Fabrication and Characterization of a Microfluidic Module for
Chemical Gradient Generation Utilizing Passive Pumping
Jonathan T.W. Kuo*, Connie Li*, and Ellis Meng, IEEE Senior MemberAbstract—We
introduceamicro-biochemical
administration module (BAM) for generating chemical
gradients for use in axonal guidance studies. The device isdesigned to be simple to use, require minimal packaging, andbe operated using only a pipette. A passive pumping
mechanism is utilized to pump liquid through a SU-8
microchannel and then the micropore on the Parylene cap ofthe microchannel. The achievable flow rate delivery throughthe micropore was characterized and manipulated by varying
the drop volumes used to passively drive fluid flow into thedevice. Biochemicals controllably delivered using this module
can be combined with neuronal cell cultures to form chemical
gradients for axonal guidance studies.

I. INTRODUCTION
Biomolecular gradients play a pivotal role withinbiological systems guiding the growth, migration,differentiation, apoptosis as well as many other functions ofcells through chemical signaling. Chemical signaling iscomplex with gradient composition, concentrations, andspatiotemporal characteristics all dynamically influencing
cellular microenvironments and thus cellular behavior.
However, studying cellular behavior with respect tochanges within the cellular microenvironment remains
difficult. Conventional tools such as puffer micropipettes, theTranswell assay, or the Zigmond chamber for gradient
generation possess limited capabilities for mimicking in vivo
conditions with relatively static chemical gradient generation
potential and high operational difficulty [1]. Oftentimes,
whole cell populations are exposed to a chemical gradient
whereas it is often desirable to precisely target specificsubpopulations or even a subcellular region.
Micromachining technologies have been exploited toconstruct microfluidic devices capable of presenting chemical
gradients in a highly controllable manner, thereby
overcoming limitations associated with conventional tools.
Agrin was focally delivered to myotubes cultured on top of a
microfluidic pore simulating in vitro synaptogenesis of aneuromuscular junction [2]. A microfluidic device capable offocally targeting a subpopulation of cells with full user
control over the chemical concentration presented wasreported [3], but was limited to the same chemical
concentration over the entire device. Another microfluidic
device consisting of multiple channels each possessing an*Both authors contributed equally to this work.
Research supported in part by University of Southern California James
H. Zumberge Award.
J.T.W. Kuo, C. Li, and E. Meng are with the University of Southern
California, Los Angeles, CA 90089 USA (phone: 213-821-3949; e-mail:ellis.meng@usc.edu).

individual pore enabled multiple focal chemical gradient
generations within the same device [4]. Microfluidic devicesconsisting of multiple input channels have also been
constructed for delivering chemical gradients to cells seeded
and cultured within the channels [5]. These devices were able
to present chemical gradients to subcellular regions bygenerating subcellular fluid streams through the exploitationof laminar microchannel flow and diffusion-limited mixingof multiple microfluidic inputs [6].
All of the aforementioned microfluidic devices requirepressure-driven flow in order to maintain steady-stategradient delivery. Pressure-driven flow is typically achieved
using a syringe pump or other off-chip pneumatic sources.
The requirement for large external equipment, electrical
power, and complicated microfluidic connections places
restrictions on microdevice portability and hampers adoption
by end-users not accustomed to such infrastructure.
Microdevices for studying axonal growth have been
constructed by Taylor et al. such that hydrostatic pressure
within the device alone was utilized to present a steady-stategradient between two compartmentalized chambers separated
by an array of microchannels [7]. Filling one chamber withmore fluid than the other and maintaining the fluid heightdifferential generated a constant hydrostatic pressure thatdrove fluid through the array of microchannels. This
technique enabled axonal growth studies by separation of thesoma from the axon and presenting a different chemical
environment between the two [8]. Moreover, this device was
not reliant on external sources of pressure-driven flow;
greatly simplifying device operation and significantly
decreasing the experimental system footprint.
For this work, the goal was to achieve a microdevice
capable of targeted focal delivery of chemicals for axonal
growth studies; this is not possible with Taylor et al.’s
microdevice paradigm which exposes the entire cell
population to a uniform chemical gradient. Our approach
specifically avoids off-chip pneumatic flow sources and all
the complex requisite packaging. Most importantly, ourapproach was selected to enable an inexpensive, simple-touse device for neuroscientists and other biologists.
II. DESIGN
A. A Modular Approach
The task of separating neuronal soma and their respective
axons is delegated to a micro-compartmented cell culture
module (CCM), a polydimethylsiloxane (PDMS) device
similar in design to Taylor et al.’s device except withaccessible compartments. This separation allows for targetedchemical delivery to either the soma or axons andsubpopulations with the use of another device, a microbiochemical administration module (BAM). BAM
placement controls the spatial characteristics of delivered
chemical gradients. Multiple BAMs can be used inconjunction, enabling experimental flexibility with usercontrolled gradient generation (Figure 1). For this work wefocus on BAM.

layer was first deposited. SU-8 2035 was then spun on (~55m). The negative photoresist was relaxed for 10 minutes
prior to softbaking on a leveled hotplate. Next, a controlled
softbake was performed (65 °C for 10 minutes followed by atemperature ramp to 95 °C and 30 minute bake). The hotplate
was turned off resulting in slow, convective cooling to room
temperature. Microchannels were patterned into the SU-8
coated wafer (UV exposure of 500 mJ). A post-exposurebake was performed in the same manner as with softbaking.
Finally, the SU-8 was developed.
A micropore for chemical gradient delivery was formed
through the base Parylene. First, thick AZ 4620 (~9.6 µm)
was spun on and patterned to serve as a Parylene etch mask.
The Parylene was then selectively removed in an oxygen
plasma environment using a reactive ion etcher (100 W: 100mT) until etched through to the silicon support wafer. The
device outline was also patterned and etched during this step.
Finally, the devices were released from the carrier wafer
by soaking in DI water and gentle separation from thesupporting substrate with tweezers. A standard clean wasthen used to remove the photoresist etch mask.

Figure 1. Illustration of modular microfluidic system that enables flexible
user-defined chemical gradient presentation for axonal guidance studies.

B. Focal Delivery
Focal delivery was achieved with fluid flow through amicromachined Parylene C pore addressed by a SU-8
microchannel. Parylene C, a United States Pharmacopeial(USP) Class VI material is widely used within biomedical
microsystems [9]. Moreover, Parylene can be micromachined
through plasma etching [10] with submicron pore diameters[11].
C. Passive Pumping
Passive pumping is exploited to drive fluid flow within
the microchannel. This approach takes advantage of thesurface tension differential present within two liquid bubbles
of different radii. When fluidically connected, e.g. through achannel, the internal pressure within the smaller bubble
exceeds that of the larger and a pressure gradient is generated
that drives fluid flow from the smaller bubble to the larger[12]. Microdevices have been constructed from PDMS for
passive pumping since a hydrophobic surface is required tomaintain flow [13]. Passive pumping enables fluid flow
through a microchannel simply by operating a pipetteobviating the need for external fluid pumps. This also makes
BAM an extremely simple-to-use device since the end-user
only needs pipetting knowledge for operation.
III. FABRICATION
A bare 3” Si wafer was used as a carrier substrate during
the fabrication process (Figure 2). A 5 m thick Parylene

Figure 2. Simplified fabrication process for BAM.

IV. PACKAGING
Following device fabrication, a clean 3 mm thick
PDMS sheet of identical dimensions was bonded to theanterior side of the device using only surface adhesion toform a watertight seal. This PDMS slab capped the initiallyopen microchannel and provided a hydrophobic surface forthe test droplets. By utilizing the transparency of PDMS, thelocation of the access ports with proper alignment with theends of the SU-8 microchannel was easily identified. Theports were then cored through the PDMS with a 14 gauge
needle to allow for fluid access to the microchannel.
V. METHODS
The passive pumping technique was characterized bymonitoring the flow rate through the Parylene film
micropore. To simulate the wet environment of a neuronalcell culture, the packaged BAM device rested on twoblocks in a manner that ensured consistent contact with thesurface of a distilled water bath. The device was filled with agreen dyed delivery fluid to facilitate visualization of theflow into the water bath.

The microchannel and ports were initially filled with dye
until the fluids in both ports were level with the PDMS sheet
i.e. a primed device. This represents the base level of dyewith which each experiment began.

the distilled water bath. It can also be observed that the fluid
flows smoothly through the Parylene film micropore toproduce an uninterrupted chemical stream into the bath.

The reservoir drop which consists of a larger volume ofdye was first pipetted onto one of the PDMS ports. A smaller
volume of dye was then applied to the other port to serve as
the pumping drop which is the source of internal pressure fordriving passive pumping (Figure 3).

Figure 3. Operating principle for BAM with the arrow indicating flow
direction.

VI. RESULTS
A. Fabrication
While previous passive pumping devices were primarily
fabricated with PDMS, we successfully utilized
microfabrication techniques to construct a passive pumpingdevice using SU-8 and Parylene. The use of Parylene
permitted the creation of a well-defined pore throughselective etching (Figure 4). In addition, the use of SU-8 wasparticularly beneficial for its increased stiffness andchemically inertness compared to PDMS. Operation of thedevice requires constant chemical exposure; use of PDMS
material for construction would result in the risk of chemicalabsorption, skewing the desired results [14].

Figure 5. Time-lapse photography (5 s between each image) captures device
operation with passive pumping. Drops of green dye are first applied via
pipette to the PDMS sheet’s access ports. The green dye is successfully
delivered into the microchannel and through the micropore to the water bathbelow without use of an off-chip flow source.

B. Fluid Delivery into Microchannel
In order to characterize how the flow rate might bemodified in accordance with user specification, different
pumping drop volumes were applied and the time it took tofor the pumping drop to completely dispense was observed(Figure 6).

Figure 4. Fabricated BAM device (left) with etched Parylene pore (right).
The slight curvature (left) is due to residual stress within the SU-8 which canbe mitigated by bonding with a flat PDMS sheet.

While the PDMS sheet acted as a cover and ahydrophobic surface in which the droplets could be
dispensed, the main working components of this device arecomprised of Parylene and SU-8. The non-permanent bond
between the PDMS and BAM enables the silicone sheet tobe replaced should undesirable absorption be observed.
Figure 5 demonstrates the dye flowing across the SU-8
microchannel, through the Parylene film micropore, and into

Figure 6. Pumping droplet size and corresponding duration of generated flow(mean ± S.E. with n = 5).

For pumping drop volumes between 5-25 L, a linear
increase in total flow time was observed. This corresponds toa consistent flow rate of ~0.63 L/s for all pumping volumes
within this regime. This result by itself was surprising since it
was expected that different pumping drop sizes would

possess different internal pressures resulting in varying flow
rates. For the pumping drop volume of 30 L, the flow ratewas ~0.46 L/s, a decrease of ~30%. This decrease wasattributed to the pumping drop being close in size to thereservoir drop (35 L), resulting in a reduction of internalpressure differential that drives passive pumping.
C. Fluid Delivery Through Micropore
To further investigate, different ratios between thepumping and reservoir drop volumes were investigated fortheir effects on flow rate through the micropore. The fullrange was explored, from a ratio of 0 in which only one drop
was pipetted, to parity between the two drops (Figure 7).

VII. CONCLUSION
With the use of passive pumping, chemical gradient
generation can be highly tailored with easy-to-use
microfluidic modules. With respect to BAM, the observed
behavior that the reservoir is the water bath points to an evenmore simplified design. However, other variables such as
micropore diameter, microchannel dimensions, andenvironmental factors must be examined more in depth. Theease of use of this device offers advantages in flexibility anduser convenience.
ACKNOWLEDGMENT
The authors are grateful to Professor Samantha Butler
and her doctoral student Supraja Varadarajan for productive
discussions on axonal guidance experimental protocols. Theauthors also thank the members of the BiomedicalMicrosystems Lab for their support.
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Figure 7. Different volume ratios between pumping and reservoir drops were
examined for possible effects on flow rate (mean ± S.E. with n = 5).

The flow rate was observed to increase with increasing
volume ratio however, at parity the flow rate decreased justas before. This suggests that chemical gradient generation
can be manipulated by varying drop volume ratios.
An interesting observation occurred at the extremes witha ratio of 0 and ratio of 1. A ratio of 0 would not be expected
to experience flow since there is no reservoir drop. However,flow did occur and the drop volume flowed completely
through the micropore into the water bath. This indicates that
passive pumping can occur within BAM without a reservoir
drop since the fluidic connection made through the micropore
can act as a reservoir with no internal pressure. Thishypothesis was further confirmed with the observation thatafter the complete dispensing of the smaller pumping drop,
the larger reservoir drop also dispensed through themicropore until it was completely gone. While gravity and a
relatively large pore diameter may be factors influencing thisbehavior, we suspect that surface tension effects associatedwith passive pumping play a more dominant role.
With a ratio of 1 in which both drops were of the same
volume, all of the dye in both drops eventually flowed
through the micropore. The decreased flow rate could be due
to opposing fluid flows within the microchannel since bothdrops would possess the same internal pressures.

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