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www.rsc.org/loc Volume 9 | Number 6 | 21 March 2009 | Pages 741–848
ISSN 1473-0197
Miniaturisation for chemistry, biology & bioengineering
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PAPER www.rsc.org/loc | Lab on a Chip
Rapid generation of spatially and temporally controllable long-rangeconcentration gradients in a microfluidic device†
Yanan Du,ab Jaesool Shim,‡ab Mahesh Vidula,ab Matthew J. Hancock,ab Edward Lo,ab
Bong Geun Chung,ab Jeffrey T. Borenstein,c Masoud Khabiry,ab Donald M. Cropekd and Ali Khademhosseini*ab
Received 15th September 2008, Accepted 13th November 2008
First published as an Advance Article on the web 10th December 2008
DOI: 10.1039/b815990d
The ability to rapidly generate concentration gradients of diffusible molecules has important
applications in many chemical and biological studies. Here we established spatially and temporally
controllable concentration gradients of molecules (i.e. proteins or toxins) in a portable microfluidic
device in an easy and rapid manner. The formation of the concentration gradients was initiated by
a passive-pump-induced forward flow and further optimized during an evaporation-induced backward
flow. The centimeter-long gradients along the microfluidic channel were shown to be spatially and
temporally controlled by the backward flow. The gradient profile was stabilized by stopping the flow.
Computational simulations of this dynamic process illustrated the combined effects of convection and
diffusion on the gradient generation, and fit well with the experimental data. To demonstrate the
applications of this methodology, a stabilized concentration gradient of a cardiac toxin, alpha-
cypermethrin, along the microchannel was used to test the response of HL-1 cardiac cells in the
micro-device, which correlated with toxicity data obtained from multi-well plates. The approach
presented here may be useful for many biological and chemical processes that require rapid generation
of long-range gradients in a portable microfluidic device.
Introduction
Concentration gradients of diffusible molecules (chemical
compounds or biomolecules) play an important role in many
chemical processes (e.g. crystal growth) as well as biological
phenomena (e.g., chemotaxis, morphogenesis and wound
healing).1–7 A variety of approaches have been developed for
generating gradients of diffusible molecules driven either purely
by diffusion or by a balance of convection and diffusion. Most of
the existing approaches for gradient generation are diffusion-
driven,8 which can be generally categorized into: (1) forming
gradients perpendicular to parallel laminar flows of varying
concentrations9–13 and (2) forming gradients along a channel by
free-diffusion between a source and a sink.14,15 The first method is
advantageous for producing stable complex gradients, but the
aCenter for Biomedical Engineering, Department of Medicine, Brigham andWomen’s Hospital, Harvard Medical School, Boston, MA, 02115, USA.E-mail: [email protected]; Fax: +1 617-768-8477; Tel: +1 617-768-8395bHarvard-MIT Division of Health Sciences and Technology,Massachusetts Institute of Technology, Cambridge, MA, 02139, USAcDraper Laboratory, Cambridge, MA, 02139, USAdU.S. Army Corps of Engineers, Construction Engineering ResearchLaboratory, Champaign, IL, 61822, USA
† Electronic supplementary information (ESI) available: Inputparameters for the computational simulations; calculation of thevolumetric flow rate of the forward flow driven by the passive-pump;calculation of the volumetric flow rate of the backward flow inducedby evaporation; effect of diffusion on the change of the gradientprofile; cytotoxicity testing of alpha-cypermethrin on HL-1 cellscultured in 96-well microplate and supplementary videos. See DOI:10.1039/b815990d
‡ Present address: School of Mechanical Engineering, YeungnamUniversity, Gyeongsan-si, Gyeongsanbuk-do, Korea.
This journal is ª The Royal Society of Chemistry 2009
experiments are not compatible with non-adherent and weakly
adherent cells and the shear/drag force generated by the flow may
alter the intercellular signaling pathways. Moreover, to generate
the laminar flows, pumping systems with external connections
(i.e. tubing and valves) are often used, which limit the portability
and ease of use of the device.16 To maintain a continuous flow,
relatively large volumes of fluid containing the materials of
interests are consumed, which constrain their applications for
precious materials (i.e. growth factors, drugs). The second
approach normally requires larger gradient generation times
and the gradient produced is hard to maintain over long time
periods.17 Gradients have also been formed parallel to the
direction of flow. Goulpeau et al. built up longitudinal concen-
tration gradients along their microchannel by using transient
dispersion along the flow.18 Kang et al. developed a device that
generated concentration gradients parallel to the direction of
flow by using a convective–diffusive balance in a counter-flow
configuration.19 Although these approaches could be used to
rapidly generate concentration gradients in less than 1 min, they
still required external components i.e. hydrostatic pumps or
valves to introduce and control the flows within the channels.
The ability to build pumpless fluidic devices that generate
controllable gradients while maintaining the portability and
scalability of microfluidic systems is of significant benefit for field
testing and high-throughput studies. Furthermore, the ability to
generate longer gradients can be used to test the effects of
molecular dose responses on cell behaviors. One approach to
eliminate the use of external pumps is by using a passive-pump
technology, which was first developed by Walker et al. as
a semi-autonomous method for pumping fluid. Passive-pump
technology only requires a device capable of producing small
Lab Chip, 2009, 9, 761–767 | 761
drops of liquid, such as a pipette.16,20 The surface tension
difference between the larger drop of solution at the outlet and
the smaller drop of solution at the inlet was used to pump the
small drop of liquid through the microchannel, which has been
shown to be a powerful high-throughput microfluidic tool for cell
culturing. Evaporation has also been used as the driving force in
‘pump-less’ microfluidic devices. Evaporation is a well-known
issue when handling small liquid volumes, especially in micro-
fluidic devices.21,22 While the loss of volume due to evaporation
may cause unwanted effects such as the change of concentrations
or osmolarity of the fluid solution; evaporation in microfluidic
devices has proven to be a useful tool in several applications,
including generating slow, steady flows in microchannels used for
chromatography,23 DNA analysis devices,24 and sample
concentration.25,26
In this study, we take advantage of a reversed flow induced by
the passive-pump and evaporation-driven sequence to rapidly
establish centimeter-long concentration gradients of molecules
along the channel of a simple and portable microfluidic device.
We applied the passive-pump technology to generate a forward
flow from the inlet to the outlet of the channel, which introduced
the molecules of interest (with volume less than 10 mL) into the
microfluidic device in a rapid and simple manner and initiated
a concentration gradient profile of the molecules due to the
parabolic shape of the front flow. An evaporation-induced
backward flow from the outlet to the inlet of the channel fol-
lowed the forward flow, which resulted in the formation of
dynamic concentration gradients of the molecule. The centi-
meter-long concentration gradients were in parallel with the flow
direction along the microfluidic channel, which could be spatially
and temporally controlled; and a particular gradient profile
could be stabilized by stopping the flow.
Our approach to generate a concentration gradient mainly
relies on the flow properties and the combined effect of convec-
tion and diffusion. Throughout the channel the flow is essentially
fully developed laminar Poiseuille flow in a rectangular channel,
a textbook example where the Navier–Stokes equations admit an
tions in temperature and humidity in the laboratory mainly affect
the gradient generation process by slightly altering the backward
flow rate induced by evaporation (see ESI);† their effects on the
forward flow and the diffusion of the molecule are negligible.
We asserted above that the flow is essentially fully developed
Poiseuille flow throughout the rectangular channel. Regions of
adjustment to the fully developed flow exist at the ends of the
channel. However, based on the Reynolds numbers (0.1 and
0.001) of the forward and backward flows, the extent of these
adjustment regions is short: approximately the channel height
[ref. 27, pp. 114, eqn 3–28].27 Thus, throughout the channel the
flow is essentially fully developed Poiseuille flow.
The concentration gradient profile in our device can be easily
altered and controlled by choosing the initial analyte concen-
tration in the applied drops and by manipulating the timing of
the forward and backward flow. Factors affecting the flow
properties, such as the fluid viscosity, the pressure difference
between the inlet and outlet, the rate of evaporation and the
geometry of the microfluidic channel are expected to affect
the gradient generation and are currently under investigation.
Conclusions
We achieved rapid generation of centimeter-long concentration
gradients of molecules using a reversed flow in a simple and
portable microfluidic device. The gradients along the micro-
fluidic channel could be spatially and temporally controlled
and stabilized. Computational simulations supporting the
This journal is ª The Royal Society of Chemistry 2009
experimental results indicate that dispersion (convection and
molecular diffusion) and the flow reversal lead to dynamic
gradient generation. In an example drug test, we applied
a stabilized gradient of a cardiac toxin concentration to test the
response of HL-1 cardiac cells. The cell morphology and
viabilities exhibited drastic differences along the microchannel,
which correlated to the concentration gradient of the toxin.
We believe that this simple and rapid approach for gradient
generation on a controllable centimeter-long scale is a promising
platform for applications such as drug testing and studying
biological phenomena, such as chemotaxis. This passive-pump
based approach can also be easily adapted to a high-throughput
platform for biological and drug discovery applications.
Acknowledgements
This research has been funded by the US Army Engineer
Research and Development Center, the Institute for Soldier
Nanotechnology, NIH, the Coulter Foundation and the Draper
Laboratory. We would like to thank Drs. Utkan Demirci, Young
Song, Won Gu Lee, Edward Haeggstrom and Ms. Tracy Chang
for the scientific and technical support. This research was sup-
ported in part by an appointment to the postgraduate research
participation program at the US Army Engineer Research and
Development Center, Construction Engineering Research
Laboratory (ERDC-CERL), administered by the Oak Ridge
Institute for Science and Education.
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