion.

Soft lithography A layer of photoresist was spun onto a

silicon wafer, covered with the photomask, and exposed to
UV light. The cross-linked photoresist hardened and the rest
was dissolved in developer.
PDMS replication PDMS base and curing agent were
mixed in a 10:1 ratio and poured over the wafers. These
were baked at 65C for at least 3 hours. After baking, holes
were punched, the slabs were silanized and tubing was
added and sealed in.
Device assembly A slab of 3% w/w room temperature
agarose was cut to match the size and shape of the PDMS
slab. These were stacked and enclosed by the Plexiglas
layers and screwed shut.

Device Characterization
Characterization experiments
5 M 5,6-carboxyfluorescein
(FAM; MW 376) was constantly
infused into the top channel while
deionized water was constantly
infused into the bottom channel
both at 15 L/min using a syringe
pump.

Introduction

PDMS device: 5M FAM constantly infused for 3 hours.

Images show all three channels at 2x magnification,
taken every 30 seconds for 3 hours.

Microfluidics can be used to study a variety of fields and present a

cost-effective way to conduct numerous experiments.
This gradient generator contains three channels (Figure A). With
constant infusion over time, diffusion occurs through the agarose and
across the center channel. This channel can contain cells that will
migrate in response to the chemical gradient. The center channel is
flow-free, allowing for migration of cells.
One disadvantage of using PDMS/agarose devices is the inability to
adhere the layers together. To create a seal between the two layers,
we silanized the PDMS layer and designed Plexiglas slabs that could
be clamped shut around the device (Figures B and C).

Viability and Migration

Device Fabrication

Brightfield image (2x

magnification) of the
three channels in a
PDMS device. A
linescan (bold red)
was drawn across the
channels for
quantitative analysis
of the gradient.

Short-term culturing experiment

C. reinhardtii were manually loaded into the center channel at a
density of 2x106 cells/mL
TAP media was continually perfused into top and bottom channels at
10 L/min by syringe pump
Cells were grown under continuous light for 48 hours
Qualitative twice daily visualization verified increase in algal cell
density and obvious swimming behavior
PDMS does not impact cell viability
Migration experiments This device was used to study C. Reinhardtii
migration. The cells were centrifuged at 4100 x g at 4C for 3 minutes,
resuspended in nitrate-free media and loaded into the center channel.
Nitrogen-rich TAP media was constantly infused into the top channel and
Nitrogen-free TAP constantly infused into the bottom channel.

Thiol-acrylate device: 5M FAM constantly infused for 3

hours. Images show all three channels at 2x magnification,
taken every 30 seconds for 3 hours.

C. Reinhardtii in a thiol-acrylate device. Images taken every 10 seconds at 10x

magnification in the center channel. Observed algae swimming behavior in the device.
The yellow and blue circles follow two algae cells swimming over a period of 50
seconds.

Porosity and permeability are different between the two devices

FITC-dextran (70,000 MW) can diffuse through agarose but not the thiol-acrylate hydrogel

Conclusion
The device was capable of producing steady gradients.
The trends for both the COMSOL model and the experimental data are
similar.
In the future, the COMSOL model can be modified for various device
conditions. Other scenarios could be examined by changing flow rates,
agarose composition, or initial concentration.
Algae is capable of migrating in this device with respect to nutrient
gradients.

Acknowledgements
One alternative is making the device out of a thiol-acrylate polymer
layer and a thiol-acyrlate hydrogel layer which bind without the use of
external force.
Thiol-acrylate replication offers advantages over standard PDMS
replication including reduced curing time performed at room
temperature.

PDMS/Agarose: Position in the center

channel vs. FAM concentration at various
time points. Initial concentration of 5M.

Thiol-acrylate: Position in the center

channel vs. FAM concentration at various
time points. Initial concentration of 5M.

COMSOL Model: Validates data

and verifies uniform concentration
in z-direction

Both sets of data correspond to the length of the center channel from 0 to 600m.
A linescan was taken at each time point and the pixel intensity correlated to a concentration of FAM using a standard curve.

Harsha Sirigireddy
Devin Manning
Todd Monroe (LSU Biological Engineering)
Supervised Undergraduate Research Experience (SURE)
grant, sponsored by the National Science Foundation
(NSF) and the Louisiana Board of Regents