micromachines

Article
On-Chip Tunable Cell Rotation Using Acoustically
Oscillating Asymmetrical Microstructures
Lin Feng 1,2,† , Bin Song 1,† , Deyuan Zhang 1,2 , Yonggang Jiang 1, * and Fumihito Arai 2,3
1 School of Mechanical Engineering & Automation, Beihang University, Beijing 100191, China;
linfeng@buaa.edu.cn (L.F.); songb@buaa.edu.cn (B.S.); zhangdy@buaa.edu.cn (D.Z.)
2 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing 100083, China;
arai@mech.nagoya-u.ac.jp
3 Department of Micro-Nano Mechanical Science & Engineering, Nagoya University, Nagoya 464-0814, Japan
* Correspondence: jiangyg@buaa.edu.cn; Tel.: +86-10-8231-6603
† These authors contributed equally to this work.




Received: 26 September 2018; Accepted: 8 November 2018; Published: 14 November 2018


Abstract: The precise rotational manipulation of cells and other micrometer-sized biological samples
is critical to many applications in biology, medicine, and agriculture. We describe an acoustic-based,
on-chip manipulation method that can achieve tunable cell rotation. In an acoustic field formed by the
vibration of a piezoelectric transducer, acoustic streaming was generated using a specially designed,
oscillating asymmetrical sidewall shape. We also studied the nature of acoustic streaming generation
by numerical simulations, and our simulation results matched well with the experimental results.
Trapping and rotation of diatom cells and swine oocytes were coupled using oscillating asymmetrical
microstructures with different vibration modes. Finally, we investigated the relationship between
the driving voltage and the speed of cell rotation, showing that the rotational rate achieved could
be as large as approximately 1800 rpm. Using our device, the rotation rate can be effectively tuned
on demand for single-cell studies. Our acoustofluidic cell rotation approach is simple, compact,
non-contact, and biocompatible, permitting rotation irrespective of the optical, magnetic, or electrical
properties of the specimen under investigation.

Keywords: micromachine; cell rotation; acoustic waves; acoustic streaming; on-chip manipulation

1. Introduction
Cell manipulations have attracted great attention in the field of modern bioscience [1,2] because
the cell is the basic structural, functional, and biological unit of all known living organisms. Cell
rotation is one of the fundamental techniques of cell manipulations, which plays a key role in various
fields, including cell observation [3], cell analysis [4–6], and drug discovery [7]. For example, diatom
cells can be set in an ordered alignment by specific rotational manipulation, which is utilized to
achieve detection in screening, diagnosis, and medicine. In addition, since spindle observation is
very important during meiosis to study its dynamic behavior and obtain other information, it should
be oriented to a specific angle via cell rotation for observation under a polarization microscope [8].
Furthermore, controllable cell rotation is also required for the removal or the transfer of nuclei in
transgenic animal research [9,10]. For instance, after enucleation by chemical treatment, an observation
via high-speed cell rotation must be performed to confirm whether the nuclei of cells have been
removed successfully, because the nucleus of an oocyte is anisotropically located at the cell edge, which
is not visible in typical translational manipulations. Thus, cell rotation has become a powerful tool for
cell observation.

Micromachines 2018, 9, 596; doi:10.3390/mi9110596 www.mdpi.com/journal/micromachines

Micromachines 2018, 9, 596 2 of 11

Different approaches have been adopted to achieve rotational manipulation of microparticles and
cells. Conventionally, two micromanipulators are used to perform motions. Under a microscope, one
micromanipulator immobilizes the target cell, while the other applies a torque for the rotation [11,12].
However, this operation requires expert operators, which reduces its applicability and repeatability.
Lin Feng et al. succeeded in cell rotation using dual-arm magnetically driven microtools, but this
technique requires a complex control system [13]. Although optical tweezers have been successfully
used to rotate cells, damage to the biological samples by laser-induced heating may be observed,
and a sufficiently strong torque to rotate larger cells (>20 µm) may not be generated [14,15]. In the
electrorotation technique, an electric field is applied to induce a torque to the target cell by patterning
multiple electrodes [16–18]. Nevertheless, these custom-designed electrode structures are rather
complex to fabricate, and this approach may destroy biological species by current-induced heating.
However, as of yet, no detailed research has been performed on high-speed rotational manipulation of
big-size cells like swine oocytes.
Acoustic waves have recently offered a promising alternative technique for force generation at
the microscale level and have been widely employed for several applications [19–21]. Acoustofluidic
devices that fuse acoustics and microfluidics have the potential to dramatically improve methods for
manipulating cells and small biological samples. For example, an oscillating solid structure placed
in a microfluidic environment generates a local acoustic flow [22–24]. Ozcelik et al. used vibrating
sharp-edged microstructures to engender two counter-rotating microvortices in the surrounding
fluid [25]. However, this acoustofluidic device merely achieved the rotational manipulation of HeLa
cell (≈20 µm) using oscillating solid structures and was unable to perform the rotation of big-size
cells like oocytes (>80 µm). Hayakawa et al. adopted micropillar patterns to accomplish the rotation
of mouse oocytes based on a vibration-induced whirling flow [26,27]. Nonetheless, the target cells
were not retrieved from the microchip, and the flexibility in adjusting the rotational speed was hard
to control. Ahmed et al. trapped air microbubbles within predefined sidewall microcavities inside a
microchannel utilized to rotate cells. A drawback of the microbubbles is their enlargement over time,
which eventually shifts their resonance frequency [28,29].
Thus, we, herein, aim to create adequately robust, stable, and controllable forces to manipulate
the high-speed rotation of big-size cells (≈100 µm) by designing custom-made structures. We propose
an on-chip cell rotation technology using acoustic waves, which integrates the rotation mechanisms of
ultrasonic vibration into a microfluidic chip. Compared with other technologies, this method is robust,
biocompatible, easy to control, and independent of the optical, magnetic, or electrical properties of the
sample. It allows an effective and precise rotation of specimens over a wide range of sizes, shapes,
and properties.
Figure 1a shows the design of the microfluidic chip driven by acoustic flows, including a
polydimethylsiloxane (PDMS) microchannel with six microstructures and a piezoelectric transducer
that provides the acoustic energy. Inlet and outlet holes were punched into the PDMS chip and used
to load and unload cells. The length, width, and depth of the microchannel were 5 mm, 1 mm, and
200 µm, respectively (Figure 1c). Considering the diameter of our experimental samples and previous
researches performed by Adem Ozcelik’s group [25], each microstructure was designed to be of a
constant length of 400 µm, a tip angle (θ) of 20◦ , and a depth of 200 µm, which was used to generate
acoustic streaming for cell rotation (Figure 1b). The piezoelectric transducer and the microfluidic
chip were bonded on a glass slide (150 µm). Our experimental objects were diatom cells (≈80 µm)
and swine oocytes (≈100 µm). Figure 1c also illustrates that, when acoustic waves are excited by a
piezoelectric transducer, vibration-induced local acoustic flows are generated in the surrounding fluid
because of viscous dissipation in the microchannel. Moreover, the response to time-harmonic forcing
is generally not harmonic because of the dissipative property of the fluid. The fluid’s response to
harmonic forcing can be viewed as a combination of a time-harmonic response, generally referred to
as acoustic response, and the remainder, referred to as acoustic streaming. The latter is a byproduct of
the acoustic attenuation caused by viscous dissipation; hence, it provides a unique method of utilizing
Micromachines 2018, 9, 596 3 of 11

Micromachines 2018, 9, x 3 of 11
the dominant viscous nature of microfluidic flows [30–33]. With the growing use of cell-on-chip tools
for
chipinvestigating microparticles
tools for investigating and cells, ourand
microparticles method
cells, isour
anticipated
method isto anticipated
be an invaluable
to be tool in biology,
an invaluable
biophysics, and biophysics,
tool in biology, medicine. and medicine.

Figure 1.
1. Design
Design of
of the rotational device using acoustic waves:
waves: (a) conceptual overview of the
microfluidic chip and (b) oscillations of the solid microstructures used for cell rotation; (c) working
working
and typical
principle and typical geometric
geometric dimensions
dimensions of
of the
the acoustofluidic
acoustofluidic rotational
rotationalmanipulation
manipulationdevice.
device.

2. Materials and
2. Materials and Methods
Methods
2.1. Theoretical Analysis
2.1. Theoretical Analysis
Numerical simulations were performed to theoretically analyze the mechanisms and properties
Numerical simulations were performed to theoretically analyze the mechanisms and properties
of acoustic streaming generation [32,33]. Vectors and scalars are represented by bold and regular fonts,
of acoustic streaming generation [32,33]. Vectors and scalars are represented by bold and regular
respectively. First, the continuity and momentum equations are as follows:
fonts, respectively. First, the continuity and momentum equations are as follows:
𝜕𝜌
∂ρ
++∇(
𝛻(𝜌𝒗) =0
ρv) = (1)
(1)
𝜕𝑡
∂t
   
4
ρ 𝜌 𝜕𝒗++(v(𝒗
∂v
·∇)
∙ 𝛻)𝒗 + 𝒗𝛻 ∙ (𝜌𝒗) = −𝛻𝑝 + 𝜁 + 3 𝜂 ··v𝒗−
v + v ∇·( ρv ) = −∇ p + ζ + η ∇×
−η𝜂𝛻 × 𝛻∇××𝒗v (2)
(2)
∂t𝜕𝑡 3
where
where ρ𝜌isisthe
themass density,v 𝒗
massdensity, is the velocity
is the velocityof of
fluid, p is𝑝the
fluid, pressure
is the of fluid,
pressure andand
of fluid, ζ and η are𝜂bulk
𝜁 and are
and
bulkshear dynamic
and shear viscosities,
dynamic respectively.
viscosities, The relation
respectively. between
The relation ρ and p𝜌 was
between andassumed to be linear:
𝑝 was assumed to be
linear:
p = c20 ρ (3)
𝑝=𝑐 𝜌 (3)
where c0 is the velocity of sound in the fluid at rest. We employed Nyborg’s perturbation technique [34]
where 𝑐 is the velocity of sound in the fluid at rest. We employed Nyborg’s perturbation technique
and expanded the field of fluid velocity, density, and pressure as follows:
[34] and expanded the field of fluid velocity, density, and pressure as follows:
𝒗=
v= v0𝒗+
𝟎+v1𝒗+
𝟏+v2𝒗+𝟐 +∙∙∙∙∙∙
······ (4a)
(4a)

𝜌=
ρ= ρ0𝜌+ ρ+1 𝜌+ ρ+2 𝜌+ ·+∙∙∙∙∙∙
····· (4b)
(4b)
p = p0 + p1 + p2 + · · · · · · (4c)
𝑝 = 𝑝 + 𝑝 + 𝑝 +∙∙∙∙∙∙ (4c)
where 𝜌 is the density of the fluid at rest and a constant, 𝒗𝟏 is the first-order sound velocity, and
𝜌 and 𝒗𝟐 are the second-order parameters. Substituting Equations (3) and (4) into Equations (1)
Micromachines 2018, 9, 596 4 of 11

where ρ0 is the density of the fluid at rest and a constant, v1 is the first-order sound velocity, and ρ2
and v2 are the second-order parameters. Substituting Equations (3) and (4) into Equations (1) and (2),
respectively, the following equations, referred to as the first- and second-order equations of acoustics,
are obtained:
∂ρ1
+ ρ0 (∇v1 ) = 0 (5)
∂t
 
∂v1 4
ρ0 = −∇ p1 + ζ + η ·v1 − η ∇ × ∇ × v1 (6)
∂t 3
and
∂ρ2
h i + ρ0 ∇·hv2 i + ∇·hρ1 v1 i = 0 (7)
∂t
ρ0 h ∂v
∂t i + h ∂t ( ρ1 v1 )i +ρ0 hv1 ·∇v1 i + ρ0 v1 ∇·v1
2 ∂
(8)
= −∇h p2 i + ζ + 34 η ·hv2 i − η ∇ × ∇ × hv2 i

where h x i denotes the time average of quantity x over a full oscillation time period. As pointed
out in Equations (5)–(8), the time averages of the continuity and momentum equations were not
zero, indicating the streaming velocity of the fluid. Moreover, note that these equations, which were
complemented with appropriate boundary conditions, were numerically solved using the commercial
finite element method ANSYS FLUENT 17.0 (Canonsburg, PA, USA) to characterize the acoustic
streaming distributions in the vicinity of the microstructures. Further details would be discussed in
the subsequent part. Consequently, the real mechanism of formation of acoustic streaming is caused
by viscous dissipation and non-linear absorption in the microchannel.

2.2. Simulation and Observed Trajectories

Finite element method (FEM) simulations for modeling the local microstreaming in the presence
of microstructures are used to confirm this unique phenomenon under the standing acoustic wave.
Figure 2a–c show the simulation results of the oscillating solid microstructure at 4.6 kHz using ANSYS
FLUENT 17.0, (type of mesh used: all triangles method, mesh size: 10 µm, boundary conditions:
outflow). With this method, two counter-rotating microvortices were generated by setting up the
oscillation function in the X–Y plane, which matched well with our previous numerical analysis.
Accordingly, 1 µm diameter fluorescent polystyrene spherical beads (PSRF01000, ZHONGKELEIMING
Co., Ltd., Beijing, China) were subjected to the experimental system to confirm the fluid field
distribution around the solid microstructures (10 µL of the particle suspension was centrifuged
and washed with deionized water three times. Then, the microsphere suspensions were diluted
to 100 µL using deionized water). Figure 3a,b show that a vibrating solid structure induced two
local acoustic flows in its vicinity when a sine wave was applied, and the driven oscillation was
harmonic with a frequency equal to 4.6 kHz (Video S1, Supplementary Materials). Moreover, the size
of the microvortices on both sides of the solid microstructure was different because of the asymmetric
design, which was utilized to perform different manipulations in a low-power acoustic field in the
following experiments.
Micromachines 2018, 9, 596 5 of 11
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Figure 2.
Figure 2. Simulation
Simulation results
Simulation results of
results of the
of the velocity vector
the velocity
velocity vector field
vector field
field inin the
in the microfluidic
the microfluidic chip
microfluidic chip using
using FLUENT:
FLUENT: thethe
arrow represents
arrow
arrow represents the
represents the direction
direction ofof motion
motion ofof (a)
(a) the
the acoustic
acoustic microstreaming
microstreaming around
around the
the oscillating
oscillating
microstructure and
microstructure
microstructure and (b)
and (b) the
(b) the flow
flow direction
direction ofof microstreaming
microstreaming from
microstreaming from the
the X–Y
X–Y plane
plane of
of the
the oscillating
oscillating
microstructure; (c)
microstructure; (c) simulation
simulation results
results of
of the
the velocity
velocity fields,
fields, unit:
fields, unit: m/s.
unit: m/s.
m/s.

Figure 3.
Figure 3. Experimentally
Experimentally observed
Experimentallyobserved trajectories
observedtrajectories of
trajectoriesof the
the 111 μm
of the μm diameter
µm diameter fluorescent
diameter fluorescent beads
fluorescent beads in
beads in our
in our
acoustically
acoustically vibrated
acoustically vibrated microstructure:
vibrated microstructure: (a)
microstructure: (a) the
(a) the driving
the driving voltage
driving voltage
voltage isis 0 V
is00 V , while
p-p,, while
Vp-p
p-p in
while in (b),
in (b), the
(b), the driving
the driving voltage
driving voltage
voltage
is 20
is
is 20 VV
20 Vp-p
p-p. ..
p-p

3. Experiments
3. Experiments and
and Results
Results

3.1. System Setup

3.1. System Setup
Figure
Figure 44 shows
shows anan overview
overview of the system
of the system components
components of the manipulation
of the manipulation platform,
platform, consisting
consisting
of the observation and driving systems. A charge-coupled device (CCD) camera (GS3-U3-23S6C-C,
of the observation and driving systems. A charge-coupled device (CCD) camera (GS3-U3-23S6C-C,
Point Grey Co., Ltd., Richmond, BC, Canada) was attached to a differential interference contrast (DIC)
Point Grey Co., Ltd., Richmond, BC, Canada) was attached to a differential interference contrast
microscope (CX41, Olympus Co., Ltd., Tokyo, Japan) to observe the manipulation better. A control
(DIC) microscope (CX41, Olympus Co., Ltd., Tokyo, Japan) to observe the manipulation better. A
personal computer was used to acquire real-time and stereoscopic images. The microfluidic chip
control personal computer was used to acquire real-time and stereoscopic images. The microfluidic
was
chip fixed on a on
was fixed stage. TheThe
a stage. other system
other included
system includedthree parts.
three parts.AAfunction
functiongenerator
generator (Wave
(Wave Station
Station
2012, LECROY, New York, NY, USA) sent a sine wave to a high-voltage amplifier (ATA-2042, Agitek,
2012, LECROY, New York, NY, USA) sent a sine wave to a high-voltage amplifier (ATA-2042, Agitek,
Xi’an, China). This output signal was then used to drive the piezoelectric transducer. Meanwhile,
Xi’an, China). This output signal was then used to drive the piezoelectric transducer. Meanwhile, a
ahigh-resolution
high-resolutionsyringe
syringepump
pump(PHD
(PHDULTRA
ULTRANANOMITE,
NANOMITE,Harvard,
Harvard, Cambridge,
Cambridge, MA,
MA, USA)
USA) was
was
applied to the inlet of the PDMS channel, which could inject micro and quantitative liquid to transport
applied to the inlet of the PDMS channel, which could inject micro and quantitative liquid to
cells steadily and accurately.
transport cells steadily and accurately.
Figure 5a shows the fabrication process of the microfluidic chip. First, the photoresist SU-8
Figure 5a shows the fabrication process of the microfluidic chip. First, the photoresist SU-8
(GM1075, Resemi, Suzhou, China) was coated on the substrate at 1000 rpm for 90 s. The SU-8 mold
(GM1075, Resemi, Suzhou, China) was coated on the substrate at 1000 rpm for 90 s. The SU-8 mold
was
was developed
developed after
after exposure
exposure (see
(see Figure
Figure 5b).
5b). A
A Sylgard
Sylgard 184
184 silicone
silicone elastomer
elastomer base
base was
was mixed
mixed with
with
Sylgard 184 silicone elastomer curing agent (Dow Corning, Midland, MI, USA) at a ratio of 10:1 and
Sylgard 184 ◦silicone elastomer curing agent (Dow Corning, Midland, MI, USA) at a ratio of 10:1 and
cured at 70 C for 2 h to form the PDMS channels. Subsequently, the surface of the PDMS channels
cured at 70 °C for 2 h to form the PDMS channels. Subsequently, the surface of the PDMS channels
and
and aa 40 mm ×
40 mm × 50 mm ×
50 mm 0.15 mm
× 0.15 (width ××length
mm (width length ××thickness)
thickness)glass
glassslide
slidewere
were treated
treated with
with oxygen
oxygen
◦
plasma for 20 s and bonded at 90 C for 10 min. Finally, a piezoelectric transducer (81-7BB-27-4L0,
plasma for 20 s and bonded at 90 °C for 10 min. Finally, a piezoelectric transducer (81-7BB-27-4L0,
Murata Electronics, Kyoto, Japan) was attached on the back of the glass and adjacent to the PDMS
channels using epoxy (84101, Permatex, Hartford, CT, USA).
 Micromachines 2018, 9, 596 6 of 11

Murata Electronics, Kyoto, Japan) was attached on the back of the glass and adjacent to the PDMS
channels using
Micromachines
Micromachines 2018,epoxy
2018, 9,9,x x
(84101, Permatex, Hartford, CT, USA). 6 6ofof1111

Figure
Figure4.4.
Figure Components
Componentsofof
4.Components the
ofthe experimental
theexperimental system.
experimentalsystem.
system.

Micromachines 2018, 9, x 7 of 11

rotational speed supports its wide utilization in single-cell studies, where the shear force for the
rotation can be tuned on-demand. In a micro/nanometer-size world, the viscous force and other
surfaceFigure
forces
Figure 5.5.
exert a dominant
5.Microfluidic
Microfluidic chip
impact(a)
chipfabrication:
fabrication: onfabrication
(a) cell manipulation,
fabrication processofof
process
while
ofthe
the the volume
microfluidic
microfluidic chipand
chip
and
and(b) inertia
(b) digital
digital
forces
Figure Microfluidic chip fabrication: (a) fabrication process the microfluidic chip and (b) digital
hardly have any
microscopic
microscopic effect.
image
image of Therefore,
ofthe
thesolid the cells rarely
solidmicrostructure
microstructure moldmaintain
mold (θ ◦ ). the rotation when the supply is cut off
(θ===20°).
20°).
microscopic image of the solid microstructure mold (θ 20
because of the strong viscosity, which could easily control the specific cell position and orientation
(3.2.
3.2.
Video
3.2. Rotational Manipulation
S1, Supplementary
Rotational Manipulation
Manipulation Materials).
Finally, we needed to assess the effect of the proposed method driven by acoustic waves on the
In
In Figure
Figure6,6,
InFigure the
6,the sound
thesound pressure
soundpressure
pressurelevel level (S.P.L.)
level(S.P.L.)
(S.P.L.)was was different
wasdifferent because
differentbecausebecausethe the vibration
thevibration amplitude
vibrationamplitudeamplitudeof of
of
viability of cells. On the basis of previous experiments by our group, we transported and rotated five
the piezoelectric
thepiezoelectric
the piezoelectrictransducertransducer
transducervaried variedwithwithchanges
changesofofthe thedriving
drivingfrequency,
frequency,
frequency,which which illustrated
whichillustrated
illustratedthat that the
thatthethe
oocytes with our combined system for five minutes. The target oocytes were collected and incubated
S.P.L.
S.P.L.was
S.P.L. was closely
wasclosely related
closelyrelatedrelatedto to acoustic
toacoustic energy.
acousticenergy.
energy.The The piezoelectric
Thepiezoelectric transducer
piezoelectrictransducer
transducerwas was driven
wasdriven
drivenby by applying
byapplying
applyingthe the
the
in a culture well at 37 °C in a 5% CO2 atmosphere for three hours. The viability of the incubated cells
voltage;
voltage;hence,
voltage; hence,
hence,the theS.P.L.
S.P.L. increased
S.P.L.increased
increasedupon upon increasing
uponincreasing
increasingthe the voltage
thevoltage
voltagewithinwithin limits
withinlimits (Figure
limits(Figure
(Figure6). 6). Therefore,
6).Therefore,
was then assessed using the LIVE/DEAD Viability Kit (L-3224, Life Technologies Japan Ltd., Tokyo,
20
2020 VV p-p sine
sinewave
sine
p-pAll
p-p wavevoltage
wave voltagewas
voltage wasapplied
was appliedon
applied ononthe the
the piezo,
piezo,
piezo, andand
and the
the frequency
the frequency
frequency ininin
highhigh
high S.P.L.
S.P.L.
S.P.L. was was
was selected
selected
selected to
Japan). experimental samples remained viable after the incubation. Thus, the proposed method
conduct
totoconduct
conduct experiments
experiments
experiments on onevident
onevident experimental
evidentexperimental phenomena.
experimentalphenomena. phenomena.
did not affect cell viability [26].
Figures7a–d
Figures 7a–dand and8a–d
8a–dshow showthe therotational
rotationalmanipulation
manipulationofofthe thediatom
diatomcell cellandandthe theswine
swineoocyte,
oocyte,
respectively(Video
respectively (VideoS1, S1,Supplementary
SupplementaryMaterials MaterialsAt Atthe thetip tipofofthe theoscillating
oscillatingsolid solidmicrostructure,
microstructure,aa
strongtorque
strong torquewas wascreated
createdatat4.6 4.6kHz
kHzand and20 20 VVp-p p-p (peak-to-peak
(peak-to-peak voltage
voltage value),
value), which
which wasused
was usedtoto
achieve cell
achieve cell rotation.
rotation. Considering
Considering different different sizes sizes ofof the the swine
swine oocyte
oocyte and and the the diatom
diatom cell,cell, the
the
rotationalspeed
rotational speedisiswell-known
well-knowntotodecrease decreaseupon uponincreasing
increasingthe thecell
celldiameter
diameterininthe thesamesamedriving
driving
voltage.InInaddition,
voltage. addition,when whenthe theapplied
appliedfrequency
frequencywas waschanged
changedtoto4.2 4.2kHz,kHz,the themicrostreaming
microstreaming
orientationwas
orientation wasswitched
switchedtotoaaclockwise clockwisedirection
direction(Video (VideoS1, S1,Supplementary
SupplementaryMaterials Materials ).).Qiang
QiangTang Tangetet
al.investigated
al. investigatedpatterns patternsofofthe theacoustic
acousticstreaming
streamingfield fieldatatdifferent
differentfrequencies
frequenciesand andproved
provedthat, that,asas
thefrequency
the frequencyincreased,
increased,the thevibrating
vibratingpattern
patternbecame becamemore morecomplex,
complex,which whichcould couldresultresultinindifferent
different
orientationsofofthe
orientations theacoustic
acousticstreaming
streamingfield field[35].[35].Such
Suchbehavior
behaviorisisattributed
attributedtotothe thechange
changeofofthe the
vibratingpattern.
vibrating pattern.At Atdifferent
differentappliedappliedfrequencies,
frequencies,the theoscillating
oscillatingmicrostructure
microstructureprovides providesdifferent
different
typesofofacoustic
types acousticwave wavepatterns
patternsbecausebecausethe theacoustic
acousticstreaming
streamingstructurestructureisisstrongly
stronglyinfluenced
influencedby by
thevibration
the vibrationsource sourcewhich whichprovidedprovidedacoustic
acousticenergy. energy.Consequently,
Consequently,the thelocation
locationofofthe theexciting
exciting
Figure 6.
Figure 6. Relationship
Relationship betweenbetweensound soundpressure
pressurelevel level(S.P.L.)
(S.P.L.)and and acoustic
acoustic frequency
frequency (distance
(distance fromfrom
the
source
source andcorresponding
and corresponding boundarieswill
boundaries willchangechangeaccording
according toto previous
previous researches
researches [36,37],
[36,37], leading
leading
the sound source:
sound source: 10 cm). 10 cm).
totolocalized
localizedreversed
reverseddirection
directionofofthe theacoustic
acousticstreaming
streamingvortex. vortex.
WeWealso alsoexamined
examinedthe therelationship
relationshipbetween betweenthe thediatom
diatomcell cellrotational
rotationalspeed speedand andthe thedriving
driving
voltage. The voltage was
voltage. The voltage was adjusted by 10 Vp-p adjusted by 10 V every time from
p-pevery time from 20 to 80 Vp-p 20 to 80 V , using an amplifier atat
p-p , using an amplifier
4.6kHz.
4.6 kHz.The Thetip tipangle,
angle,channel
channelwidth, width,and anddepth depthwere wereexperimentally
experimentallyoptimized optimizedtoto20°, 20°,1000,
1000,and and
200 μm, respectively. Figure 9 illustrates that the rotational speed
200 μm, respectively. Figure 9 illustrates that the rotational speed of the diatom cells increased with of the diatom cells increased with
theincreasing
the increasingvoltage, voltage,which whichcould couldbe beasaslargelargeas, as,approximately,
approximately,1800 1800rpm. rpm.The Theflexibility
flexibilityofofthe the
 (Video S1, Supplementary Materials).
Finally, we needed to assess the effect of the proposed method driven by acoustic waves on the
viability of cells. On the basis of previous experiments by our group, we transported and rotated five
oocytes with our combined system for five minutes. The target oocytes were collected and incubated
in a culture2018,
Micromachines well9,at59637 °C in a 5% CO2 atmosphere for three hours. The viability of the incubated7 cells
of 11
was then assessed using the LIVE/DEAD Viability Kit (L-3224, Life Technologies Japan Ltd., Tokyo,
Japan). All experimental samples remained viable after the incubation. Thus, the proposed method
Figures
did not affect7cell
andviability
8–d show the rotational manipulation of the diatom cell and the swine oocyte,
[26].
respectively (Video S1, Supplementary Materials). At the tip of the oscillating solid microstructure,
a strong torque was created at 4.6 kHz and 20 Vp-p (peak-to-peak voltage value), which was used to
achieve cell rotation. Considering different sizes of the swine oocyte and the diatom cell, the rotational
speed is well-known to decrease upon increasing the cell diameter in the same driving voltage.
In addition, when the applied frequency was changed to 4.2 kHz, the microstreaming orientation was
switched to a clockwise direction (Video S1, Supplementary Materials). Qiang Tang et al. investigated
patterns of the acoustic streaming field at different frequencies and proved that, as the frequency
increased, the vibrating pattern became more complex, which could result in different orientations of
the acoustic streaming field [35]. Such behavior is attributed to the change of the vibrating pattern.
At different applied frequencies, the oscillating microstructure provides different types of acoustic
wave patterns because the acoustic streaming structure is strongly influenced by the vibration source
which provided acoustic energy. Consequently, the location of the exciting source and corresponding
boundaries will change according to previous researches [36,37], leading to localized reversed direction
Figure 6. Relationship between sound pressure level (S.P.L.) and acoustic frequency (distance from
of thethe
acoustic streaming vortex.
sound source: 10 cm).

Figure
Micromachines 2018, 9, x 7.
Figure 7. (a–d)
(a–d) Rotational
Rotational manipulation
manipulation of
of the diatom cell
the diatom cell at
at 4.6
4.6 kHz
kHz and
and 20 p-p. .
20 VVp-p 8 of 11

Figure
Figure8.8.(a–d)
(a–d)Rotational
Rotationalmanipulation
manipulationof
ofthe
theswine
swineoocyte
oocyteat
at4.6
4.6kHz
kHzand
and20 Vp-p
20 V p-p..

We also examined the relationship between the diatom cell rotational speed and the driving
voltage. The voltage was adjusted by 10 Vp-p every time from 20 to 80 Vp-p , using an amplifier at
Micromachines 2018, 9, 596 8 of 11

4.6 kHz. The tip angle, channel width, and depth were experimentally optimized to 20◦ , 1000, and
200 µm, respectively. Figure 9 illustrates that the rotational speed of the diatom cells increased with
the increasing voltage, which could be as large as, approximately, 1800 rpm. The flexibility of the
rotational speed supports its wide utilization in single-cell studies, where the shear force for the
rotation can be tuned on-demand. In a micro/nanometer-size world, the viscous force and other
surface forces exert a dominant impact on cell manipulation, while the volume and inertia forces
hardly have any effect. Therefore, the cells rarely maintain the rotation when the supply is cut off
because of the strong viscosity, which could easily control the specific cell position and orientation
Figure 8. (a–d) Rotational manipulation of the swine oocyte at 4.6 kHz and 20 Vp-p .
(Video S1, Supplementary Materials).

Figure 9. Relationship between the rotation speed of the diatom cells and the voltage, showing that the
Figure 9. Relationship between the rotation speed of the diatom cells and the voltage, showing that
former can be tuned by the applied peak-to-peak voltage.
the former can be tuned by the applied peak-to-peak voltage.
Finally, we needed to assess the effect of the proposed method driven by acoustic waves on the
3.3. Cell Trapping
viability of cells. On the basis of previous experiments by our group, we transported and rotated five
oocytes Figure 10 shows
with our combined cell system
trappingfor during the transportation
five minutes. by adjusting
The target oocytes different
were collected andfrequencies,
incubated
inwhich effectively
a culture well at 37 ◦
settled
C in the
a 5%problem about transporting
CO2 atmosphere the cell
for three hours. Thetoviability
a certain position
of the with cells
incubated high
precision
was (Video S1,
then assessed using the LIVE/DEAD
Supplementary ). The cell
Viability
Materials Kit was trapped
(L-3224, in a special trajectory
Life Technologies driven
Japan Ltd., Tokyo,by
acoustic
Japan). Allflows when wesamples
experimental applied remained
a vibrationviable
with after
the frequency of 4 kHz
the incubation. at the
Thus, the voltage
proposedof method
40 Vp-p .
did not affectthe
Especially, cellright-side
viability structure
[26]. was able to form stronger acoustic streaming than the other side,
which was utilized to trap cells in a lower voltage. Figure 11 shows that the time required for
3.3. Cell Trapping
successfully trapping a cell at a distance of 400 μm was only approximately 1.5 s and would continue
to reduce
Figurewith the increasing
10 shows cell trappingdriving voltage.
during the transportation by adjusting different frequencies, which
effectively settled the problem about transporting the cell to a certain position with high precision
(Video S1, Supplementary Materials). The cell was trapped in a special trajectory driven by acoustic
flows when we applied a vibration with the frequency of 4 kHz at the voltage of 40 Vp-p . Especially,
the right-side structure was able to form stronger acoustic streaming than the other side, which was
utilized to trap cells in a lower voltage. Figure 11 shows that the time required for successfully trapping
a cell at a distance of 400 µm was only approximately 1.5 s and would continue to reduce with the
increasing driving voltage.
Micromachines 2018, 9, 596 9 of 11
Micromachines
Micromachines 2018,
2018, 9,
9, xx 99 of
of 11
11

Figure
Figure10.
Figure 10. (a–i)
10. (a–i)Microscopic
(a–i) Microscopicimages
Microscopic imagesof
images of the
of the multiple
the multiple diatom
multiple diatom cells
diatom cells trapped
cells trapped by
trapped by adjusting
by adjusting the
adjusting the frequency.
the frequency.

Figure 11.
Figure 11. (a–d)
(a–d) Microscopic
Microscopic images
Microscopic imagesshowing
images showingthe
showing thetime
the timenecessary
time necessaryto
necessary tototrap
trapaa adiatom
trap diatomat
diatom atatthe
thethedistance
distance
distanceof
ofof
400 μm.
μm.
400 µm.

4.
4. Conclusions
Conclusions
This
This study
study describes
describes aa rotational
rotational manipulation
manipulation method
method using
using acoustic
acoustic waves
waves inin aa microfluidic
microfluidic
chip. We
chip. We also
We also successfully
also successfully demonstrated
successfully demonstrated the
demonstrated the feasibility of non-invasive
the feasibility
feasibility of and
of non-invasive non-contaminated
non-invasive and trapping
and non-contaminated
non-contaminated
and rotation
trapping
trapping andofrotation
and diatomof
rotation cells
of and swine
diatom
diatom cells
cells andoocytes
and swine
swineat oocytes
differentat
oocytes atvibration
differentmodes.
different Themodes.
vibration
vibration torqueThe
modes. created
The by
torque
torque
acreated
specially
created by designed
by aa speciallyoscillating
specially designed microchannel
designed oscillating shape
oscillating microchannel with triangles
microchannel shape
shape with was controlled
with triangles
triangles wasby adjusting
was controlled the
controlled by
by
voltage applied
adjusting the to the
voltagepiezoelectric
applied to transducer,
the which
piezoelectric can be used
transducer, for various
which applications
can
adjusting the voltage applied to the piezoelectric transducer, which can be used for various be used by
for choosing
various
the required rotational
applications
applications by
by choosingspeeds.
choosing the Our future
the required
required work will
rotational
rotational be focused
speeds.
speeds. Our on achieving
Our future
future work
work willa three-dimension
will be
be focused
focused on
on
rotation
achievingof oocytes with
achieving aa three-dimensionincreased
three-dimension rotation precision
rotation of and
of oocytes motion
oocytes with stability.
with increased
increased precision
precision and
and motion
motion stability.
stability.
Supplementary
Supplementary
Supplementary Materials:The
Materials: Thefollowing areare
The following
available online
following are available
available online
at http://www.mdpi.com/2072-666X/9/11/596/
online atat www.mdpi.com/xxx/s1,
www.mdpi.com/xxx/s1, Video Video S1:S1: Cell
s1, Video S1: Cell Materials:
manipulation. Cell
manipulation.
manipulation.
Author Contributions: L.F. and B.S. equally contributed to this work. L.F., B.S., and Y.J. conceived and designed
Author
the
Author Contributions:
experiments; L.F.
L.F. and
and B.S.
B.S. performed
Contributions: theequally
B.S. contributed
experiments;
equally contributedL.F.,to this
this work.
D.Z.,
to and F.A.
work. L.F., B.S.,
B.S., and
analyzed
L.F., Y.J.
andthe conceived
Y.J.data; and
and designed
L.F., Y.J.,
conceived and D.Z.
designed
the
the experiments; B.S. performed the experiments; L.F., D.Z., and F.A. analyzed the data; L.F., Y.J., and
experiments;
contributed B.S. performed the
reagents/materials/analysis experiments;
tools; L.F. L.F.,
and D.Z.,
B.S. and
wrote F.A.
the analyzed
paper. the data; L.F., Y.J., and D.Z.
D.Z.
contributed
contributed
Funding: reagents/materials/analysis
reagents/materials/analysis
This tools;
tools;
research received no external L.F.
L.F. and
funding. and B.S.
B.S. wrote
wrote the
the paper.
paper.
Funding:
Funding: This
This research
Acknowledgments: Thereceived
research no
no external
authors would
received funding.
like to
external thank Xiaoxiang Hu, Xiaojuan Liu, and Yuexin Lin (Department of
funding.
Biological Sciences, China Agricultural University, Beijing, China) for their help with swine oocytes preparation.
The authors gratefully acknowledge the support in diatom cells experiments from Jun Cai and Zhenghu Wang
Micromachines 2018, 9, 596 10 of 11

(Department of Mechanical Engineering and Automation, Beihang University, Beijing, China). This work
was supported by the Natural Science Foundation of Beijing (Grant No. 17L20128) and Beihang University
(ZG216S1751 and ZG226S188D).
Conflicts of Interest: The authors declare no conflict of interest.

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