Samudra Sengupta, Michelle M. Spiering, Krishna K.

Dey, Wentao Duan, Debabrata Patra,

Peter J. Butler,,* R. Dean Astumian,,* Stephen J. Benkovic,,* and Ayusman Sen,*

ARTICLE

DNA Polymerase as a Molecular Motor

and Pump

Department of Chemistry and Department of Bioengineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States, and
Department of Physics and Astronomy, The University of Maine, Orono, Maine 04469, United States

ABSTRACT

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using uorescence correlation spectroscopy,
we demonstrate that the diusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into
the growing DNA template. The diusion coecient of the complex also shows a strong dependence on its inorganic cofactor, Mg2 ions. When exposed to
gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of
higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered
uid pumps. These miniature pumps are capable of transporting uid and tracer particles in a directional manner with the pumping speed increasing in the
presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature uid pumps using enzymes as engines.
KEYWORDS: enzyme . DNA polymerase . catalysis . diusion . nanomotor . chemotaxis . pump

ropulsion of objects at the nano- and

microscale can be achieved by utilizing the chemical free energy of the
surrounding medium.1!13 In most cases,
the energy is derived by catalytic turnover
of a fuel or substrate in the vicinity of the
motor to generate mechanical work for
performing useful functions.1!13 In particular, there has been signicant interest in
developing catalytic motors because the
ability to rationally design synthetic and
hybrid nano/micromotor systems will facilitate the emergence of novel smart devices.
Upon immobilization, these catalytic motors can also impart their energy to the
surrounding uid. Using this concept, a
number of articial micropumps have been
designed, which are capable of driving both
uid and small particles alike.14!31
SENGUPTA ET AL.

The major inspiration behind designing

articial nano/micromotors and micropumps is to mimic the precision and eciency of the naturally occurring biomotors
like kinesins, myosins, and dyneins. Enzymedriven biological motors are responsible for
driving the biased motion of organisms in
response to specic stimuli (chemicals or
light).32!36 Due to their vast diversity, enzyme-based articial motors become an
obvious choice for the next generation of
intelligent devices that may enable nanotechnological and medical applications,
such as dynamic self-assembly of superstructures, drug delivery, and lab-on-a-chip
devices.2!4,10,11 Recently, we have reported
a substrate-concentration-dependent increase in diusion of both catalase and
urease in the presence of their respective
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* Address correspondence to
pbutler@psu.edu,
astumian@maine.edu,
sjb1@psu.edu,
asen@psu.edu.
Received for review November 18, 2013
and accepted February 27, 2014.
Published online March 06, 2014
10.1021/nn405963x
C 2014 American Chemical Society

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Figure 1. Schematic overview showing nucleotide incorporation on a DNA template by a DNA polymerase motor.

substrates, hydrogen peroxide and urea.37,38 These

enzyme molecules can also sense a gradient in substrate concentration and exhibit collective migration
toward a higher substrate concentration. Schwartz and
co-workers also reported a similar behavior with T7
RNA polymerase.39
Biological motors perform specic functions in
living systems.40!42 The T4 DNA polymerase is a wellcharacterized biomotor that is responsible for the
sequential incorporation of nucleotides to synthesize
DNA (Figure 1).40 The polymerase adds deoxynucleotide triphosphates (dNTP) to the growing DNA strand,
and the energy released from the removal of the
pyrophosphate group results in the formation of a
phosphoester bond that covalently links the deoxynucleotide monophosphate (dNMP) to the growing DNA
chain. The polymerase motor binds to the DNA template inducing an internal conformational change in
the polymerase.
The wild-type DNA polymerase has two active
sites;the polymerase site that incorporates bases
with high precision, and the exonuclease site, which
acts as an error correction site to remove any mismatched nucleotides incorporated in the DNA primer
strand. The motor can switch the DNA primer strand
from the polymerase site to the exonuclease site
whenever a mismatched nucleotide is identied and,
after the error correction, rapidly revert the DNA strand
back to the polymerase site for the replication process
to continue.
In the presence of an excess of the single nucleotide
templated next for incorporation, the polymerase
is restricted to an idling state. During the idling!
turnover process, the protein switches the DNA primer
strand back and forth between the polymerase and the
exonuclease active sites, incorporating and removing
the nucleotide, thereby converting dNTP to dNMP until
the nucleotide is completely depleted. With an exonuclease site mutant of DNA polymerase (exo-), a single
nucleotide is incorporated, but subsequent cycles of
incorporation and removal are prohibited. Both the
polymerase site and the exonuclease site require the
inorganic cofactor of two Mg2 ions per site for activity.
These unique functions of the DNA polymerase encouraged us to explore its possible role as a nanomotor
outside biological systems and to investigate its further
potential as a micropump when immobilized onto a
surface.
SENGUPTA ET AL.

Figure 2. Diusion studies of the DNA polymerase molecular complex. The diusion coecient of the wild-type polymerase DNA molecular complex in the idling!turnover
mode showed an increase of 46% in the presence of 5 mM
dATP (substrate nucleotide) and Mg2 ions (cofactor) in the
buer over the diusion coecient of the DNA polymerase
(exo-) complex. The increase in the diusion coecient of
the molecular complex was lowered in the absence of Mg2
ions in the buer. The three diusion coecients are
signicantly dierent from each other with a signicance
value of P < 0.015. Error bars represent standard deviations.
The means and standard deviations were calculated for
10 dierent measurements.

In this study, we show that the diusive movement

of a molecular complex of DNA template and T4 DNA
polymerase is enhanced in the presence of a nucleotide, 20 -adenosine triphosphate (dATP). The molecular
complex shows a collective directional movement
in the presence of a dATP concentration gradient.
Further, in the presence of a concentration gradient
of the Mg2 cofactor, it migrates toward areas of higher
Mg2 concentration. Finally, by immobilizing the molecular complex on a patterned surface, uid and tracer
particles can be pumped in a directional manner with
the pumping speed increasing in the presence of
cofactor in the surrounding uid.
RESULTS AND DISCUSSION
The diusive mobility of a wild-type T4 DNA polymerase complexed with DNA was measured in the
presence of a substrate nucleotide in an idling!
turnover mode. We used uorescence correlation
spectroscopy (FCS), an ultrasensitive technique, for
diusion studies.43,44 The primer strand of the DNA
was labeled with a uorescent tag for monitoring by
FCS. Diusion of the DNA polymerase (exo-) complex
was monitored as a control (Figure 2). The measured
diusion coecient of the complex was 1.22 #
10!6 cm2/s. The diusion coecient of the wild-type
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polymerase DNA complex increased to 1.77 #

10!6 cm2/s in the presence of 5 mM dATP in buer
with Mg2, a 46% increase in diusion (Figure 2). This
increase in diusion was attenuated in the absence of
Mg2 in the buer (Figure 2). The slight increase in
diusion of the complex molecules in the absence of
cofactor can be attributed to some residual or contaminant Mg2 ions present in the buer in which the
protein was stored. FCS traces for a wild-type polymerase DNA molecular complex in buer (with and without Mg2 ions) in the presence of 5 mM dATP (red and
blue, respectively) and DNA polymerase (exo-) molecular complex in buer (with Mg2 ions) in the presence
of 5 mM dATP (green) are shown in Figure 3. A shift
in the FCS trace to the left corresponds to a faster
diusion rate.
We studied the collective migration behavior of the
molecular complex in the presence of a dATP concentration gradient. We utilized a three-inlet microuidic
device (see Supporting Information, Figure S1A) to
generate a dATP substrate nucleotide gradient. DNA
in buer with Mg2 was injected through the center
inlet, anked by DNA polymerase and 10 mM dATP in
buer with Mg2 on one side and buer with Mg2 on
the other. As a control, the DNA was injected through
the center inlet, anked by DNA polymerase in buer
with Mg2 through one side inlet and buer with Mg2
through the other side inlet. Using a syringe pump, a
linear laminar ow rate of 1.85 # 104 m/s was maintained in the main channel. The collective spreading
behavior was studied using a uorescence imaging
setup. Lateral uorescent spreading of the polymerase
DNA complex was measured across the channel
40 mm downstream in the main channel. The uorescence intensity proles were normalized between
0 (minimum) and 1 (maximum) to quantify the shift
at a dened value of uorescence intensity. We observed that the normalized uorescent intensity prole
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Figure 3. Fluorescence correlation spectroscopy traces. FCS

traces for DNA polymerase complex molecules showing a
plot of the normalized autocorrelation as a function of time.
The traces shown are for wild-type polymerase DNA complexes in buer with and without Mg2 ions in the presence
of 5 mM dATP (red and blue, respectively) and polymerase
(exo-) DNA complexes in buer with Mg2 ions in the
presence of 5 mM dATP (green).

of the molecular complex showed a lateral spreading

of 9 m toward the substrate nucleotide in the presence of dATP as compared to no substrate (Figure 4A).
Broadening of the uorescence intensity prole was
observed in the non-normalized plots in the presence
of dATP as compared with no nucleotide, which is a
direct consequence of the collective migration behavior (Supporting Information, Figure S2B); a similar
eect was observed with urease and catalase.37 As
expected, a smaller shift in the normalized uorescence intensity prole of 4.5 m was observed when
the laminar ow rate was doubled to 3.70 # 104 m/s
(Figure 4B). The non-normalized plots once again
showed the anticipated broadening of the uorescence intensity prole (see Supporting Information,
Figure S2).
Further, we explored the response of the polymerase DNA complex to a concentration gradient of its
cofactor, Mg2 ions. As discussed before, a uorescence imaging setup was utilized for studying the
spreading behavior, and a two-inlet microuidic channel (see Supporting Information, Figure S1B) was used
for generating a Mg2 ion concentration gradient. A
mixture of polymerase DNA complex, 10 mM dATP,
and buer without Mg2 ions was injected through
one of the inlets, and buer with 20 mM Mg2 ions was
injected through the other inlet. A lateral shift in the
uorescence intensity prole of 9 m toward the
region with the cofactor as compared to no Mg2 ions
was observed 40 mm down the channel (Figure 5).
On the basis of the observed enhanced diusion of
polymerase DNA complex in the presence of dATP, we
hypothesized that the complex when immobilized on a
surface should create ow in the surrounding uid
due to symmetry breaking. Accordingly, a circular gold
pattern was designed on a PEG-coated glass surface
using an e-beam evaporator. Utilizing Au!thiol chemistry, a quaternary ammonium-terminated ligand (see
Supporting Information, Figure S3 and Scheme S1) was
functionalized onto the Au pattern. The ligand formed
a self-assembled monolayer (SAM) on the gold surface.
The negatively charged backbone of the DNA bound
selectively to the SAM-functionalized gold surface via
electrostatic interactions. The DNA polymerase forms a
complex with the DNA bound to the gold surface
(Figure 6). The selective functionalization of the
DNA polymerase complex on the SAM-modied Au
pattern was veried by incubating the surface with
uorescent-labeled DNA. Then the surface was monitored under a uorescence microscope. Fluorescence
signal was observed only on the Au pattern, conrming
the presence of DNA on the Au pattern (see Supporting
Information, Figure S4). No uorescence intensity was
detected on the PEG-coated glass surface. To demonstrate the pumping ability of immobilized enzymes, a
spacer was placed on top of the enzyme-patterned
surface. Sulfate-functionalized polystyrene beads

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Figure 4. Collective migration of an ensemble of DNA polymerase complex molecules in response to a concentration gradient
of substrate nucleotide. (A) Plot of mean normalized (1 corresponds to the maximum, and 0 corresponds to the minimum)
uorescence intensity (a.u.) prole as a function of the lateral position along the width of the channel shows a 9 m shift for
an ensemble of DNA polymerase molecular complex toward 10 mM dATP (red; right side of the plot) as compared to buer
with Mg2 ions (blue; right side of the plot) when viewed 40 mm down the channel and at a ow rate of 400 L/h. The shift in
the plot (red) to the right corresponds to a shift toward the dATP channel. (B) Plot of mean normalized uorescence intensity
(a.u.) prole as a function of the lateral position along the width of the channel shows a 4.5 m shift for an ensemble of DNA
polymerase molecular complex toward 10 mM dATP (red; right side of the plot) as compared to buer with Mg2 ions (blue;
right side of the plot) when viewed 40 mm down the channel and at a ow rate of 800 L/h. The shift in the plot (red) to the
right corresponds to a shift toward the dATP channel.

suspended in a buered solution of dATP with Mg2

ions were used as tracers to monitor the uid ow. In
the presence of 10 mM dATP in buer with Mg2, the
tracer particles moved in toward the gold surface at an
average speed of 1.4 m/s (Figure 7; see Supporting
Information, video 1). When the concentration of dATP
was lowered to 1 mM, the average uid pumping
speed decreased to 1.0 m/s (Figure 7). However, in
the absence of Mg2 ions in the buer, reduced uid
pump speeds of 0.4 m/s were observed both in
10 mM dATP and 1 mM dATP (Figure 7; see Supporting
Information, video 2). No uid pumping was detected
in the absence of the dATP substrate (see Supporting
Information, video 3). Since the micropump design
utilized here is a closed system, uid owing toward
the Au disk near the PEG-coated glass surface should
ow away from it in another plane by uid continuity.
Indeed, the uid was found to ow away from the gold
surface in another plane.
We considered a number of possibilities for the
observed increase in diusivity of the polymerase
DNA molecular motor. The presence of substrate had
a negligible eect on the solvent viscosity. The viscosity of buer containing either 5 or 10 mM dATP was not
signicantly dierent. Bulk rise in solution temperature
SENGUPTA ET AL.

due to enzymatic catalysis of urea and hydrogen

peroxide by urease and catalase, respectively, has been
estimated and reported to be in the micro-Kelvin
range.37,38 Similarly, the possibility of a rise in solution
temperature due to nucleotide incorporation in the
DNA was estimated and found to be too small to account
for the observed increase in diusion (see Supporting
Information). Active transport of catalytic particles in
uids can be achieved by various local and global force
elds.1!13,45!54 Recently, enhancement in diusion of
single-molecule urease, catalase, and RNA polymerase
enzymes in the presence of their respective substrates
has been reported.37!39 The observed increase in diusion occurred irrespective of the charge on the reaction
products (neutral or ionic). Incorporation of a nucleotide
into the DNA by the polymerase is accompanied by the
release of pyrophosphate, the release of which can drive
the molecular complex by a self-electrophoretic mechanism.47,53 However, such a mechanism is unlikely to
play a prominent role due to the very fast rotational
diusion of the DNA polymerase complex, which will
normalize any concentration gradient of pyrophosphate
generated in the vicinity of the complex.
An alternative explanation for the enhanced diusion is based on a cycle of nonreciprocal conformational
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Figure 5. Collective migration of an ensemble of DNA

polymerase complex molecules in response to a concentration gradient of cofactor. A plot of mean normalized
(1 corresponds to the maximum, and 0 corresponds to the
minimum) uorescence intensity prole (a.u.) as a function
of the lateral position along the width of the channel shows
a 9 m shift for an ensemble of DNA polymerase molecules toward buer with 20 mM Mg2 ions (red) as
compared to buer with no Mg2 ions (blue) when viewed
40 mm down the channel.

Figure 6. Schematic of a DNA polymerase-powered micropump. Au is patterned on a PEG-coated glass surface.

The patterned surface is functionalized with a quaternary
ammonium thiol, which forms a SAM (self-assembled monolayer) on the Au surface. The negatively charged backbone
of the DNA binds selectively to the SAM-functionalized
Au-patterned surface via electrostatic interactions. The
DNA polymerase forms a complex with the DNA bound to
the gold surface. The schematic is not drawn to scale.

changes.55 It has been proposed that enzymes can

propel themselves in solution during catalytic turnover
by going through a sequence of nonreciprocal conformational changes that encompass substrate binding and product release.56!59 During the nucleotide
incorporation process, DNA polymerase molecules go
through a series of conformational steps which are
well-documented.60!63 It is possible that a cycle of
asymmetric conformational changes can overcome
random Brownian diusion to cause autonomous motion of the molecular complex. During idling!turnover
of the DNA polymerase motor, the repetitive incorporation of dATP molecules and their subsequent
removal from the DNA template also involves a cycle
of nonreciprocal conformational changes.
SENGUPTA ET AL.

Figure 7. Fluid pumping in a DNA polymerase complexpowered micropump. The pumping speed of a DNA polymerase complex-powered pump increased with increasing
the dATP concentration from 1 to 10 mM in the presence
of Mg2 ions. Fluid pumping is highly attenuated in the
absence of Mg2 ions. No uid pumping was detected in the
absence of dATP. Error bars represent standard deviations.
The means and standard deviations are calculated for 30
tracer particles. The pumping velocities at dierent dATP
concentrations and in the presence and absence of Mg2
are statistically dierent (P < 0.01).

We propose that the collective spreading of an

ensemble of molecular complexes toward higher substrate concentration results from an enhanced diusion mechanism.37,39,64,65 As the ows of DNA in the
center inlet and polymerase/dATP in one of the side
inlets diuse into each other under laminar ow conditions, the polymerase binds to the DNA to form a
molecular complex (dissociation constant of the molecular complex, KD = 70 nM66). As the molecular
complex moves across the channel, it encounters a
higher nucleotide concentration and experiences a
higher diusion coecient, which results in a greater
spreading of the complex molecules toward higher
nucleotide concentration. This is analogous to the substrate concentration gradient acting like a Brownian
ratchet.67!70 As an eect of this collective migration,
the broadening of uorescence intensity proles was
observed in the non-normalized plots (Supporting
Information, Figure S2). Collective movement in a
chemical gradient, an exceptional phenomenon outside biological systems, has been reported for ensembles of urease, catalase, and RNA polymerase enzymes.37!39,54,64,65 We propose a similar mechanism,
which is stochastic in nature, for the directional spreading behavior of the DNA polymerase complex toward
higher Mg2 cofactor concentration. The spreading
behavior of the molecular complex observed in our
experiments is dierent from biological chemotaxis as
shown by bacteria, which requires temporal memory
of the concentration gradient of their food.
An equally intriguing observation is the uid pumping action exhibited by these molecular complexes
when immobilized on a patterned surface. It was
shown previously that catalysts supported on a surface can generate uid pumping through catalytic
turnover,21!27,30,71 resulting from phoretic mechanisms such as diusiophoresis,45 osmophoresis,54 and
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self-electrophoresis.53 Transport of uid and tracers in

DNA polymerase-powered pumps may arise from an
electrolyte diusiophoretic mechanism since nucleotide incorporation into the DNA generates charged
reaction products due to dATP hydrolysis.46,72 The
zeta-potential of tracer particles determines the direction of transport in electrolyte diusiophoresis, with
tracers with opposite charges moving in opposite directions. However, we observed that the direction of
movement of positively charged amine-functionalized
tracers was exactly the same as their negative sulfatefunctionalized counterparts (see Supporting Information, video 4).
The uid ow monitored with both positive and
negative tracers reverses direction relative to the glass
surface when the pump setup is inverted (Figure 8; also
see Supporting Information, video 5). This is possible
only when the ow is driven by a dierence in density.
Importantly, the energy released per second in the
polymerase reaction is estimated to be nearly 10!9 W,
while that required to drive uid convection of the
order of 1 m/s (observed magnitude of ow) within
the experimental chamber is nearly 10!16 W (see
Supporting Information). The chemical energy available in the system is therefore more than sucient to
power the observed convective movement of the uid.
However, the mechanism by which the density gradient is established remains an open question. To estimate the role of reaction exothermicity, multiphysics
simulations were performed in COMSOL, coupling the
physics of heat transfer and laminar ow of liquid (see
Supporting Information for details). The simulations
conrmed that, in order to establish a convective
ow of the order of 1 m/s through a purely thermal
gradient, the system needs to have a power input of
approximately 10!4 W, which is far larger than the
energy released in the polymerase reaction. Thus, we
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Figure 8. Fluid pumping in upright and inverted micropump setups. The direction of uid ow monitored with
both positive and negative tracers reverses direction relative to the glass surface when the pump setup is inverted (B)
as compared to an upright setup (A). The uid ow switches
direction relative to the glass surface in the upright
and inverted micropump setups due to a density-driven
mechanism.

conclude that the density gradient and accompanying

uid convection generated in the present system are
nonthermal in origin, suggesting the possibility that
the observed phenomena may be due to nonreciprocal conformational changes occurring at the immobilized enzymes. This is reminiscent of the direct conversion of metabolic energy to kinetic energy that is
responsible for uid motion observed in the presence
of free swimming bacteria.73 The driven conformational changes of the polymerase molecules may
increase the mean free path between water molecules
near the surface, eectively reducing the density at
that position. Alternatively, the conversion of dATP to
dAMP PPi may result in a net decrease in uid density
near the pump surface.71 Either one of these proposed
mechanisms is consistent with our experimental observations. In the upright setup in which the polymerase pattern is at the bottom, the uid ow is directed
upward from the pump, and near the bottom glass
surface, the ow is toward the Au pattern due to uid
continuity. In the inverted setup where the pattern is at
top of the device, the uid ow is reversed because the
less dense uid prefers to occupy the upper layers and
spreads along the glass surface away from the polymerase pattern. The observed energy conversion eciency of the micropump (10!7) is comparable to
that of previously reported synthetic autonomous
systems that also transduce the energy from chemical
reactions to mechanical motion.74,75
ATP powers most of the biological pumps found in
nature. While articial micropumps have been designed,15,76 this is the rst example of a polymerasepowered pump. This pump has the unique capability
to sense and respond to specic triggers: dATP and
Mg2 ions. The ability of enzymes and biomotors to
pump uids, small molecules, and particles in a directed fashion may enable the development of smart
micro- and nanoscale devices that turn on in response
to specic analytes and contain sophisticated levels of
control over the location and ow rate of uids.
CONCLUSION
In conclusion, we show that the incorporation of
nucleotides into a DNA template by DNA polymerase is
responsible for an enhancement in diusion of the
complex. It is possible that nonreciprocal conformational changes in this biomolecular complex play a role
in the observed increase in diusive mobility. Furthermore, in the presence of substrate and cofactor concentration gradients, an ensemble of these complexes
spreads toward areas of higher concentration, which
is a direct consequence of the enhanced diusion exhibited by these complex molecules. Finally, by immobilizing DNA polymerase complexes on a patterned
surface, uid pumps can be designed and fabricated.
The ndings reported in this paper can be a stepping
stone in the design of novel bioinspired motors and
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diusion and pumping, possibly due to nonreciprocal

conformational changes, are generic and may play a
fundamental role in biological motility and transport
by biomolecular machines.

METHODS

Videos were recorded and analyzed using Image J software.

In each experiment, the mean uorescence intensity was calculated from a collection of 2500!3000 images. A region of
interest (ROI) was selected along the channel, and the stackaveraged uorescence intensity was plotted as a function of the
width of the channel.
Lateral shifts in normalized uorescence intensity were
calculated as follows.
Two-Inlet Microfluidic Device. The central pixel was selected
inside the ROI, and its normalized fluorescence intensity was
measured in the absence of substrate. Then, in the presence of
Mg2 cofactor ions, the ROI was investigated again to find a
pixel with an equivalent fluorescence intensity to the one
previously measured. The lateral distance of this new pixel from
the center of the channel was taken to be the lateral shift. The
central pixel was chosen to calculate the lateral shifts in our
measurements because the DNA polymerase molecules will
experience the maximum gradient in cofactor concentration at
this point.
Three-Inlet Microfluidic Device. A pixel was selected inside
the ROI, which was midpoint between the DNA polymerase flow
region and the substrate nucleotide flow region, and its normalized fluorescence intensity was measured in the absence of
substrate. Then, in the presence of 10 mM dATP substrate, the
ROI was investigated again to find a pixel with an equivalent
fluorescence intensity to the one previously measured. The
lateral distance of this new pixel from the one measured in
the absence of substrate was taken to be the lateral shift. The
pixel midpoint between the polymerase flow region and the
nucleotide flow region was chosen to calculate the lateral shifts
in our measurements because the DNA polymerase molecules
will experience the maximum gradient in nucleotide concentration at this point.
Micropump Design, DNA Polymerase Immobilization, and Particle
Tracking. Using an e-beam evaporator, Au was patterned on a
PEG-coated glass surface (MicroSurfaces). The surface was
cleaned thoroughly with isopropyl alcohol followed by acetone
and dried under a nitrogen stream. Previously synthesized quaternary ammonium thiol79 was used for self-assembled monolayer formation on the Au surface. The ligand was dissolved in a
methanol/water mixture, and the surface was incubated in it
overnight at room temperature under an inert atmosphere.
Later, the surface was washed several times with a methanol
mixture and dried under an inert atmosphere. The SAMmodified surface was incubated with a DNA solution for 4!5 h.
The negatively charged backbone of the DNA molecules bound
selectively to the Au-patterned surface via electrostatic interactions. The DNA polymerase forms a complex with the DNA
molecules bound to the Au surface.
To monitor the uid ow, functionalized polystyrene microspheres (2 m) were introduced as tracers in our experiments.
Videos were captured using an optical setup, which comprised
a microscope (Olympus BX60M) with a halogen lamp (100 W)
and a 50# objective lens (LMPlanFLN 50#/0.5 BD /0/FN26.5,
Olympus). Videos were recorded using a CCD camera attached
to the optical microscope. For measuring uid pumping speed,
30 particles were tracked using PhysVis software.

Preparation of Fluorescent-Tagged DNA Sample. Fluorescentlabeled DNA primer oligomer (MW = 4402.2, TM = 53.8 !C,
100 nmol, HPLC purified, 50 -/5Cy3/TCG CAG CCG TCC A-30 )
and DNA template oligomer (MW = 6,127, TM = 62.2 !C, 25 nmol,
purified by standard desalting technique, 50 -AAA CCC TTG GAC
GGC TGC GA-30 ) were purchased from Integrated DNA Technologies. Using a UV!vis spectrophotometer, the concentrations
of stock solutions of primer oligomer ( = 122 100 M!1 cm!1)
and template oligomer ( = 190 700 M!1 cm!1) were measured
as 56.92 and 91.61 M, respectively. The absorbance was
recorded at 260 nm at a path length of 1 cm. From the stock
solutions, 10 M solutions of both primer and template oligomers were prepared in 10 mM Tris, pH 8.0 buffer. The mixture
was incubated at 65 !C in a water bath for 3 min and allowed to
cool slowly to room temperature. The resulting fluorescenttagged DNA sample was stored in aliquots of 50 L at !20 !C.
Wild-type T4 DNA polymerase was purified as previously described77 and stored at !80 !C. Both the DNA and DNA polymerase samples were thawed on ice before experiments were
performed.
Single-Molecule Diffusion Measurements with Fluorescence Correlation
Spectroscopy (FCS). See Supporting Information for detailed FCS
measurement conditions.
Microfluidic Device Fabrication. The microfluidic device was cast
in polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning)
using standard soft lithography protocols.78 A 100 m deep
master pattern, which contains the inverse-shaped microfluidic
device, was created on a silicon wafer (Silicon Quest) by spin
coating a thin layer of positive photoresist, SPR-220 resist
(Microposit). The wafer containing the photoresist was treated
and structured by using photolithography methods. This was
followed by deep reactive ion etching (DRIE, Alcatel Speeder
100 deep silicon reactive ion etch) to generate the master
stamp. The master silicon wafer was exposed to 1H,1H,2H,2Hperfluorooctyltrichlorosilane (Sigma Aldrich) in a vacuum desiccator to minimize adhesion of PDMS to the wafer during the
peeling step. The PDMS was prepared by thoroughly mixing the
PDMS base polymer and the curing agent in a 10:1 (weight/
weight) ratio. The mixture was then degassed in a desiccator for
about an hour and baked at 60 !C in an oven for 30 min. The
PDMS was then cooled and peeled off. Pinholes acting as inlets
and outlets were drilled open with a drill press (Dremel Model
220 WorkStation), a rotary tool (Dremel 300 Series), and drill bits
(Drill Bit City). To ensure permanent bonding between the
PDMS and the glass substrate, the device was sealed to a No.
1 glass coverslip (VWR) with a high-frequency generator (model
BD-10AS, Electro-Technic Products, Inc.). Polyethylene tubing
(Becton Dickinson and Company) was inserted into the inlets
and outlet of the microfluidic device. Fluid flow through the
microchannel was generated by syringe pumps (KDS 200 and
220, KD Scientific).
Fluorescence Imaging Setup. Collective migration of DNA polymerase complex molecules in the presence of a dATP concentration gradient was characterized using fluorescence imaging.
The optical setup comprised an inverted microscope (Zeiss
Axiovert 200 MAT) with a halogen lamp (12 V max, 100 W).
Excitation light was passed through the appropriate filter cube
(Zeiss) depending on the excitation/emission wavelengths of the
fluorescent tag. It was then focused on to the sample through
a 20# objective (EC Epiplan-NEOFLUAR 20#/0.5 HD DIC /0,
Zeiss). Fluorescence emission was collected by the objective,
passed through interference filters, and finally detected by a
high-sensitivity Flea 3 CCD camera (FL3-U3-32S2C-CS, Point Grey)
with a resolution of 2080 # 1552 pixels at 60 frames per second.

SENGUPTA ET AL.

ARTICLE

micropumps that will have a wide range of applications

from nano- and microscale sensing devices to cargo
delivery vehicles. It has also not escaped our notice
that the mechanisms of directed motion, enhanced

Conflict of Interest: The authors declare no competing

nancial interest.
Acknowledgment. We gratefully acknowledge nancial
support by the Penn State MRSEC under NSF Grant DMR0820404 and, in part, by the Defense Threat Reduction Agency
(HDTRA1-13-1-0039). This publication was also supported by
the Pennsylvania State University Materials Research Institute

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Supporting Information Available: Single-molecule diusion

measurements with FCS, viscosity measurements, generation of
nucleotide and cofactor concentration gradients, calculations
for bulk temperature variations, synthesis of quaternary ammonium thiol ligand, supporting gures and supporting videos
are provided. This material is available free of charge via the
Internet at http://pubs.acs.org.

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