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DLD pillar shape design for efficient separation of

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spherical and non-spherical bioparticles†

Cite this: Lab Chip, 2014, 14, 4250
Shashi Ranjan,‡a Kerwin Kwek Zeming,‡a Roland Jureen,b Dale Fishercd
and Yong Zhang*aef

Received 16th May 2014,
Accepted 26th August 2014
DOI: 10.1039/c4lc00578c
www.rsc.org/loc
Particle sorting methods in microfluidic platforms are gaining momentum for various biomedical
applications. Bioparticles are found in different shapes and sizes. However, conventional separation
techniques are mainly designed for separation of spherical particles. Thus, there is a need to develop new
methods for effective separation of spherical and non-spherical bioparticles for various applications. Deter-
ministic lateral displacement (DLD) microfluidic methods have become popular for high separation resolu-
tion, simplicity, and predictability. However, shape sorting in the DLD separation methods is not well
researched. Recently, we explored this area and found that pillar shapes in DLD significantly affect bio-
particle separation. In this work, we designed a group of different pillar shapes with protrusions and groove
structures with the hypothesis that pillar protrusions will induce particle rotation while pillar grooves will
confine the particle rotational movement in a directed path for effective separation in a DLD pillar array.
Using combinations of protrusions and grooves, 3-dimensional spherical particles, 2-dimensional planar
disc-shaped red blood cells and 1-dimensional rod-shaped bacteria were separated and two interesting
phenomena were observed. Firstly, the arrangement of pillar protrusions and grooves induces inertial
movements, enhancing the separation of spherical particles. Secondly, non-spherical particles experience
dominant rotational movements due to the protrusions and grooves which help in changing their orienta-
tions. This gives an opportunity to perform efficient separation based on the desired orientation (the
longest dimension of the particles) by restricting or containing their movement within a specific DLD path.

Introduction motions and random orientations in a fluid medium. Conven-

Rapid separation of bioparticles such as bacteria from patho-
logical samples can facilitate faster disease detection, thus
eliminating the reliance on laborious and time-consuming
techniques like culture-based methods.1,2 Bioparticles exist in
a myriad of different shapes and sizes. Unlike spherical parti-
cles, non-spherical bioparticles have complex 3-dimensional
a
Department of Biomedical Engineering, Faculty of Engineering, National
University of Singapore, 9 Engineering Drive 1, Singapore 117576.
E-mail: biezy@nus.edu.sg; Tel: +65 65164871
b
Department of Laboratory Medicine, National University Hospital, 5 Lower Kent
Ridge Road, Singapore 119074
c
Department of Medicine, National University Health System, 1E Kent Ridge
Road, Singapore 119228
d
Department of Medicine, Yong Loo Lin School of Medicine, National University
of Singapore, 5 Kent Ridge Road, Singapore 119074
e
NanoCore, National University of Singapore, 5 Engineering Drive 1,
Singapore 117576
f
NUS Graduate School of Integrative Sciences and Engineering, National
University of Singapore, 28 Medical Drive, Singapore 117456
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4lc00578c
‡ These authors contributed equally to this work.
tional particle separation techniques such as centrifugal and
size-exclusion technology are based on a spherical particle
model and would not effectively separate these biological
components based on their shape.3–6 The separation of bacte-
ria based on shape may provide early clues to their identifica-
tion; circulating tumor cells are found to have varying
deformability resulting in a different degree of sphericity, and
hence, their separation based on sphericity may aid in the
prognosis of cancer.7 Red blood cells (RBCs) are uniquely
disc-shaped and effective separation based on their shape
may increase the efficiency of blood component analysis in
clinical pre-diagnosis processes. Briefly, separation of biologi-
cal components based on shape and size would greatly
enhance medical pre-diagnostics, therapeutics and analysis of
the biological entities. Hence, there is a strong practical need
for investigating shape-sensitive particle separation techniques.
Shape-based sorting for bioparticles has recently gained
traction in microfluidic devices, which are platform technolo-
gies for point-of-care medical devices. Pinch flow fraction-
ation (PFF) methods were explored to separate a range of
non-spherical particles including synthetic particles and
bioparticles such as RBCs.8–10 Kersaudy-Kerhoas et al. studied

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the movements of RBCs within the hydrodynamic fluidic device
and achieved maximum separation efficiency of 60.8% for
human RBCs.11 While working to separate liver cells using
hydrodynamic filtration, Yamada and his team noticed that
non-spherical shape caused by aggregated cell attachment
would affect the separation efficiency of the cells. 12 This
observation of non-spherical particle separation was further
investigated by Sugaya et al., and they showed how fluidic
forces can play a role in the separation of spherical and
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non-spherical particles via flipping motion induction.13
Masaeli et al. studied the orientation and motion of synthetic
spherical, elliptical and rod-shaped particles of approximately
30 μm and tested their separation in PFF separation
techniques.14 The particle shape changes due to deformability
have been utilized for separation. The separation via
deformability of soft body was performed by separation of
malaria-infected blood cells, bacteria-infected blood and
blood component separation.15–17 While various groups have
performed shape-based separation, most of these methods
are limited by separation resolution and restrictions on
channel dimensions required for effective separation of
particles. It is also very challenging to enable distinct separa-
tion of these non-spherical particles within the microfluidic
devices as there are particle separation spectrum overlaps.13
A precise and distinct particle separation method with
high separation resolution is deterministic lateral displace-
ment (DLD).18,19 Briefly, DLD consists of a pillar gradient
array which has a specific critical size for particle separation
with respective pillar spacing and array gradient. Particles
larger than the critical size are bumped off their flow path
and displaced laterally from their original fluid stream to
follow the pillar gradient, while smaller particles will
continue the fluid path unaffected by the DLD pillar array.
Holm and Beech et al. have performed RBC orientation-
based separation in a DLD device using conventional circular
pillars.20,21 They found that the RBC orientation is important
for the separation. If the RBC's orientation is flat to the sur-
face of the device, the separation critical diameter is 7 μm,
but if it leans to the side of the pillars, its critical separation
diameter is less than 3 μm. They effectively separated RBCs
by creating a channel of depth 4 μm to orientate the RBC flat
to the surface. The restriction on channel depths reduces the
throughput and is limited to disc-shaped bioparticles. For
rod-shaped or spiral-shaped bacteria, tuning the orientation
using similar methods would have been impractical as it
would result in a channel depth of less than 1 μm with
increased challenge to control the orientation of nearly
1-dimensional rod-shaped bacteria. Recently, we explored the
separation of disc-shaped RBCs in a DLD device by inducing
rotation or flipping of RBCs.22 The rotating RBC would have
a similar effect to an increase in hydrodynamic diameter,
while the principle of inducing flipping motions is similar to
the hydrodynamic filtration method developed by Sugaya
et al. except that the use of DLD techniques enables precision in
separation resolution and predictability of separation size as
well as increased control over the flipping and rotational
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motions of non-spherical bioparticles.13 The study also
suggested that the pillar protrusions and grooves in DLD
devices could be critical in controlling the orientation and
hence the separation of non-spherical particles.
In this work, new pillar shapes with varying protrusions
and grooves were used in DLD devices to study the separation
of particles with various spatial dimensions, namely,
3-dimensional microspheres, 2-dimensional disc-shaped
RBCs and 1-dimensional rod-shaped bacteria. We hypothe-
size that in a DLD microfluidic device, the protrusions of the
pillars perpendicular to the flow of the particles will act as an
extended arm for inducing particle rotation, while the
grooves within the pillars will contain the rotation. The com-
bination of both pillar protrusions and grooves brought
about two interesting phenomena. First, spherical particles
experience inertial effects which induce translational momen-
tum changes of the particle within the novel DLD pillar
shapes, resulting in enhancement of the separation. Second,
non-spherical particles experience dominant rotational
momentum changes which allow more effective separation
via controlled rotation motion. Such movements provide
opportunities to perform separation based on the desired ori-
entation (the one presenting the longest dimension). Thus,
new pillar designs are explored here which can take advan-
tage of changing the orientation to restrict or contain particle
rotational movements and direct its motion along the DLD
pillar gradient array. We qualitatively term it as “containment
effect”, which is the effect of restricting the movement of a
rotating particle using physical barriers and selectively direct
it in a particular determined path of motion. Such effect can
result in more selective and distinct particle separation. This
study on how novel pillar shape designs can affect particle
shape sorting provides additional dimension for the
increased selectivity and potential separation of bioparticles.
Compared to our previous study, where we showed that
increased rotation of particle can enhance the separation effi-
ciency of non-spherical particles, this paper introduces a new
mechanism to restrict the rotational momentum of the parti-
cle in order to contain the particle on the desired flow path
for effective separation. Thus, we studied how a pillar design
can help in increasing the containment of rotating/flipping
particles to allow efficient separation. Moreover, the study
also provides a novel insight into utilization of inertial trans-
lational movements for separation of spherical particles. We
believe that the results shown here will not only be restricted
to DLD methods but may also be applicable to the inertial
microfluidics such as PFF, hydrophoretic filtration and flu-
idic sheath flow techniques which may aim to separate non-
spherical particles by introducing particle rotation.
Experimental design
Pillar design
All pillars were designed to fit in a square (as shown in
Fig. 1a and e). Some portions are cut out of this square to
produce grooves and protrusions (Fig. 1a). The cut-out
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Fig. 1 Schematics of particle separation experiments: (a) different pillar shape designs with varying protrusions and grooves. Darker shading
indicates the pillar design, while lighter shade denotes the groove. The minimum protrusion length is indicated here by a double-headed arrow
located between the green and red dotted lines. (b and c) Pillars designed for studying proposed “containment effect”. (b) L-shaped pillars can
contain a particle due to obstruction caused by the vertical edge, while (c) the inverted L-shaped pillar would have minimum containment. The
overall device design can be seen in (d) where the sample inlet stream (shown in green) is sandwiched between two buffer streams. The device
channel length is 14 mm, while the width is 700 μm. The output channels will show how far the samples have separated from the input sample
stream. The sample undergoes a pre-filter process to remove larger unwanted debris. The device has pillars which are designed to fit within a
15 μm by 15 μm square region shown in (e). The deviation of pillars is 2.87°, while the gaps are fixed at 10 μm. The output channel is subdivided
into 26 channels for separation characterization.

portion leaves a gap in the design which is called groove in
this work. The portion protruding out from the maximum
depth of cut is considered as protrusion in this work. The
length of the portion measured from the maximum depth of
the cut is the length of the protrusion and the minimum
length of the protrusion is calculated for the smallest protru-
sion (Fig. 1a). As such, three DLD pillar parameters have
been selected for tests, namely, number of protrusions,
length of protrusions (minimum protrusion to length of
pillar ratio) and orientation. For simplicity of presentation,
we have not included change in groove as a parameter.
However, by changing the number of protrusions and the
length of protrusions, the shape of the groove is changed
(Fig. 1a). Thus, combinations of these three parameters intro-
duce a variety of pillar shapes to be tested, as shown in
Table 1. While the number of combinations and permuta-
tions of the parameters can be non-exhaustive, 7 pillar shape
designs were selected to test very specifically the workings
of protrusions and pillar orientation. The type of groove
has been fixed as a curved groove to allow smooth transition
between protrusion interactions. I-shaped pillars were
designed with two protrusions and a fully formed

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Table 1 Experimental parameters of the various pillar shapes to be tested. The table shows pillar shapes with varying DLD pillar parameters: number of
protrusions, length of protrusion and orientation
Type Pillar shape
(i)
I-shape
(ii)
Inverted anvil shape
(iii)
Anvil shape
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(iv)
Inverted T-shape
(v)
T-shape
(vi)
L-shape
(vii)
Inverted L-shape
semicircular groove between them (enclosed groove). Anvil-
shaped and inverted anvil-shaped pillars were designed by
reducing the length of one of the protrusion distorting the
semicircular groove as compared to the I-shaped design (par-
tially enclosed groove). Completely removing one of the pro-
trusions from the I-shaped design results in T-shaped and
inverted T-shaped pillars with arc-like grooves (open grooves).
L-shaped pillars were designed with a single protrusion-like
T-shape, while the vertical edge was shifted to one end
(Fig. 1a). It was designed to maximize the chances of physical
containment of particles (Fig. 1b). Inverted L-shaped pillars
were designed for minimum containment (Fig. 1c).
Device design
For performing the separation study for the different pillar
shape designs, microfluidic devices were designed for two
buffer streams sandwiching the sample stream. We employ a
negative pressure-driven process from the output of the
device. Fluids are loaded in the reservoirs located at input
regions. The gaps (horizontal and vertical) between pillars
were fixed to 10 μm and the gradient of the DLD array was
set at 1/20. Based on these parameters, the critical separation
diameter (Dc) for the DLD array is calculated and experimen-
tally tested to be around 3.32 μm.22,23 The output channels
were subdivided into 26 channels to enable greater accuracy
in characterization. The channel number represents the
amount of overall lateral displacement of particles caused by
Separation index 
 mean output location  mean input location   100
 total no. of channels used for the separation  mean innput location 
the pillar array. Subchannels 1 to 5 contain the input buffer
stream and subchannels 5 to 10 contain the input sample
stream (Fig. 1d). A simple size-exclusion filter is designed
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Minimum protrusion
Protrusions length-to-pillar length ratio Orientation
2 0.333 —
2 0.167 Inverted
2 0.167 Upright
1 0.333 Inverted
1 0.333 Upright
1 0.833 Upright
1 0.833 Inverted
into the sample input region to filter away bigger unknown
particles or dust.
For the bacterial separation study, a DLD microfluidic device
containing an I-shaped pillar array was fabricated (Fig. S1†).
A pillar array was designed with 4 μm of gap size (horizontal
and vertical), 1.6° of pillar shift gradient and 6 μm of pillar
length. The array was flanged by 50 subchannels for inlets
and outlets numbered from 1 to 50 from left to right, which
were designed for easy visualization and quantification of the
bacterial separation. Input sample stream was located
between channels 5 and 10, whereas DI water flowed in the
other subchannels. DI-water flow between channels 10 and
54 was designed for the collection of separated bacteria,
while the flow between channels 1 and 5 acted as sheath flow
to prevent their flow near the edge of the device where DLD
effect may not be observed.
Results
Pillar shape design for the separation of spherical particles
In this study, the separation of spherical microspheres was
studied for different pillar shapes as designed here to find
out the separation effectiveness of each design and to study
the underlying separation mechanism. To quantitatively
denote the separation effectiveness, we have introduced an
index called ‘separation index’ for comparison of separation
results among different pillar designs. It is defined as
follows:
This index is used as a standard for general comparison
of efficiency and separation quality between various devices.
The strength and quality of separation is expressed as the
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magnitude of the index, while the resolution is denoted as

the standard deviation. The advantage of using an index is to
have a standard method of comparison between various
devices across all DLD experiments having specific critical
diameters regardless of the number of output channels or
widths of lateral displacement. Hereafter, we would discuss
the result in terms of separation index (I S) and would con-
sider an IS value of 50 or more as distinct or clear separation.
Traditionally, a term called critical diameter (D c) is used to
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indicate the spherical size of a particle that can be separated

or deviated using a particular DLD array. However, it does
not indicate the amount of deviation in the array. In this
study, the Dc of a DLD array would be defined for an IS value
of at least 50.
The DLD parameters such as gap between pillars and
angle of gradient were chosen here based on a theoretical Dc
value of 3.32 μm (calculated from the current DLD formula
as published for the conventional circular pillar array,24
suggesting that microspheres of size less than 3.32 μm would
not be separated in a conventional circular pillar array).
Fig. 2 shows a comparison graph for the displacement of
3.0 μm (smaller than the theoretical D c) microspheres with
an array of different pillar shape designs including conven-
tional circular pillar design. There are observable differences
in the displacements of microspheres from the initial sample
flow for different pillar shapes. This indicates that the shape
of the pillars affects the separation of spherical particles.
Also, it can be noted that 3.0 μm beads would not be sepa-
rated with a conventional circular pillar array of the same
DLD parameters (I S = 17.4), but all the pillar shapes tested
here have shown some degree of separation, i.e. I S greater
than that of the circular design.22,24 Moreover, three of the
pillar designs have achieved distinct separation with an I S
value greater than 50. This result is interesting and points
towards some other effects that have been neglected in the
conventional DLD models.
The current DLD model is based on the fluid flow profile
and accounts for effects of the viscous (fluidic) forces
neglecting any inertial effects.24 Based on this model, the
separation of spherical particles due to different pillar shapes
should not be different and it should be similar to theoretical
values (calculated for the conventional circular pillar array)
as the flow profile at any point between protrusions would be Fig. 2 Output separation of spherical beads for various pillar shapes.
The figure depicts the output distribution of 3.0 μm beads in various
similar to the parabolic flow profile of circular pillars (except
pillar shape designs. The output distribution spread between channels
for L-shaped pillars).18,24,25 However, the results do not 5 and 26 with a corresponding separation index ranging from 0 to 100.
match with this assumption. This suggests that other factors The values above the error bars are the mean output channel, and
such as the role of inertial effects may arise due to fluid flow error bars indicate the standard deviation from the mean. The total
through grooved structures of the pillar design. This proposi- number of beads counted per set of data range from 600 to 1000.
#
The data from circular pillars were extracted from previous studies
tion is based on the similarities of I-shaped pillar protrusion
and correlated using the separation index.22 Conversion of the
and groove structure with the orifice channel device investi- separation data to IS can be found in Fig. S2.†
gated by Park et al.26 The literature explains that a flowing
particle experiences certain types of inertial forces such as
wall-effect lift force,27,28 shear-gradient lift14,29 and dean-drag momentum changes (due to changes in velocity) and may
forces30–32 due to the flow in curved geometry. It has been generate inertial force on the particle.29 Such inertial force
suggested that with sudden expansion or contraction of fluid may allow a particle to escape the fluid stream, which may
streams, the particle may experience sudden translational cause transverse migration of particles away from their

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original flow stream, resulting in a lateral displacement.
Small minute displacements may accumulate over a large
number of DLD pillars (~1500). Recently, different micro-
fluidic devices have been developed for separation of parti-
cles based on inertial effects. 26,27,33 The I-shaped design
should show maximum inertial effects due to fully formed
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protrusions and grooved structures, which also correlate with
the separation result (I S value of 91.6). Microspheres mostly
follow the pillar gradient (separation path) for an I-shaped
pillar array (Fig. 3a and Movie S1†). As the grooved structures
are compromised, the effectiveness of separation also
decreases, as seen for anvil- and inverted anvil-shaped pillars

Fig. 3 The sequential positions of 3.0 μm beads in various pillar arrays and the changes in fluid motion. The beads in (a) I-shape, (b) inverted anvil
shape and (c) anvil shape have shown separating displacement motion, while (d) inverted T-shape, (e) T-shape, (f) L-shape and (g) inverted L-shape
show the tracked bead slipping past the pillar. The flow mechanisms are depicted in the schematics with the bold arrow showing strong fluid
motion and the narrow red arrow showing weaker fluid motion. Scale bars in all the figures show a length of 10 μm.

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(I S values of 67.7 and 64.1, respectively). It is further

decreased with the removal of grooved structures in T-shaped
and inverted T-shaped pillars (I S values of 37.2 and 51.3,
respectively). Fig. 3 shows the movements of spherical
particles in different pillar designs. Some of the micro-
spheres are laterally displaced for anvil- and inverted anvil-
shaped pillars (Fig. 3b and c), while most of the beads slip
away for T-shaped and inverted T-shaped pillars, resulting in
less effective separation (Fig. 3d and e). Moreover, it can be
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noted that changing the orientation of the pillars does not

affect the separation results. The separation results seem
to correlate with the number of protrusions, which is a key
parameter for inertia effects and influences particle separa-
tion through the generation of rapidly changing flow patterns
during the expansion and contraction phase cycle. Pillars
with uneven expansion and contraction phases result in less
effective separation which can be explained with unbalanced
inertia forces.
The L-shaped pillars were designed for better containment
of particles which may allow better DLD separation. However,
the result for DLD separation using L-shaped pillars does not
show any improvement over other pillar designs (Fig. 3f).
Moreover, the DLD separation result for L-shaped pillars is
not significantly different from the inverted L-shaped design
which is supposed to have weaker particle containment
(Fig. 2 and 3g). This indicates that separation of spherical
particles is possibly dependent on the number and length of
protrusions which induce translational momentum changes
of spherical particles during the expansion and contraction
flow phases. Thus, it further supports the role of the inertial
effects as the suitable explanation for the obtained results.

Pillar shape design for the separation of

non-spherical particles
Most of the bioparticles are non-spherical in shape, and thus,
it is important to develop new methods for their separation.
Such methods should take into account the shape of particles
as the effective separation size of non-spherical particles
would depend on their shape. For example, the criteria
for complete separation of RBCs (width ~2 μm and length
~8 μm) are usually performed based on their width, whereas
the separation would be more effective if it is performed
based on their diameter. To take into account the shape of a
particle, it is important to allow separation based on the cor- Fig. 4 Output separation of red blood cells for various pillar shapes.
rect orientation of a particle to allow separation based on its The figure depicts the output distribution of RBCs for various pillars.
The output distribution spread between channels 5 and 26 with a
longest dimension.
corresponding separation index ranging from 0 to 100. The values
To achieve the abovementioned goal, our idea is to induce above the error bars are the mean output channel and error bars
rotation/flipping of particles in the desired direction so that indicate the standard deviation from the mean. The total number of
different orientations of particles are presented. Rotation/flip- cells counted per set of data ranges from 600 to 1000. #The data from
ping of particles increases their effective separation size.22 It circular pillars were extracted from previous studies and correlated
using the separation index.22
would also be important to contain the correctly oriented par-
ticles in the direction of the array gradient to achieve effi-
cient DLD separation. Based on this idea, separation of RBCs
(as the model for non-spherical bioparticles) was studied showed that the I-shaped and the L-shaped pillars are the
using different pillar shapes (Fig. 4). The separation results most effective design for the separation of RBCs compared to

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all other designs (IS values of 95.3 and 92.3, respectively). The
movements of RBCs were observed in the high-speed movie
obtained for all pillar designs. Although all these designs
were able to induce flipping of RBCs due to protrusions and
grooves, separation results were not the same for all of them.
This variation in the result can be attributed to the contain-
ment effect, i.e. the ability of a pillar shape design to contain
or restrict movement of a particle in the direction of the pillar
gradient array. Interestingly, the L-shaped pillars produce two
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contrasting results for non-spherical particle separation. The
design with better containment effect should be the most
effective in the separation, as seen here for the L-shaped
design, while the inverted L-shaped design with the worst
containment effect is the least effective (IS value of 20.9).
The I-shaped pillar design was developed to induce rota-
tion of non-spherical particles, which increases dynamic
radius, whereas the L-shaped pillar was designed for the
physical containment of rotating particles (Fig. 1a). To fur-
ther verify the containment effect, the movements of RBCs
for three types of pillar designs, namely, I-shaped, L-shaped
and inverted L-shaped, were studied from high-speed movies
(Movies S2–S4†). Various sequential images from the movie
were overlaid to obtain a picture to depict the movement of a
RBC for all three types of pillar arrays (Fig. 5a–c). It can be
noted that as a RBC reaches the protrusion of the pillar it
flips and moves along the pillar gradient (Fig. 5a and Movie
S2†). If it does not flip here, it may slip at this point. The flip-
ping of RBCs is caused by the mechanical and fluidic torque
created by the protrusion and the grooved structure of the
I-shaped pillar design. The rotation/flipping of RBCs prevent
them from slipping at a pillar and keep them moving in the
direction of the pillar gradient array, enhancing their DLD
separation. For other pillar shape designs, RBCs do not flip
as frequently compared to the I-shaped pillar design and
slip at a pillar compromising the separation. Even for the
Fig. 5 RBC movement tracking in overlay video frames. All three images consist of a sequential overlay of frames to track the movement of a RBC
in (a) I-shape, (b) L-shape and (c) inverted L-shape. The COMSOL computational fluid streamlines are subsequently superimposed over the image.
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L-shaped pillar design, RBCs just perform a single flip due to
lack of a second perpendicular protrusion and complete
groove (unlike the I-shaped pillar). The flipping motion of
RBCs provides enough rotational momentum for them to
cross the distance from the end of a pillar to other adjacent
pillars in the same column, and as they reach the second pil-
lar, they are contained by the vertical edge of the L-shaped
pillar in the correct orientation (Fig. 5b and Movie S3†).
Although the mechanism of containment is different for the
I-shaped pillars and the L-shaped pillars, the overall effect is
the same. It shows that containment of particles is an impor-
tant factor besides their rotation/flipping. This point is fur-
ther verified by studying the movements of RBCs through an
inverted L-shaped pillar array. In contrast to the L-shaped pil-
lar, the inverted L-shaped design does not contain the flipped
RBCs, which results in their slipping (Fig. 5c and Movie S4†).
Although both of the designs induce flipping of RBCs in a
similar way, the results are strikingly different due to the dif-
ference in their ability to contain a flipping particle. This sug-
gests that the rotation/flipping should be accompanied by
the containment of the flipped particles for achieving the
effective separation of non-spherical particles in microfluidic
devices.
The role of inertial effects can also be argued at this junc-
ture. In our previous study, we have shown that the separa-
tion of RBCs is due to their rotation and not just due to
reduction in D c.22 We believe, although inertial effects may
contribute to the separation, that the rotational and contain-
ment effects would be much larger in the case of non-
spherical particles due to a large difference in their sizes
based on their orientation. It can also be verified from the
results here. L-shaped pillars do not show good separation
for spherical particles compared to the I-shaped pillar design
but perform equally well for the separation of non-spherical
particles.
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Bacterial separation study
It has become clear from the above study that the I-shaped
pillar design is the best design among all designs studied
here for the separation of spherical as well as non-spherical
particles. Thus, this design was chosen for further study on
complete separation of bacteria which are found in different
shapes. This study is important as movements of non-
spherical (rod-shaped) bacteria would be more complicated
compared to RBCs, and the results obtained for RBCs may
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Separation of the bacteria was studied using the bacterial
separation device mentioned under the device design section.
Fig. 6 shows the full view of the I-shaped pillar array (channel
numbers are indicated in this figure) with merged fluores-
cence image of flowing bacteria (E. coli in this figure). It can
be seen that bacteria enter in the sample stream between
channels 6 and 12 (Fig. 6a), deterministically displaced
towards channel 50 (Fig. 6b) and are concentrated in channel
50 (Fig. 6c). Fig. 6b clearly shows the displacement path of

not be extrapolated for bacteria. Rod-shaped bacteria are
nearly one-dimensional and may take various orientations or
may move differently into a three-dimensional space. This
study is also important as bacterial separation is a more chal-
lenging and clinically relevant problem.
Separation of different types of bacteria
The aim of this work is to study the separation of different
shapes of bacteria. For this purpose, four bacterial species
were chosen: E. coli (rod-shaped or bacillus, width ~0.5 μm
and length ~2 μm), S. epidermidis (spherical or coccus, size
~0.7 μm), K. pneumoniae (rod-shaped, width ~0.6 μm and
length ~1.8 μm) and P. aeruginosa (rod-like or coccobacillus,
width ~0.6 μm and length ~1.6 μm). Here, two rod-shaped
bacteria (with some differences in morphology), spherical
bacteria and rod-like bacteria (lie between rod shape and
spherical shape) are chosen to represent various types of
bacteria. Moreover, these are clinically relevant bacteria.
the bacteria through the I-shaped pillar array in a snapshot,
which is different from the conventional circular design as
shown in Fig. 6d.
For quantitative analysis, the percentage of bacteria that
passed through each input channel and each output channel
was calculated for all types of bacteria using the same device
(Fig. 7). 100% of bacteria are deviated to channel 1 for rod-
shaped and rod-like bacteria (Fig. 7a–c). Moreover, it helps
in the concentration of bacteria as they can be located in a
single stream (Fig. 6c and 7a–c). Clear separation of spherical
bacteria was also achieved (Fig. 7d).
Movement comparison study for different particles in
I-shaped pillar design
After establishing I-shaped pillar design as the most effective
design for bioparticle separation, the movements of different
types of particles, 3-dimensional microspheres, 2-dimensional
disc-shaped RBCs and 1-dimensional rod-shaped bacteria,

Fig. 6 Bacterial separation study. Optical and fluorescence images show the separation of bacteria (green fluorescent E. coli) from the input
stream. Bacterial sample is flowed in the device containing an I-shaped pillar array (a). Green fluorescence indicates bacteria in the sample.
The bacteria in the sample are laterally displaced in the device using an I-shaped pillar array to achieve their separation from the sample (b). Once
deviated away from the original sample stream, bacteria are concentrated in a single channel (c). The deviation path as obtained for circular pillar
array is shown in (d), which shows that the majority of bacteria is not deviated in this array.

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Fig. 7 Separation of different species of bacteria. Fluorescence images of different types of bacteria: (a) E. coli, (b) K. pneumoniae,
(c) P. aeruginosa and (d) S. epidermidis. The plot shows the percentage distribution of different species of bacteria in different input and output
channels indicating their separation from input stream to output stream.
were studied to get better insight into the separation mecha-
nism. These movements were studied from high-speed
movies (Movies S1, S2 and S5†). The movements of the
microspheres show simple translational movements through
the pillar gradient array (Fig. 8a). RBCs move in continuous
rotational motion, hitting pillars. The rotation allows
accounting for the shape of RBCs as the projected size of
RBCs perpendicular to fluid flow, which represents the effec-
tive separation size, is based on their diameter (Fig. 8b). The
rod-shaped bacteria represent a complex system for DLD sep-
aration due to a large number of orientations they can take
in fluid medium. Similar flipping is observed for bacteria as
observed for RBCs when they hit an I-shaped pillar. Such flip-
ping mostly helps in changing the orientation of bacteria to
allow them to flow horizontally (Fig. 8c). Also, bacteria do not
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hit pillars frequently while moving through the I-shaped pil-
lar array. Although the pillar design is the same, the move-
ments of the bacteria are slightly different from that of RBCs
due to the difference in their shape. RBCs are disc shaped
and have a large circular face which is exposed to fluid when
there is any change in their orientation. For this reason, flu-
idic forces acting on RBCs would increase substantially due
to the large surface area exposed in the direction perpendicu-
lar to the direction of fluid flow. Thus, viscous drag force
from fluid causes the expected rotational movements of
RBCs. On the other hand, the open faces of one-dimensional
rod-shaped bacteria are small, which may not be enough to
frequently cause the rotation of the bacteria. Interestingly,
the bacteria continue to move horizontally, with their length
perpendicular to the direction of fluid flow, thus increasing
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Fig. 8 Movement of different particles in the I-shaped pillar array: movement of (a) 3D microsphere, (b) 2D disc-shaped RBCs and (c) 1D
rod-shaped bacteria through the I-shaped pillar array.
their effective separation size and allowing the separation
based on their length (longest dimension). The basis of such
phenomenon is currently unknown; however, a good guess
could be the interaction between the surface of the E. coli
and the DLD pillar. Nevertheless, its length-based separation
is relatively more effective as compared to that of the disc-
shaped RBCs.
Discussion
DLD microfluidic techniques have gained popularity recently.
Conventionally, the role of inertial effects has been neglected
in the current DLD models as in other microfluidic analysis
for the flow at low Reynold's number. However, inertia effects
in DLD microfluidics have been reported to have a possible
role in separation of particles.34 Such forces may be small
but may produce a measurable effect in a long fluidic run as
in the case of DLD devices. Thus, novel pillar shapes were
designed in this study to enhance the separation of spherical
particles based on inertial effects. The I-shaped pillar design
showed the most effective separation of spherical particles
with a clear decrease in Dc as compared to conventional cir-
cular pillar arrays with the same DLD parameters (a maxi-
mum of about 50% reduction in the Dc was noted due to the
I-shaped pillar design; microspheres of about 0.5 μm size
were separated using this I-shaped array with a theoretical Dc
value of 1.0 μm as calculated by assuming a circular pillar
array, Fig. S3†). Such differences should not occur if the
movements of particles are dominated by fluidic (viscous)
effects only. Thus, it indicates that DLD separation may be
affected by the inertial effects which may reduce the Dc of a
DLD array. An earlier attempt to reduce the Dc was based on
the fluidic forces where an asymmetric fluid profile was
4260 | Lab Chip, 2014, 14, 4250–4262
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created using a triangular pillar in such a way that the width
of the flow stream was reduced at the tip of the triangle.25
This helped in the reduction of Dc as compared to the circu-
lar pillar array. Compared to that work, the flow profile was
kept symmetrical similar to the circular pillar, yet reduction
in D c was observed in this study. This further supports the
role of inertial effects in the DLD separation. This study high-
lights that different pillar shapes can be designed to enhance
the effectiveness of DLD separation based on inertial effects.
Unlike spherical particles, the orientation of non-spherical
particles is an important factor for determining their effective
separation size. To achieve separation based on the longest
dimension of a non-spherical particle, it would be important
to precisely control the particle's orientation. In this work,
different pillar shape designs are studied based on these two
aspects. The pillar shape can induce rotation/flipping for pre-
sentation of different orientations of a particle. The separa-
tion is based on the correct orientation by containing or
forcing the movement of the particle in the direction of the
gradient array. It is found that containment is an important
aspect of DLD separation besides particle rotation/flipping.
All 7 designs of pillar shape could induce flipping of RBCs.
Such flipping can help in changing the stream as a diametric
face (longest dimension) of a RBC opens up. Some degree of
separation was achieved for all the pillar shape designs,
which are better compared to the conventional cylindrical pil-
lar design (Dc of less than 3.0 μm). However, the best separa-
tion was noted for I-shaped and L-shaped designs which
contained RBCs better than other designs due to rotational
containment and physical containment, respectively. This
point was verified by comparing the separation of RBCs
by L-shaped pillars with that of inverted L-shaped pillars.
Flipping of RBCs was almost similar in both designs. Also,
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Lab on a Chip
computational analysis showed that the fluid forces acting
on RBCs were directed in a similar direction (Fig. S4†), but
the separation results were strikingly different with one
having the best separation and the other having the worst
separation. This supports the theory that the ‘containment
effect’ is crucial for DLD pillar design and opens a new
dimension for designing DLD microfluidic devices for the
separation of non-spherical particles.
Among the pillar shape designs studied here, the I-shaped
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design is the best for the separation of spherical as well as
non-spherical particles. In the meantime, it should also be
noted that the L-shaped pillar design is equally as good as
the I-shaped design for the separation of non-spherical parti-
cles, but it is not an effective design for the separation of
spherical particles. These points are important for designing
microfluidic devices for particle separation as they would
help in the selection of the pillar design for a particular pur-
pose. For example, the L-shaped pillar design may be better
compared to the I-shaped pillar design as it enhances the
separation of non-spherical particles but not spherical parti-
cles. The inverted L-shape is the worst in containing the rota-
tion of RBCs; however, relatively it is better for spherical
particle separation from a sample of soft deformable RBCs.
Materials and methods
Detailed experimental methods have been provided in
section 2 of the ESI.† Briefly, the layout of the device design
was drawn using AutoCAD and was printed as a chrome-
quartz photomask. Devices were fabricated by standard
lithography and a deep reactive ion etching process. PDMS
covers containing inlets and outlets were used for sealing
devices. Pre-treatment of devices was performed using a sur-
face coating of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-
trichlorosilane (Sigma, Singapore) and a pre-flow treatment
of pluronic F-127 solution (Sigma, Singapore) to avoid attach-
ment of bioparticles to the device surface. Bacterial adhesion
to the device surface was one of the roadblocks that was
solved by pluronic treatment on the silane-treated devices
(section 3 of the ESI†). Samples and buffers were introduced
into the device using a negative pressure-induced syringe
pump. The output data and the movement of the particles
were captured using a high-speed camera. Counting of bio-
particles in each subchannel was manually performed using
a high-speed video.
Conclusions
Different novel pillar shape designs have been presented
for studying the separation of bioparticles, spherical and
non-spherical, based on their shape. These pillar designs
contained protrusions and groove structures to enhance
the DLD separation of bioparticles with different shapes.
Three types of particles, 3-dimensional microspheres,
2-dimensional disc-shaped RBCs and 1-dimensional rod-
shaped bacteria, were used as model particles for this study.
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We found two interesting DLD phenomena that have not
been studied before: first, the role of inertial effects in the
DLD microfluidic devices for the enhanced separation of
spherical particles and, second, containment effect, i.e. sepa-
ration based on containing or restricting the particle motion
in a particular direction, for the enhanced separation of non-
spherical particles. The I-shaped design was the most effec-
tive design among all other designs studied here for the sepa-
ration of spherical and non-spherical particles. Additionally,
the L-shaped pillar design was equally effective as the
I-shaped pillars in segregating RBCs. Highly efficient separa-
tion (100%) of rod-shaped bacteria was noted, while clear
separation of spherical bacteria was also achieved using the
I-shaped pillar design.
Acknowledgements
We acknowledge the funding support from Singapore Minis-
try of Education AcRF tier 1 funding (R-397-000-183-112). We
also acknowledge infrastructural support from the National
University of Singapore.
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