Regular Papers: Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture Woo-Jin Chang,

Regular Papers: Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip for Bacterial Culture Woo-Jin Chang,
Biomedical Microdevices 5:4, 281±290, 2003
# 2003 Kluwer Academic Publishers. Manufactured in The Netherlands.
Regular Papers:
Poly(dimethylsiloxane) (PDMS) and Silicon Hybrid Biochip
for Bacterial Culture
Woo-Jin Chang,1,2 Demir Akin,1,2 Miroslav Sedlak,2,3
Michael R. Ladisch,2,3 and Rashid Bashir1,2,*
1
Laboratory of Integrated Biomedical Micro/Nanotechnology and
Applications, Birck Nanotechnology Center, School of Electrical and
Computer Engineering
2
Department of Biomedical Engineering
3
Department of Agricultural and Biological Engineering, Purdue
University, West Lafayette, IN 47907
E-mail: [email protected]
Abstract. In this study, a novel PDMS/silicon hybrid micro¯uidic
biochip was fabricated and tested for the long-term batch culture of
bacterial cells. The PDMS ( poly(dimethylsiloxane)) cover with
3-dimensional micro-channels for ¯ow was fabricated using Te¯on
tubing and hole-punch techniques, without photolithographic
methods. The PDMS/silicon hybrid biochip was prepared by
bonding of PDMS cover and a silicon chip that had electrodes
and micro-¯uidic channels de®ned. The absorption of liquid into
PDMS cover was characterized and conditions to prevent drying of
nutrient media within the micro-chamber were shown. The
absorption of liquid from micro-chambers into the PDMS cover
was reduced up to 2.5 times by changing the mixing ratio of PDMS
and curing agent from 10 : 1 to 2.5 : 1. In addition, pre-saturation of
the PDMS cover with media prior to the incubation resulted in the
preservation of liquid in the micro-chambers for up to 22 hours.
Optimization of the mixing ratio and pre-saturation of the PDMS
cover reduced the drying time 10 times when compared to the
unsaturated PDMS cover composed of 10 : 1 ratio of PDMS and
curing agent. Listeria innocua and a strain of Escherichia coli,
expressing green ¯uorescent protein (GFP), were successfully
cultured in batch mode within the PDMS/silicon hybrid biochip.
Key Words. PDMS/silicon hybrid biochip, bacterial culture,
fabrication of 3-dimensional micro-channel, absorption in PDMS
1.
Introduction
As a merger of microelectronics and biological sciences,
BioMEMS is one of the most promising research ®elds
and has the potential of solving important problems in a
wide variety of areas such as micro¯uidics (Beebe et al.,
2000), genomics (Kopp et al., 1998), proteomics
(Mouradian, 2001), and metabolomics (Yasukawa et
al., 2002). Microscale bioreactors and fermentors based
on the BioMEMS principles constitute an important part
of these applications, not only as a tool for biological
techniques such as manipulation of cells (Beebe et al.,
2002), but also for mimicking in vivo conditions to
investigate intercellular interactions and behaviors
(Bhatia et al., 1998).
Long-term cultivation using perfusion in a micro¯uidic device has been reported recently (Leclerc et al.,
2003); however, batch culture is another important
concern for applications such as infectious agent
detection and cell-based screening of drug candidates.
Speci®cally, the detection of small quantities of
pathogenic bacteria or toxigenic substances in food,
bodily ¯uids, tissue samples, soil, etc, are a few of the
most important applications of biochips (GoÂmez et al.,
2001). These applications necessitate not only the
counting of the total number of bacteria but also
detection of the number of viable cells in the samples.
A silicon-based microscale biochip for electrical detection of bacterial growth was recently demonstrated for
this type of application (GoÂmez et al., 2002). The
detection of bacterial growth was accomplished by
measuring the changes of impedance in the medium
due to generation of ionic metabolites during bacterial
growth in batch culture.
The advantages of polymer micro¯uidic devices when
compared with the silicon, glass and quartz based devices
are their low cost and rapid fabrication (Hong et al.,
2001). Moreover, the mechanical ¯exibility of poly
(dimethylsiloxane) (PDMS) allows the creation of valves
and pumps within the micro¯uidic structures (Unger
et al., 2000). PDMS is a promising material for micro-
*Corresponding author.
281
282
Chang, Akin and Bashir
¯uidic devices for cell cultivation due to its excellent
biocompatibility, low cost, easy fabrication and more
importantly its permeability which can provide oxygen to
the media without additional equipment or set-up
(Leclerc et al., 2003). Micro¯uidic devices made of
PDMS have already been applied for applications related
to cell culture such as endothelial cell (Borenstein et al.,
2002) and liver cells (Powers et al., 2002). In spite of the
merits, the lack of rapid, simple and reliable fabrication
methods to make electrodes on PDMS makes PDMS/
silicon (or glass) hybrid biochips a viable solution for
biomedical applications. Bonding of metal and PDMS
has been reported previously in a few articles using
evaporated metal (Bowden et al., 1998), Si-H bond on
metal (Hirayama et al., 1999). Embedding of carbonbased electrodes in micro-channel was suggested as
substitute of metals (Gawron et al., 2001). Nevertheless,
rapid fabrication of electrodes on PDMS in a reproducible fashion has not been reported so far.
There have been some reports on the characterization
of permeation of organic and aqueous solvents in siliconbased elastic polymers (elastomers). Swelling of the
PDMS network with solvent was observed with different
content and molecular weight of siloxane oligomer
precursors (Sivasailam and Cohen, 2000). Permeability
of gas through a composite membrane made of PDMS
and other polymers was measured for the separation of
gases (Kimmerle et al., 1991). The absorption and
diffusion properties of organic (Blume et al., 1991) and
water vapors (Favre et al., 1994) in the PDMS network
have also been reported. The absorption of liquid in
PDMS can cause major problems in cell culture
applications due to drying of a minute volume of the
liquid media. For long term observation ranging from a
few hours to more than 12 hours, which encountered for
the cell growth and also detection of changes in
environmental condition of cell culture inside the
chips, this problem becomes critical. For this reason,
prevention or delay of the absorption of liquid into
PDMS covers is a fundamental concern for micro-scale
biochips made of PDMS. There are no reports to date on
bacterial cell culture inside nano-liter volume chambers
within micro-¯uidic devices.
In this study, a novel PDMS/silicon hybrid micro¯uidic biochip was fabricated and tested for the longterm batch culture of bacterial cells. The 3-dimensional
micro¯uidic channels of the PDMS cover were made
without photolithographic methods, using Te¯on tubing
and hole-punch. The hybrid biochip was assembled by
bonding of PDMS cover and the silicon biochip, which
had electrodes and micro-wells. Based on the electrical
measurements of absorption of liquid into PDMS cover,
the optimum ratio of PDMS and curing agent composition and operating conditions for long-term cultivation of
cells were recommended. Fluorescently labeled Listeria
innocua and green ¯uorescent protein (GFP) producing
Escherichia coli were successfully cultured for more
than 20 hours in these devices.
2. Device Fabrication
Micro¯uidic devices are made of at least two layers, one
layer that contains the micro¯uidic channels and
chambers, and the other layer being used as the cover.
Due to the soft characteristics of PDMS, it is very easy to
treat and alter its shape even after it is cured, for example,
by cutting and boring using a blade or punch,
respectively. We decided to use a silicon wafer to make
the channels and wells, due to ease of integration with
metallization and future electronics, and a PDMS layer to
make the cover and the layer for the connecting ports. In
addition, a horizontal channel was used to connect the
nano-liter volume channel to ¯uidic ports, as discussed
below. Thus, the input and output ports were placed far
enough from each other to enable viewing of the wells
from the top with high magni®cation objectives (206±
406) in epi-illumination con®guration with a ¯uorescent microscope (Eclipse E600, Nikon).
2.1. Fabrication of silicon chip
The electrodes and micro-channels/micro-chambers
were fabricated in a silicon wafer. Detailed dimensions
and fabrication methods for silicon biochip have been
described before (GoÂmez et al., 2001). Brie¯y, microchannels and micro-chambers were fabricated on silicon
wafers with a thickness of 450 mm and (1 0 0) orientation.
The depth of channels and chambers were 12 mm and it
was etched by potassium hydroxide (KOH) using the
thermally grown SiO2 as a hard mask. The electrodes
were de®ned over the oxide by lift-off, using a 5 mm thick
photoresist layer (AZ4620, Clariant Co., New Jersey,
USA). The metallization is formed by evaporation of a
Ê thick layer of gold over a 300 A
Ê thick ®lm of
300 A
titanium. These electrodes were used to measure the
impedance in the chamber for electronic observation of
the drying of liquid from chamber. The size of silicon
biochip and micro-chamber for cell culture were
9.1 mm 6 8.9 mm and 530 mm 6 850 mm, respectively.
The volume of the chamber in which cells were grown
was 5.3 nl. The chamber has two interdigitated electrodes
with ®ve ®ngers each. The exposed area of each ®nger is
580 mm 6 70 mm, and distance between the two ®ngers is
10 mm.
2.2. Fabrication of PDMS cover
Three-dimensional channels were needed for the connection of hybrid biochip and macro-scale ¯uidic
PDMS and Silicon Hybrid Biochip For Bacterial Culture
283
hole-punch. As shown in Figure 1a, a mixture of PDMS
(Sylgard 184, Dow-Corning) and curing agent was
poured, approximate thickness of 500 mm, onto a
silanized 3 inch silicon wafer mold and heat cured at
120 C for 10 minutes. The surface of silicon wafer mold
was silanized with vapor of trichloro(3,3,3-tri¯uoropropyl)silane (Sigma-Aldrich Co., Missouri, USA) for 2
hours in a desiccator to prevent the bonding of cured
PDMS. The reagent was vaporized by application of
vacuum using vacuum pump. For the construction of 3dimensional micro-channels, a 7 mm long piece of Te¯on
tubing (Cole Parmer Co., Illinois, USA) that had outer
diameter of 360 mm and inner diameter of 50 mm, was
placed on the ®rst PDMS layer. This tubing was used as a
mold to create a horizontal channel to connect the
channel in silicon with the output port in the second
layer, as shown in Figure 1b. Another PDMS mixture
was added on this ®rst layer as shown in Figure 1c. After
curing, the PDMS cover embedded with the Te¯on
tubing was peeled off from the mold, and cut into the
adequate size for the silicon chip. The embedded Te¯on
tubing was pulled out of the PDMS cover from one open
end of horizontal channel and then it was blocked with
PDMS as shown in Figure 1d. Holes for vertical inputand output-¯ow of liquid were punched from both sides
of the cover as shown in Figure 1e.
2.3. PDMS/silicon bonding
PDMS can be bonded on an oxidized silicon chip
reversibly without any treatment, and it is reported that
this bond can withstand pressures up to 20 psi. However,
reversibly bonded cover could not withstand pressure
even less than 10 psi for a few hours. Thus, a stronger
bond was necessary for achieving higher ¯ow rates and at
least a few hours of ¯ow. In this work, the PDMS cover
layer and the oxidized silicon chip were permanently
bonded to each other after oxidation of both surfaces by
exposure to oxygen plasma at 200 W for 15 seconds.
Te¯on tubing was inserted into the inlet and outlet holes,
sealed with PDMS mixture around the tubing, and cured
at 90 C oven for 10 minutes. The cross-section of ®nal
device is shown in Figure 1f. The Te¯on tubing could not
be permanently bonded on the PDMS, however, this
bond could easily withstand 15 psi. Figure 1g shows an
optical micrograph of the ®nal device.
Fig. 1. Process ¯ow cross-sections (a±e), schematic drawing (f ) an
optical micrograph (h) of the hybrid PDMS/Silicon biochip.
components and for appropriate distance between the
ports. A two-layered PDMS cover was made without
photolithographic methods using Te¯on tubing and a
2.4. Hybrid biochip with input reservoir
In another version of the process, a reservoir was added
at one of the ports (input port for example), where a
sample can be dispensed and then pulled through the
micro¯uidic channel by applying a vacuum at the output
port. Instead of inserting an input tubing, a third thick
layer of PDMS was used to form a reservoir. The
284
Chang, Akin and Bashir
Fig. 2. Partial process ¯ow cross-section (a) and optical micrograph
(b) of the hybrid PDMS/Silicon biochip with an input reservoir.
schematic drawing of fabrication method is shown in
Figure 2a. The conical shape of reservoir was made using
yellow tip for micro-pipette and molding of PDMS. The
200 ml yellow tip was positioned perpendicular to the
surface, and followed by pouring and heat curing
processes as general PDMS molding technique. The
contact of yellow tips and silicon mold was secured
during curing by addition of weight on top of them. A
piece of reservoir layer was cut into appropriate size after
removal of yellow tip from the PDMS layer and bonded
on top of PDMS cover. It was permanently bonded by
application of PDMS solution. The height and diameter
of reservoir was roughly 2 mm and 1 mm, respectively.
The volume of the reservoir was about 0.5 ml. Figure 2b
shows an optical image of this device.
3.
Absorption of Liquid in PDMS
3.1. Absorption study in PDMS slabs
The PDMS covers, made of different ratio of PDMS
polymer solution and curing agent solution, were used to
investigate the characteristics of absorption of the liquid.
The cured polymer with different ratio of PDMS and
curing agent is expected to give different hardness and
permeability, and pressure actuated valve and pumps
made by these materials have been reported (Unger et al.,
2000), using this.
The PDMS elastomer is composed of a pre-polymer
(de®ned as the solution of PDMS before it is cured) and a
cross linker. The physical and chemical characteristics of
polymer changed with the ratio and properties of these
components. Normally, a pre-polymer that has long chain
length makes polymer more ¯exible, and higher
concentration of cross linker makes it more rigid.
PDMS is a silicon-based polymer, and its ¯exibility is
derived from the length and distance among each PDMS
chains, connected by cross linker. The mixing ratio of
pre-polymer and curing agent also affects the rigidity.
Higher concentration of curing agent leads to larger
number of linking between pre-polymer and curing
agent, resulting the formation of more rigid cured
polymer.
The gaps between polymer chains allow liquid and
solutes to draw closer in the polymer network. Even
though PDMS is hydrophobic in nature, moisture and
solutes are known to be absorbed by it. Manufacturer's
suggested ratio of PDMS solution and curing agent is
10 : 1, respectively. In our experiments, PDMS covers
with different hardness were made by varying the mixing
ratios of PDMS solution and curing agent. The
absorption rates of water into PDMS slabs that had
various mixing ratios of PDMS polymer and curing agent
were tested by a spectrophotometer (DU Series 600,
Beckman, California, USA). The PDMS slabs were
saturated with deionized water during a 12 hour long
immersion step. The optical absorbance of the slab was
scanned between 350 nm to 900 nm. Maximum change in
the absorbance was observed at 590 nm. All of the
consecutive measurements were performed at this
wavelength. The optical absorbance of PDMS slab is
changed due to an increase of the amount of water
absorbed.
Five PDMS slabs were made with the ratio of 2 : 1,
2.5 : 1, 3 : 1, 7.5 : 1, 10 : 1 of PDMS solution and curing
agent. The thicknesses of slabs were 1 mm. Each slab
was cut into 1 cm62 cm size using a razor blade after
peeling off from the mold, and then placed inside the
cuvette, close to the opening of the light path. The
opening of the cuvette was sealed with para®lm during
the measurement. The absorption of liquid in six
different samples was measured simultaneously.
Control cuvettes were used that had PDMS slab without
deionized water and deionized water without PDMS slab.
Any variations in the data from the controls were
subtracted from the actual data points.
The initial rates of increase of absorbance were
similar in all cases. The amount of water absorbed into
PDMS and the time when the absorbance reaches the
maximum point …tmax† decreased with increased content
PDMS and Silicon Hybrid Biochip For Bacterial Culture
285
Fig. 3. The optical absorbance of PDMS slab submerged in deionized water. The ratios of PDMS polymer and curing agent were 10:1 (n), 7.5:1
(h), 3:1 (s), 2.5:1 (e), and 2:1 (6).
of curing agent. The trend is clear for the 10 : 1 and 7.5 : 1
ratio while the differences among the three lower ratios
were minor. As the ratio of the PDMS solution to curing
agent was changed from 10 : 1 to 2 : 1, tmax changed from
12 hours to 5 hours. A lower tmax implies an earlier
saturation and hence a reduction of absorption. The
PDMS slab with 2 : 1 ratio of solutions showed the lowest
absorption. The PDMS slab that had 2.5 : 1 ratio was
chosen for further experiments, because relatively larger
content of excessive curing agent in 2 : 1 mixture
sometimes leads to damage on the silanized surface of
mold, resulting the irreversible bonding of cured PDMS
onto the mold.
3.2. Absorption of liquid in PDMS/silicon hybrid
biochip
The ¯ow path in PDMS/silicon hybrid biochip was
sterilized to prevent the in¯uence of potential bacterial
contamination within the biochip on the electrical
measurements of absorption. The PDMS/silicon hybrid
biochip was sterilized with 70% (v/v, in deionized water)
ethanol solution for more than 2 hours. The ethanol was
washed out by circulation of sterilized deionized water
and LB media for 1 hour and 15 minutes, respectively.
Pressurized nitrogen gas was used to facilitate liquid
¯ow. Biochips were made with different PDMS covers
that have different ratios of PDMS polymer and curing
agent based on the results of absorption characteristics of
the PDMS slabs. The ratios of normal and harder covers
were 10 : 1 and 2.5 : 1 of PDMS polymer and curing
agent, respectively. Since the goal is to determine and
maximize the time that the LB media would stay within
the nano-liter volume chambers, electrical impedance
measurements were used as a means to determine when
the liquid has been absorbed from the chambers. A rapid
increase of impedance reaching the air capacitance
would indicate the drying of the ¯uid. The composition
of low conductivity LB media was yeast extract (5 g/L),
tryptone-peptone (10 g/L), and dextrose (3.3 g/L). The
impedance at 1.1 kHz with a 50 mV A.C. signal was
measured with HP4284A LCR meter (Hewlett Packard
Co., California, USA).
The biochips were also tested with as-is unsaturated
PDMS covers and PDMS covers saturated with the LB
media. The nitrogen pressures used for unsaturated and
saturated PDMS were 2 and 15 psi, respectively. Often,
unwanted air-bubbles were trapped inside the channels
and wells. These bubbles can be removed by blocking the
output port and pressurizing the ¯ow path, prior to the
incubation. The bubbles can be removed using this
technique because of the permeability of PDMS cover to
air and ¯uid. The incubation was started immediately
when the bubbles disappeared. The temperature was set
at 37 C using a micro-heater positioned under the
biochip. The A.C. electrical impedance curves were
measured as a function of time and results are shown in
Figure 4. The impedance started to increase when the
liquid in the chamber of interest (where the impedance
measurement is being performed) started to be absorbed.
The impedance reached the maximum threshold of
1:06106 O when all of LB media in the chamber was
absorbed into PDMS cover.
286
Chang, Akin and Bashir
Fig. 4. The impedance change in PDMS/silicon hybrid biochip by absorption of LB media into PDMS cover. The frequency was 1.1 KHz. PDMS
covers were made of 10 : 1 (s) and 2.5:1 (h) ratio with saturated (solid line) and unsaturated (dotted line) modes before incubation started.
As shown in Figure 4, in the case of the unsaturated
PDMS cover, the complete absorption was delayed from
2.7 to 6.3 hours by the increase of the mixing ratio of
curing agent from 10 : 1 to 2.5 : 1. Recalling the optical
data from Figure 3, the absorption reached a maximum at
around 5 hours in all PDMS slabs of ratio 2 : 1, 2.5 : 1, and
3 : 1. The reason why the absorption of unsaturated 10 : 1
completed in about 2.7 hours is actually due to the
amount of liquid in 5.3 nl volume chamber. The amount
of liquid was a lot less than the experiment described in
Figure 3, and, hence, could be absorbed in a shorter
amount of time. In the case of unsaturated 2.5 : 1 cover,
the absorption was delayed up to 5 hours. This is
expected to be due to the different absorption rate of
moisture in the PDMS covers with different compositions. The lower concentration of PDMS polymer is
expected to lead to smaller gaps among PDMS chains,
larger numbers of bonds between PDMS polymers and
curing agents, and a reduction of mechanical ¯exibility.
The change of physical properties is expected in not only
¯exibility but also in hydrophobicity and porosity,
affecting the absorption of moisture.
The largest delay in the absorption was achieved using
the saturated PDMS cover. It took 19 and 23 hours for
10 : 1 and 2.5 : 1 PDMS covers, respectively, for the media
to evaporate. The difference in the baseline of impedance
in Figure 4 is due to chip-to-chip variation. The absorption
was delayed around 20% by increasing the content of
curing agent from 10 : 1 to 2.5 : 1, even the cover was
saturated before incubation started. When the cover is
saturated before the incubation, the limiting factor for
drying is evaporation of moisture from the surface of
PDMS to the outside ambient. The interaction between
water molecules and PDMS chains would be changed
with different content of curing agent. However, even if
the evaporation rates from PDMS were same for both of
the covers made of 10 : 1 and 2.5 : 1 ratios, the differences
of permeability and water content of those covers are
expected to be resulted in different absorption times.
3.3. Discussion on absorption in PDMS
PDMS is a well-known material for its use in separation
of ethanol from aqueous solution. Diffusion of solvents
through PDMS membranes has been characterized and
reported, however, there are large differences among
these reported data. Characteristics of solvent diffusion
in PDMS were summarized and compared (Watson and
Baron, 1996) and a new apparatus was suggested (Yeom
et al., 1999) for a more precise measurement of
permeation. The hydrophilic impurities within PDMS
were claimed as the main source of large differences in
permeation of liquids in PDMS. We used Sylgard 184,
which is a commonly used PDMS material for
micro¯uidic devices, however, to our knowledge there
have been no reports on parameters for diffusion of liquid
in this material.
PDMS and Silicon Hybrid Biochip For Bacterial Culture
The main polymer and cross linker composing the
PDMS can have a large variation depending upon
molecular weights and mixing ratio of polymer and
cross linker, amount of additives, and etc. The absorption
of ¯uid in PDMS is very important for cell culture
applications when the solution is sustained in the microchambers for long time. Diffusion coef®cient of water in
PDMS, solubility of water in PDMS, porosity of PDMS,
interaction between water and PDMS chains, effect of
temperature on diffusivity, etc. are essential to obtain a
fundamental understanding of absorption in PDMS based
micro¯uidic devices, and more detailed study than what
is presented here, needs to be undertaken. We present
some simple analysis and explanation below.
Typically, mass transfer between different phases is
described using Fick's diffusion law as J ˆ D…Dc=Dz†.
In this equation, J represents the linear ¯ux of molecule
per unit area …g=s ? m2 †, which can be calculated using
the diffusion coef®cient D…m2/s†, difference of concentration Dc…g=m3 †, and distance Dz…m†. The measured
weight and calculated volume of PDMS cover used in
this experiment was approximately 0.06 g and 64 mm3,
respectively. The water content of RTV 615 PDMS
membrane was suggested as 0.38% (w/w) by Blume et al.
(1991). PDMS cover used in this experiment would
absorb around 0.228 ml of water, if similar amount of
water could be absorbed as in the RTV 615 (General
Electric) PDMS membrane.
Assuming the PDMS cover was completely dried
before culture started, rc between saturated and
unsaturated PDMS cover used in this experiment is
3.6 /m3, obtained based on the maximum content of
water mentioned above. The volume of micro-chamber
for cell culture was 5.3 nl, and it is only 2.3% of the
amount of water that could be absorbed in the PDMS
cover. The diffusion coef®cient of water (25 C) through
1.1 mm thick PS342.5 (Fluorochem Ltd., Old Glossop,
UK)
PDMS
membrane
was
measured
as
2:0610 9 m2 =s (Watson and Baron, 1996). However,
the diffusion coef®cient can vary signi®cantly, an order
of magnitude, with different sources of PDMS and
measuring conditions. If the diffusion coef®cient of
Sylgard 184 is assumed to be in the range of 2:0610 10
and 2:0610 8 m2 =s, then the ¯ux can be calculated to
be from 7:1610 7 l=s ? m2 to 7:1610 5 ? m2 . This
would correspond to anywhere from 2.8 minutes to 280
minutes (4.6 hours) for the absorption of 5.3 nl of water
into PDMS through 0.45 mm2 of contact area. The
contact area is simply calculated from the size of microchamber, 530 mm6850 mm. Considering the drying time
of LB media in unsaturated 10 : 1 PDMS cover, as shown
in Figure 4 was 2.7 hours, Sylgard 184 is expected to
have around ®ve times lower diffusion coef®cient than
PS342.5.
287
The total area of our micro-channels and microchambers contacting the PDMS cover was 2.8 mm2. The
amount of water that can pass through 2.8 mm2 of PDMS
for 3 hours is calculated to be about 215 nl. This is close
to the amount of water which saturates the PDMS cover
used in this experiment, even without the consideration
of the pressure that was applied for ¯ow. Thus, PDMS
cover is expected to be saturated with liquid before
incubation starts, when higher pressure was used for the
¯ow.
It should be noted that the transfer of molecules
through polymer is affected by many factors, including
temperature, viscosity of the liquid, characteristics of
polymer, interaction between polymer and liquid, etc.
The estimation performed in this experiment was
extremely simpli®ed, because of the lack of data on
Sylgard 184. The LB medium also has a higher viscosity
than water because of various substances dissolved in the
solution. The solutes absorbed in PDMS cover can affect
the permeability of liquid as well. Considering all these
factors, further investigations are required to characterize
the actual behavior of liquids in PDMS in order to
provide a better understanding of polymer-based micro¯uidic devices.
4. Culture of Bacteria in the Hybrid
Biochips
Listeria innocua (ATCC F4248) and GFP recombinant
Escherichia coli (Invitrogen Life Technologies,
Carlsbad, CA) were cultured in the PDMS/silicon
hybrid biochips saturated with moisture. Low conductivity LB media was used for all the culture steps, from
stock culture to main culture.
The L. innocua cells were stained with Vybrant DiI
carbocyanine dye (Molecular Probes, OR) before they
were introduced into the biochip. This lipophilic,
carbocyanine dye diffuses laterally within the plasma
membrane of the cells, resulted in staining of the entire
cell. The dye is also transferred into daughter cells after
proliferation, however, the concentration reduces upon
each cell division. The maximum wavelength of
absorption and emission of DiI dye is 549 nm and
565 nm, respectively, thus it gives orange ¯uorescence.
The GFP E. coli was transformed with pQBI T7-GFP
plasmid (Quantum Biotechnologies). Ampicillin was
added (50 mg/ml) in low conductivity LB media for
GFP E. coli culture. The ampicillin resistance gene is
included in GFP E. coli to prevent the contamination of
other microorganisms and to maintain GFP gene encoded
on the plasmid. The stock culture was stored in a
refrigerator at 4 C after 20 hours of culture in 37 C
288
Chang, Akin and Bashir
Fig. 5. Fluorescence image of the cultivation of Listeria innocua in a well within the PDMS/silicon hybrid biochip.
incubator. The cell culture solution was prepared by the
following steps. The 100 ml of stock culture was
inoculated into 2 ml of fresh medium as seed culture
and incubated at 37 C in static incubator without
mixing. Another 100 ml of this seed culture was
transferred into fresh medium again after 16 hours of
growth as main culture. Actively growing cells are
obtained through these two steps of cultivation by using
the cells in exponential growth phases. The culture in
biochip was started after injection of another diluted
solution of cells prepared by addition of 100 ml of main
culture solution into a new medium already set at 37 C
to avoid heat shock. Two hours of pre-culture period was
given to this solution for the adaptation of cells to the
new atmosphere. The images of cells grown inside the
chip were taken with a Kodak digital camera (DSC240)
connected to a Macintosh computer (PowerMac G4,
400 MHz) having Photoshop software.
Fig. 6. Fluorescence image of the cultivation of GFP Escherichia coli in a well within the PDMS/silicon hybrid biochip.
PDMS and Silicon Hybrid Biochip For Bacterial Culture
The number of ¯uorescent L. innocua cells increased
with incubation as shown in Figure 5. The brighter dots
are ¯uorescently labeled cells in the micro-chamber. The
size of cells appears different because of the differences
in the intensity of ¯uorescence and the vertical position
in the 12 mm deep micro-chambers of the biochip.
However, increased number of ¯uorescent cells were
not observed clearly after 5 hours of culture, despite the
fact that the higher concentration of cells was optically
con®rmed by bright-®eld micrographs (data not shown).
This was due to a loss of intensity of ¯uorescence during
culture. The loss of ¯uorescence coming from not only
the reduced amount of dye in each cell by proliferation,
but also due to quenching of the dye. To alleviate this
problem, culture of GFP E. coli in the chip was
performed. GFP E. coli is expected to have a more
stable ¯uorescence for long-term culture, comparing
with ¯uorescence labeled microorganism. The plasmids
coding green ¯uorescence protein are inserted in this
strain. The cell gives green ¯uorescence when this
protein is expressed and concentrated with cell growth.
The pictures of the culture of GFP E. coli are shown in
Figure 6. Since the synthesis of GFP inside the cells is a
slow process, the daughter cells do not ¯uoresce 2±3
hours after the cell division has occurred. Thus, the
number and intensity of ¯uorescence of cells slowly
increased with cell growth. Normally, doubling time of
E. coli is 30 minutes, and hence the concentration of cells
after 20 hours of culture is thought to reach to more than
1.0 6 108 CFU/ml. The higher concentration of cells
was optically con®rmed. Consequently, the culture is
believed to be at the stationary or death phase of growth
with this concentration. There is also a possibility of the
growth of non-¯uorescent cells due to the loss of the
plasmids because of the depletion of ampicillin in the
medium. These non-¯uorescent cells dominantly grow
when ampicillin is depleted, because the growth rate of
reversed cell is faster than the cells with the plasmids.
However, considering the increase of the number of
¯uorescent cells and intensity of the ¯uorescence after 18
hours of culture, in this case, the production and
accumulation of GFP is believed to be still in progress.
Cell growth was observed for up to 22 hours, thus,
proving the feasibility of the device for long-term batch
culture of bacterial cell inside hybrid polymer-based
micro-devices.
5.
Conclusions
A novel PDMS/silicon hybrid micro¯uidic biochip was
fabricated and tested for the long-term batch culture of
bacterial cells. The PDMS cover that had 3-dimensional
micro-channels for ¯ow was fabricated without photo-
289
lithographic methods. Liquid was kept in microchambers for up to 22 hours with the PDMS covers
made of 2.5 : 1 ratio of PDMS and curing agent when
they are pre-saturated with liquid before the start of the
incubation. In this case, the drying was delayed by more
than ten times longer than biochip made of unsaturated
PDMS cover composed of 10 : 1 ratio of PDMS and
curing agent. Electrical impedance measurements were
used to determine the onset of drying inside the microchambers. Batch culture of L. innocua and E. coli were
performed in this PDMS/silicon hybrid biochip, demonstrating the possible utility of these devices in a wide
variety of biomedical sensing, diagnostics, drug
screening and biotechnological applications.
Acknowledgments
Dr. Woo-Jin Chang was partially supported by the ERC
for the Advanced Bioseparation Technology, KOSEF,
Korea, and by an NIH grant R21EB000982±1. Dr. Demir
Akin was supported by NIH grant R21 EB000778±1. The
authors would like to thank Rafael GoÂmez and Haibo Li
for their help in the fabrication of silicon chip and
measurement set up. The authors would also like to thank
Prof. A. Bhunia (Food Science, Purdue University) for
providing the Listeria innocua.
References
D.J. Beebe, J.S. Moore, Q. Yu, R.H. Liu, M.L. Kraft, B.-H. Jo, and C.
Devadoss, P.N.A.S. 97, 13488 (2000).
D. Beebe, M. Wheeler, H. Zeringue, E. Walters, and S. Raty,
Theriogenology 57, 125±135 (2002).
S.N. Bhatia, U.J. Balis, M.L. Yarmush, and M. Toner, Biotechnol. Prog.
14, 378±387 (1998).
I. Blume, P.J.F. Schwering, M.H.V. Mulder, and C.A. Smolders,
J. Membrane Sci., 61, 85 (1991).
J.T. Borenstein, H. Terai, K.R. King, E.J. Weinberg, M.R. KaazempurMofrad, and J.P. Vacanti, Biomed. Microdev. 4, 167±175 (2002).
N. Bowden, S. Brittain, A.G. Evans, J.W. Hutchinson, and G.M.
Whitesides, Nature 393, 146±149 (1998).
E. Favre, P. Schaetzel, Q.T. Nguygen, R. CleÂment, and J. NeÂel,
J. Membrane Sci. 92, 169 (1994).
A.J. Gawron, R.S. Martin, and S.M. Lunte, Electrophoresis 22, 242±248
(2001).
R. GoÂmez, R. Bashir, and A.K. Bhunia, Sensors & Actuators: Part B:
Chem. 86, 198 (2002).
R. GoÂmez, R. Bashir, A. Sarikaya, M.R. Ladisch, J. Sturgis, J.P.
Robinson, T. Geng, A.K. Bhunia, H.L. Apple, and S. Wereley,
Biomed. Microdevices 3, 201 (2001).
M.K.N. Hirayama, W.R. Caseri, and U.W. Suter, Appl. Surface Sci. 143,
256±264 (1999).
J.W. Hong, T. Fujii, M. Seki, T. Yamamoto, and I. Endo.,
Electrophoresis 22, 328±333 (2001).
K. Kimmerle, T. Hofmann, and H. Strathmann, J. Membrane Sci. 61, 1
(1991).
290
Chang, Akin and Bashir
M.U. Kopp, A.J. de Mello, and A. Manz, Science 280, 1046 (1998).
E. Leclerc, Y. Sakai, and T. Fujii, Biomed. Microdev. 5, 109±114
(2003).
S. Mouradian, Curr. Opinion in Chem. Biol. 6, 51 (2001).
M.J. Powers, K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A.
Capitano, A. Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R.
Kamm, and L.G. Grif®th., Biotechnol. & Bioeng. 78, 257±269
(2002).
K. Sivasailam and C. Cohen, J. Rheol. 44, 897 (2000).
M.A. Unger, H.-P. Chou, T. Thorsen, A. Scherer, and S.R. Quake,
Science 288, 113 (2000).
J.M. Watson and M.G. Baron, J. Membrane Sci. 110, 47 (1996).
T. Yasukawa, A. Glidle, J.M. Cooper, and T. Matsue, Anal. Chem. 74,
5001 (2002).
C.K. Yeom, B.S. Kim, and J.M. Lee, J. Membrane Sci. 161, 55
(1999).
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Related manuals

Download PDF

advertisement