Integrated Microfluidic System for DNA Analysis
V. R. Dukkipati and S. W. Pang
Department of Electrical Engineering and Computer Science
The University of Michigan
Ann Arbor, MI, 48109
ABSTRACT — In this paper, we report the fabrication of
a Si based microfluidic system, which is integrated with
electrodes and polymer structures. Low temperature Si to
glass bonding using PMMA as an intermediate layer has
been developed to integrate electrodes and PMMA structures
with Si channels. The electrodes in the microchannel are
used for electrokinetic-based DNA stretching. DNA
molecules are stretched using high frequency ac electric
fields inside Si channels that are 100 µm wide and 20 µm
deep.
Index Terms — Microfluidic, DNA, PMMA, bonding,
eletcrokinetic
I. INTRODUCTION
Electrokinetic techniques such as dielectrophoresis and
electro-rotation have been utilized for many years for
manipulation, separation and analysis of cellular-scale
particles [1]. Proteins as small as 5 nm have been
manipulated using both attractive [2] and repulsive forces
[3]. Dielectrophoresis, concerned with manipulation of
molecules in solution, where wet environments are
necessary for bio-molecules, requires microfluidic system
with integrated electrodes [4].
Recently DNA manipulation has been demonstrated
using ac electrokinetics [5]. The integration of electrodes
inside channels involved UV glue bonding of glass
channels. This bonding technique cannot be used in case
of submicrometer channels since the UV glue will seep
into and block the channels. We have developed a low
temperature PMMA bonding technique to integrate
electrodes inside Si channels. These electrodes can be
used to manipulate biological molecules such as DNA and
proteins inside very small channels.
Stretching of DNA molecules inside microchannels by
combing has been demonstrated [6]. The disadvantage of
this technique is that the DNA molecules are dried from
the solution in the channel, which would result in DNA
not being biologically active. We have used electrokineticbased DNA stretching technique to demonstrate that DNA
immobilization and stretching can be achieved inside
sealed microchannels.
II. FABRICATION TECHNOLOGY
Integrating electrodes inside Si channels requires
bonding of Si and glass using PMMA as an adhesive
layer. The procedure is illustrated in Fig 1. To ensure good
bonding, the wafer surface has to be free of particles and
impurities. The 100 µm thick glass is immersed in 1:1
H2O2:H2SO4 solution for 20 min, followed by dehydration
at 200 °C for 1 h in an oven. 20/50 nm thick Cr/Au layer
is deposited on glass using electron beam evaporation.
Photoresist is spun and patterned on the glass using optical
lithography. The pattern is then transferred to metal layers
by wet etchants with photoresist as the mask. Photoresist
is then removed using PRS 2000 heated at 100 °C, leaving
Cr/Au electrodes on the glass, as shown in Fig. 1(a). It is
very important to ensure that there are no PR residues left
on the glass since this will result in poor bonding. The
glass wafer is dehydrated again at 200 °C for 1 h in an
oven. 6% 950K PMMA is spin-coated on the glass wafer
containing the electrodes at 2.5 krpm, resulting in 600 nm
thick layer, as shown in Fig. 1(b). 10 nm thick Ti layer is
evaporated on the PMMA. To define areas over which the
PMMA is etched, photoresist is spun and patterned on the
Ti layer, as shown in Fig. 1(c). The thin Ti layer prevents
the solvents in the resist and the PMMA layers from
interacting with each other. Following photoresist
patterning, Ti layer is etched in 20:1:1 H2O:H2O2:HF
solution [7]. The exposed PMMA is then etched in O2
plasma with 100 W rf power, 250 mTorr, 100 sccm of O2
for 12 min as shown in Fig. 1(d). Photoresist is then
removed in PRS 2000 at room temperature, since removal
in higher temperature solvent would result in PMMA
peeling off from the glass surface. The last step involves
Ti layer etched in 20:1:1 H2O:H2O2:HF solution for 10 s,
leaving Cr/Au electrodes and patterned PMMA on glass as
shown in Fig. 1(e).
The channels are etched in Si using plasma etching. The
etch process is switched between the SF6 etch step and the
C4F8 passivation step to obtain fast Si etch rate and vertical
etch profile. A source power of 75 W and stage power of
12 W are used at 26 mTorr with 105 sccm SF6 in the 14-s
etch cycles. A 75 W source power is applied with no stage
power at 16 mTorr and 40 sccm C4F8 in the 4-s passivation
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cycles. Fluidic ports of 300 µm diameter to access the
channels are defined by etching through the 550 µm thick
wafer using plasma etching. A source power of 75 W and
stage power of 20 W are used at 26 mTorr with 160 sccm
SF6 in the 13-s etch cycle. A 75 W source power was
applied with no stage power at 16 mTorr and 85 sccm C4F8
in the 7-s passivation cycles. The total etching time is 3 h.
The Si wafer with etched channels and fluidic ports is
cleaned in 1:1 H2O2:H2SO4 before bonding.
of 45 cycles of switching is carried out with a total etch
time of 15 min. The area over which the PMMA is etched
is illustrated in the figure.
Figure 3 shows a fluorescent micrograph of PMMA
structure integrated inside a 100 µm wide and 1 µm deep
Si channel. The fabrication process is similar to the one
described above. PMMA is spun and patterned on the
glass. Instead of Ti, Au is used as an intermediate layer
between PMMA and photoresist. The bonding conditions
used are similar to the one mentioned above.
Si Channels
GLASS
Cr/Au Electrode
Cr/Au Electrode
Si
PMMA on Glass
Etched PMMA
20 µm
Fig. 2. Cr/Au electrodes integrated in 3 and 10 µm wide Si
channels.
Fig. 1. Fabrication process flow of the integrated device.
The wafers are first aligned and then transferred to the
bonding tool for bonding. Bonding of the wafers is carried
out in vacuum at 75 Torr pressure. This prevents the
formation of air gaps and helps to improve the contact
uniformity across the wafer. The bonding is performed
above the glass transition temperature of PMMA (109 °C)
for 2 h at a pressure of 0.4 MPa, resulting in electrodes
integrated inside fluidic channels as illustrated in Fig. 1(f)
[8].
III. INTEGRATED ELECTRODES AND PMMA STRUCTURES
IN SI CHANNELS
Figure 2 is an optical micrograph showing electrodes on
a 100 µm thick glass aligned and bonded to Si wafer
containing channels perpendicular to each other.
Conditions used for bonding the wafers are 110 °C, 0.4
MPa for 2 h in 75 Torr vacuum. The Cr/Au electrodes on
the glass are 20/50 nm thick and the channels in Si are 30
µm deep, 3 and 10 µm wide. To etch the channels, a total
10 µm
Fig. 3. 5 µm wide PMMA pattern integrated in Si
channel.
IV. DNA MANIPULATION IN CHANNELS
Electrodes in the channel are used to stretch the DNA
molecules using ac electric fields. Channel leak is tested
before applying the electric field. It is important there are
no leaks because the presence of leaks drives the fluid and
DNA molecules inside the channel by capillary force,
which results in disturbing the position of the DNA
molecules inside the channel with respect to the applied
electric field.
The forces acting on the DNA molecules and the fluid
by ac electric fields have been described by Ramos et al
[9]. The electrokinetic stretching of DNA molecule in a
fluidic medium involves several forces acting on the
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elongation of the DNA molecules. After DNA has
stretched to around 11.5 µm, the length remains constant,
indicating that the DNA might be stretched to the full
contour length. The actual length of the fully stretched
DNA would be longer than measured because the attached
end of DNA could be located a distance away from the
electrode edge.
Straight edge
Cr/Au Electrode
Pointed
Cr/Au Electrode
30 µm
Si Channel
Fig. 4. λ-DNA immobilized and stretched using
electrokinetics in a 100 µm wide and 20 µm deep Si
channel. The applied ac voltage is 100 KHz sinusoidal and
10.8 V peak-to-peak.
12
DNA STRETCHED LENGTH (um)
DNA. One of them is the dielectrophoretic force caused
by the induced dipole along the backbone of the DNA
molecule. This force is directed towards the electrode
edges. The second force is the torque exerted on the
induced dipole by the electric field. For a DNA molecule
with one end immobilized on the electrode, the torque
tends to elongate the DNA parallel to the electric field.
The third force is the electrothermal force acting on the
fluid medium. This force arises from the thermal gradients
in fluid created by the variations of the electric field. The
electrothermal force induces a circulatory motion of the
fluid near the electrodes. The fluid medium used in this
case is 1:1 DI water:morpholinoethanesulfonic (MES)
buffer (50 mM, 5.5 pH). When DI water alone is used as
the fluid medium, the electrothermal force acting on the
fluid is high due to its low conductivity. The flip side is,
the probability that one end of the DNA molecule would
get immobilized on the electrode in DI water is low. When
MES buffer alone is used as the fluid medium, the
electrothermal force acting on it is comparatively lower
due to its higher conductivity, but the probability that one
end of the DNA would be immobilized on the electrode is
higher. Taking the above factors into account, the fluid
medium chosen is a mixture of equal amounts of DI water
and MES buffer.
3 ng/µl of λ-DNA in 1:1 DI water:MES buffer is
introduced into the channels. 100 KHz sinusoidal ac
voltage starting from 0.5 V is slowly ramped up. The
DNA molecules start moving towards the electrodes at
around 2.2 V due to the combination of dielectrophoretic
force and the circulating fluid. During this process some
of the DNA molecules that get attracted to the electrode,
have one of their ends immobilized on the electrode. After
ramping to about 6 V the tethered DNA molecules start
stretching out as shown in Fig 4. The Cr/Au electrodes
consist of straight edge electrode on the left that is
separated by 30 µm from the tip of the pointed electrode
on the right. These electrodes are integrated in a 100 µm
wide and 20 µm deep Si channel. The thickness of the
Cr/Au electrodes is 20/50 nm. More DNA molecules are
immobilized on the pointed electrode due to higher
electric field gradient at the pointed tip. After DNA
immobilization, the voltage is ramped is up, which results
in torque and electrothermal force exerted on the attached
DNA molecules. The torque acting on the molecule
combined with the electrothermal fluid flow contribute to
the elongation of DNA [10]. The DNA molecules attached
to the electrodes are stretched out as seen in the Fig 4.
Figure 5 shows the variation of stretched lengths of
DNA molecule with applied voltage. The DNA stretched
length corresponds to a DNA molecule that is
immobilized on the electrode and stretched from the tip of
the electrode. With increase in voltage, the electric field
increases, resulting in an increase in torque and
electrothermal force. The increase in forces result in
10
8
6
4
2
5
6
7
8
9
10
11
12
VOLTAGE (VP-P)
Fig. 5. Variation of stretched lengths of a λ-DNA
molecule due to applied ac fields. The length is measured
from the tip of the electrode to the edge of the stretched
DNA molecule.
V. CONCLUSION
We have demonstrated the fabrication of Si based
microfluidic system integrated with electrodes and
polymer structures. A low temperature PMMA bonding of
Si channels to glass has been developed which allows us
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to integrate electrodes and PMMA structures inside Si
channels. The electrodes in the microchannel have been
used for electrokinetic-based DNA stretching. DNA
immobilization to electrodes and stretching have been
demonstrated using high frequency ac electric fields.
Maximum DNA stretched length of 11.5 µm has been
obtained in microchannels that are 100 µm wide and 20
µm deep.
th
[4]
[5]
[6]
VI. ACKNOWLEDGMENTS
The authors would like to acknowledge the assistance
from E. Sung and J. Kim. This project is supported by
National Science Foundation under grant number BES0304316.
REFERENCES
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[8]
[9]
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