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An on-demand femtoliter droplet dispensing system based on a gigahertz acoustic resonator

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An on-demand femtoliter droplet dispensing system based on a gigahertz acoustic resonator

Abstract and Figures

On-demand droplet dispensing systems are indispensable tools in bioanalytical fields, such as microarray fabrication. Biomaterial solutions can be very limited and expensive, so minimizing the use of solution per spot produced is highly desirable. Here, we proposed a novel droplet dispensing method which utilizes a gigahertz (GHz) acoustic resonator to deposit well-defined droplets on-demand. This ultra-high frequency acoustic resonator induces a highly localized and strong body force at the solid–liquid interface, which pushes the liquid to generate a stable and sharp “liquid needle” and further delivers droplets to the target substrate surface by transient contact. This approach is between contact and non-contact methods, thus avoiding some issues of traditional methods (such as nozzle clogging or satellite spots). We demonstrated the feasibility of this approach by fabricating high quality DNA and protein microarrays on glass and flexible substrates. Notably, the spot size can be delicately controlled down to a few microns (femtoliter in volume). Because of the CMOS compatibility, we expect this technique to be readily applied to advanced biofabrication processes.
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PAPER
Cite this: DOI: 10.1039/c8lc00540k
Received 29th May 2018,
Accepted 17th July 2018
DOI: 10.1039/c8lc00540k
rsc.li/loc
An on-demand femtoliter droplet dispensing
system based on a gigahertz acoustic resonator
Meihang He,
a
Yangchao Zhou,
a
Weiwei Cui,
a
Yang Yang,
a
Hongxiang Zhang,
a
Xuejiao Chen,
a
Wei Pang*
b
and Xuexin Duan *
a
On-demand droplet dispensing systems are indispensable tools in bioanalytical fields, such as microarray
fabrication. Biomaterial solutions can be very limited and expensive, so minimizing the use of solution per
spot produced is highly desirable. Here, we proposed a novel droplet dispensing method which utilizes a
gigahertz (GHz) acoustic resonator to deposit well-defined droplets on-demand. This ultra-high frequency
acoustic resonator induces a highly localized and strong body force at the solidliquid interface, which
pushes the liquid to generate a stable and sharp liquid needleand further delivers droplets to the target
substrate surface by transient contact. This approach is between contact and non-contact methods, thus
avoiding some issues of traditional methods (such as nozzle clogging or satellite spots). We demonstrated
the feasibility of this approach by fabricating high quality DNA and protein microarrays on glass and flexible
substrates. Notably, the spot size can be delicately controlled down to a few microns (femtoliter in vol-
ume). Because of the CMOS compatibility, we expect this technique to be readily applied to advanced bio-
fabrication processes.
Introduction
Dispensing and manipulating micro- or nanoscale droplets
are essential in developing a highly integrated lab-on-a chip
system for bioanalytical applications.
14
For example, chemi-
cal regents,
5
biomolecules,
6
and cells
7
are deposited on target
substrates at precise locations to form a microarray, which be-
comes an indispensable tool in drug discoveries, genomics,
proteomics, and cell analysis.
810
Different droplet or liquid
dispensing methods have been developed in recent years.
Generally, they can be categorized as contactand non-
contactapproaches according to whether there is a physical
contact between the device and substrate.
8
At present, pin
printing
1113
and inkjet printing
1416
are the two major tech-
niques which represent the contact and non-contact methods,
respectively. Biomaterial solutions can be very limited and ex-
pensive, so minimizing the use of solution per droplet pro-
duced is highly desirable. The printed spot resolution of the
current system is 50 μm in diameter.
10
On average, about 1
nL of solution is deposited per spot. Thus, developing a
higher resolution liquid dispensing system with consumption
of fewer samples is one of the driving forces for the develop-
ment of advanced microarray fabrication techniques. In the
past decade, acoustics-based techniques have been developed
as alternative approaches for liquid handling and dispens-
ing.
17,18
In general, acoustic methods utilize acoustic radia-
tion forces induced by vibration of piezoelectric materials to
atomize liquid
19
or eject liquid droplets out of the airliquid
interface,
20,21
thus microarraying in a non-contact manner. A
variety of biomaterials have been successfully patterned on
different substrates with high biocompatibility.
2224
The
acoustic techniques do not require any pins or nozzles, which
avoid the risks of damaging the substrate
25
or clogging noz-
zles.
26
However, they still suffer from spot uniformity issues,
such as forming satellite spots because the ejected droplets
tend to settle randomly in any given direction before landing
on the substrate.
27,28
Such a settling effect also limits the spot
resolution and may induce misalignment of the spots.
Here, in this work, we reported a new concept of an
acoustic-based on-demand droplet dispensing technique
which is between the contact and non-contact methods. It
utilises a microfabricated gigahertz (GHz) acoustic resonator
to induce a highly localized and strong body force at the
solidliquid interface, which then pushes the liquid upwards
to overcome its surface tension and generates a stable and
sharp liquid needle.
29
By coming into contact with the tar-
get substrate, controlled liquid droplets can be fabricated on
the target substrate through transient contact between the
liquid needle and the substrate. 3D simulation is applied to
Lab ChipThis journal is © The Royal Society of Chemistry 2018
a
State Key Laboratory of Precision Measuring Technology & Instruments, Tianjin
University, Tianjin 300072, China. E-mail: xduan@tju.edu.cn
b
College of Precision Instrument and Optoelectronics Engineering, Tianjin
University, Tianjin 300072, China. E-mail: weipang@tju.edu.cn
Electronic supplementary information (ESI) available: Device fabrication pro-
cess and microarray printing process. See DOI: 10.1039/c8lc00540k
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theoretically understand the formation of the liquid needle.
The performance of the spotting system was demonstrated by
printing DNA and protein microarrays on glass and flexible
substrates. Notably, the spot size of the printed droplets can
be well modulated from several microns to hundreds of
microns, corresponding to liquid volumes from femtoliters to
nanoliters, which shows one order of magnitude smaller than
those of current commercial methods. The liquid needle is
formed in an open well and is always vertical to the device,
thus, there is no concern about clogging or droplet misdirec-
tion issues. Besides, since resonators are fabricated with a
CMOS compatible process, they can be easily integrated as a
multiplexed spotting system for high throughput large-scale
fabrication processes.
Materials and methods
Reagents
Cy3-labelled goat anti-human IgG was obtained from Biosyn-
thesis Biotechnology Co., Ltd. (Beijing, China). The fluores-
cein isothiocyanate (FITC) modified oligonucleotide: 5FITC-
CAG TCG CGA GTA-3was obtained from Sangon Biotech Co.,
Ltd. (Shanghai, China). TrichloroIJ1H,1H,2H,2H-perfluorooctyl)-
silane was obtained from Energy Chemical (Shanghai, China).
The polydimethylsiloxane (PDMS) precursor and curing agent
(Sylgard 184) were obtained from Dow Corning. Poly-L-lysine
(PLL) was obtained from Sigma-Aldrich (USA).
Setup construction
Fig. 1A illustrates the setup of the automatic droplet dispens-
ing system. The acoustic resonator is excited using a radio
frequency signal (RFS) source (Keysight, N5171B) combined
with a power amplifier (Mini-Circuits, ZHL-5W-422+). A reser-
voir connected with the microchannel made from PDMS was
fixed on top of the resonator to store and supply liquid sam-
ples. The target substrate is located above the sample reser-
voir which is precisely controlled with an XYZ precision posi-
tioning stage. To observe and characterize the liquid
dispensing dynamics, a camera with a speed of 240 fps was
utilized to record the dispensing process in real time. A
custom-developed LABVIEW program was used to control the
system including the XYZ stage, camera and RFS source.
The acoustic resonator is fabricated via standard MEMS
technology. The fabrication process has been reported in pre-
vious literature studies
30,31
and briefly described in the ESI
(Fig. S1 and Table S1). As shown in Fig. 1C, the resonator is
composed of a 1.1 μm aluminum nitride piezoelectric (PZ)
layer sandwiched between 0.15 μm and 0.17 μm molybdenum
as the top and bottom electrodes, respectively, a 0.2 μm sili-
con dioxide passivation layer on the surface, and multilayers
of alternately deposited molybdenum (0.64 μm) and silicon
dioxide (0.65 μm) serving as Bragg reflectors at the bottom of
the sandwich structure. Fig. 1B is the top view of the resona-
tor, where the square region is the Bragg reflectors, the mid-
dle pink electrode is the top electrode and the electrodes on
both sides are the bottom electrodes. The area where the top
and bottom electrodes overlap, i.e. the pentagon, is the reso-
nant region. Fig. 1D presents an assembled printing head.
Pretreatment of glass slides and the PDMS substrate
Microscope slides were firstly activated with O
2
plasma under
120 W for 5 minutes, and then placed in a desiccator together
with an open vial containing several droplets of
trichloroIJ1H,1H,2H,2H-perfluorooctyl)silane. The desiccator
was evacuated and left overnight. Finally, the glass slides were
taken out and baked at 85 °C for 0.5 h. The PDMS substrate
was soaked for 0.5 h in HEPES buffer with 3% PLL, rinsed in
ultra-pure water, and dried with a stream of nitrogen.
Results and discussion
Principle and theory
The resonator is activated via an inverse piezoelectric effect,
which can generate vertically propagated acoustic waves in
thickness-extension mode. The resonator is designed to be
pentagonal in shape so that the transverse spurious modes
can be further eliminated. To investigate the acoustic field in-
duced by the resonator, we carried out simulations using
multi-physics finite element analysis software (COMSOL 5.0).
The displacement magnitude of the resonator vibration U=
(x,y,z,t) is as follows:
2
2
2
2
10UU
ct
R
(1)
where c
R
represents the speed of the thickness-extensional
acoustic waves in the piezoelectric layer of the resonator. As
the displacement magnitude of the vibration at the boundary
of the resonant region is zero, the boundary condition can be
expressed as:
U|
At Boundary
= 0 (2)
Fig. 1 (A) Schematic illustration of the automatic droplet dispensing
system. (B) Top view of a fabricated acoustic resonator. (C) Schematic
sectional view of section AA'. (D) The assembled dispenser head.
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At the resonatorair interface (z= 0), the displacement
magnitude of the vibration is as follows:
U(x,y,0,t)=u(x,y)e
iωt
(3)
where uIJx,y) is the vibration displacement magnitude at the
resonator surface. Substituting eqn (3) into eqn (1) and eqn
(2), the numerical solution of uIJx,y) can be obtained, as
shown in Fig. 2A. It is shown that the intensity of the acous-
tic field reaches the maximum at the center and sharply de-
creases around the center, which indicates that the acoustic
energy is highly concentrated at the center of the resonant
area. It is notable that the acoustic energy can be precisely
tuned by adjusting the amplitude of the RFS without chang-
ing its distribution.
When the acoustic field interacts with liquid, the gener-
ated acoustic waves will propagate into the liquid and attenu-
ate within a short distance. The coefficient of the attenuation
is given by
32
b
cL
2
3
2(4)
where ωis the angular frequency of the acoustic waves, ρis
the liquid density, c
L
is the sound speed in the liquid, and
b
4
3

B, where μand μ
B
are the viscosity and bulk viscos-
ity of the fluid, respectively. As the attenuation coefficient
scales with the square of acoustic frequency, the GHz acous-
tic beam generated by the resonator attenuates within a very
short distance.
Due to the nonlinear attenuation of the propagating
acoustic beam, a finite time-averaged momentum flux is gen-
erated, exerting a body force (F
B
) on the liquid to drive a di-
rected fluid motion along the propagation path of the acous-
tic waves.
33
The body force induced by the acoustic waves is
expressed as:
F
B
(x,y,z)=2ρβω
2
u
2
(x,y)e
2βz
(5)
The induced body force decreases exponentially along the
zdirection. As for an acoustic frequency of 2.45 GHz, Fig. 2B
presents the maximum body force as a function of the dis-
tance from the resonatorliquid interface, revealing an enor-
mous body force on the scale of 10
8
Nm
3
generated within
a very short distance. The decay length, which is defined as
the distance where the body force magnitude attenuates to
1/eof its initial value, is 1.36 μm. In the (x,y) plane, since the
body force also scales with the square of uIJx,y), the magni-
tude of the body force has a similar distribution to the vibra-
tion field of the resonator, i.e., the maximum value is located
at the center of the resonator and decreases sharply towards
the edges. The inset in Fig. 2B shows the calculated body
force magnitude distribution of the plane 1 μm above the res-
onatorliquid interface. A locally concentrated body force is
formed without the aid of any additional acoustic focus strat-
egy. Fig. 2B also reveals that the size of the body force distri-
bution region is determined by the area of the resonator.
Moreover, as the body force scales with ω
2
, a higher acoustic
wave frequency provides a more efficient approach to gener-
ate great body force instead of applying high acoustic power.
Under the effect of the highly center-concentrated body
force, the liquid is pushed upward, producing similarly
Fig. 2 (A) 3-D simulation result of the acoustic field generated by the GHz acoustic resonator, wherein the pentagon indicates the resonant region
of the resonator. (B) Calculated maximum body force magnitude along the zdirection. Inset: The planar distribution of the body force magnitude
of the plane 1 μm above the resonatorliquid interface. (C) Liquid needles generated by resonators with different sizes, where the area of the reso-
nant region of the device is 0.4k μm
2
,2kμm
2
and 5k μm
2
, respectively. Scale bar is 100 μm.
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center-concentrated momentum. From the perspective of en-
ergy, the coupled acoustic energy converts into kinetic energy
of the fluid and thermal energy. The locally confined body
force drives the liquid towards the liquidair interface,
whereas the liquid surface tension and gravity are directed to
the opposite direction. Consequently, a sharp liquid needle
is stably formed. Fig. 2C presents the liquid needles gener-
ated by resonators with different sizes (i.e., the area of the
resonant region). The resonator with a larger size can gener-
ate a larger liquid needle, primarily because the body force
distribution region is determined by the area of the resona-
tor. Therefore, in addition to RFS power, the size of the reso-
nator is another alternative variable for effective control of
the liquid needle size.
Droplet dispensing process
When the tip of the liquid needle comes into contact with
the target substrate, the latter will be wetted due to the inter-
molecular attraction forces between these two phases. When
the RFS is turned off, the liquid needle will retract to the res-
ervoir immediately under the effect of surface tension and
gravity. Consequently, the needle will be cut off and a droplet
will be deposited on the substrate due to the surface tension
between the liquid needle and the substrate. Hence, in our
system, the acoustic resonator works intermittently. The dis-
pensing process is presented by a sequence of images cap-
tured using a high speed camera (Fig. 3). The images show
that once being actuated by the acoustic resonator, the liquid
surface deforms into a needle shape and grows high enough
to come into contact with the substrate within a very short
time (17 ms). It is notable that the shape of the liquid nee-
dle fits well with the distribution of the acoustics-induced
body force presented in Fig. 2B. The liquid needle finally re-
mains very stable during its contact with the substrate which
is very important to fabricate uniform droplets. After turning
off the RFS, the liquid needle disappeared immediately, and
the residual liquid that was left on the substrate formed a
droplet. The whole process of liquid dispensing is completed
in a very short time (33 ms), enabling a fast and high effi-
ciency droplet dispensing system.
During the deposition process, the device has no physical
contact with the substrate, while the liquid needle is directly in
contact with the substrate, thus this method is between contact
and non-contact printing. Compared to conventional microar-
ray fabrication methods which utilize pins or nozzles to transfer
liquid samples, this acoustic method utilizes a liquid needle as
a virtual pin to deposit droplets from an open source well.
Meanwhile, the virtual pin is flexible to installor dismantle,
thus eliminating concerns such as nozzle clogging, being
hard to rinse, and being prone to damage due to pins.
Spot size control
For microarray fabrication, it is of great importance to gener-
ate uniform and size-controllable spots. In our system, the
size of the printed spot can be well controlled by different
factors, including the hydrophobicity of the substrate, device
size, RFS power and RFS duration. Here, we focused on the
physical effects that determine the spot size. For a given dis-
tance between the substrate and the liquid samples, the
acoustic power is optimized and set at a fixed value (200 to
500 mw). We investigated the influence of the device size and
the RFS duration on the spot size. For this purpose, Cy3 con-
jugated goat anti-human IgG solution was deposited onto the
glass slide surface as an example by using three different de-
vices (resonator sizes ranging from 0.4k μm
2
to 10k μm
2
).
Meanwhile, the RFS duration time was varied from 30 ms to
150 ms with an interval of 20 ms. The printing results are
presented and summarized in Fig. 4. It clearly shows that
both the device size and RFS working time have a profound
effect on the printed spot size. The spot size is proportional
to the device size. And for a fixed device, the spot size shows
a linear relationship to the RFS duration (Fig. 4D). These re-
sults are consistent with the theoretical analysis. In sum-
mary, the spot size can be delicately modulated by adjusting
the device size and RFS duration.
Viscosity effect
In addition, we also investigated the dispensing of viscous
liquid using the acoustic device. In principle, viscous liquid
can be deposited with a higher RFS power, which provides a
larger body force. However, the maximum power that the cur-
rent device can bear is around 1.5 W, thus it has a limitation
on handling highly viscous liquid. Glycerol was mixed with
ultra-pure water at different ratios to achieve liquids with dif-
ferent viscosities. The printing results are shown in Fig. S2.
It clearly shows that the acoustic device can handle liquids
with a wide range of viscosity and liquid droplets with a vis-
cosity as high as 15.2 mPa s were successfully printed.
Biomaterial printing and patterning
Before printing, the glass slides were treated to become hydro-
phobic for better adsorption of DNA and protein.
3436
FITC la-
belled DNA and cy3 conjugated goat anti-human IgG solution
Fig. 3 Droplet dispensing process captured using a CMOS camera,
where the time interval between two adjacent images is 4.1 ms.
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were respectively diluted in HEPES buffer at concentrations of
10 μM and 0.1 μM. 5% (v/v) glycerol was added to improve the
spot morphology.
37
We fabricated 4 ×4 DNA and protein
microarrays with a spacing of 250 μm×250 μm on the glass
slide in a series of spot diameters (Fig. 5, Video S1). Since
the system works in an intermittent manner, the temperature
of the liquid sample is below 26 °C during the printing pro-
cess, which is acceptable for most biomaterials. To prevent
evaporation of the dispensed droplets, the humidity was kept
at 50%. The spot diameter of the four 4 ×4 microarrays in
Fig. 5 is 8 μm, 60 μm, 120 μm and 180 μm, respectively. It is
notable that the smallest spot we have achieved so far is only
8μm in diameter, corresponding to a droplet volume on the
femtoliter scale. Therefore, the system has great potential in
making microarrays with very high spot density, which is very
important for high-throughput applications.
Accurate quantitative analysis of the printed microarrays
is only possible if spot uniformity (i.e., spot-to-spot size and
shape repeatability) and positional accuracy are achieved.
38
As the step motor of the precision positioning stage has a
precision of 5 μminXYZ directions, the droplets can be
spaced out and arrayed accurately. In the pin printing pro-
cess, since a pin can only carry a limited amount of liquid at
a time, repetitive reloading of samples is required during the
printing process, making the pin-generated spots grow
smaller in size after each transfer. This is not an issue for the
method we present here since the liquid sample is supplied
in real time. For reported acoustic jet printing, satellite drop-
lets are usually produced due to the difficulty in precisely con-
trolling the settling position of the ejected droplets.
27,28
Such
misalignment due to the droplet settling effect is not an issue
in our system since the liquid needle is well aligned vertically
to the device and is directly in contact with the targeted sub-
strate. To accurately characterize the spot uniformity, we
conducted fluorescence intensity (FI) analysis through a
MATLAB program, and the FI analysis results confirm the
high uniformity of the fabricated microarrays (Fig. 5).
In addition to uniformity, morphology is another impor-
tant issue for the array technology. Since most microarray
fabrication methods rely on the dispensing liquid, the perfect
spot shape is round. In fact, some reported results suffer
from spot morphology issues, e.g. the coffee ringeffect,
where the sample concentration is greater at the outer circle
than in the middle,
39
or bullseyespots, where the sample
concentration is highest in the center.
40
These defects greatly
affect the analysis accuracy. In our experiment, none of the
coffee ringor bullseyespots appear, which is due to the
hydrophobic treatment of the substrate surface.
Besides DNA and protein arrays, we also tested the dis-
pensing of living cells in a droplet array with the acoustic de-
vice. It shows that cells can be successfully printed on the tar-
get substrate with retained bioactivity (Fig. S3). These
results indicate that the system is capable of dispensing a va-
riety of different biomaterials with high biocompatibility.
Fig. 4 IJA)(C) Variable-size spots dispensed by using resonators of different sizes and RFS duration periods. The spots grouped in A, B, and C were
dispensed by resonators with areas of 400 μm
2
, 2000 μm
2
, and 10 000 μm
2
, respectively. For every group, the RFS duration of each spot varies
from 30 ms to 150 ms with an interval of 20 ms. (D) Summarized relationship of spot diameter versus RFS duration and resonator size.
Fig. 5 Fluorescence images of 4 ×4 DNA (A) and (B) and protein (goat
anti-human IgG) microarrays (C) and (D). The fluorescence intensity anal-
ysis was acquired along the white dashed line, which gave quantification
of the printed spot size and uniformity. Scale bar in the inset is 5 μm.
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Printing on a flexible substrate
A recent trend in micro/nanotechnology is developing flexible
electronics and analytical devices, which requires technolo-
gies to pattern materials on flexible substrates.
4143
Polydi-
methylsiloxane (PDMS) is a type of mouldable silicone rub-
ber, which has attracted growing attention over the past
decade as a flexible material for microarray applications or in
situ synthesis of biomaterials (e.g. through soft lithogra-
phy),
44,45
because of its intrinsic hydrophobicity, transpar-
ency, flexibility and biocompatibility. Here, we demonstrated
the direct spotting of large-scale protein arrays on a flexible
PDMS substrate. The PDMS substrate was first coated with
PLL by electrostatic adsorption to improve the affinity with
the protein which facilitates the spotting.
46
A protein micro-
array comprising 200 spots (20 ×10) was then successfully
deposited on the PDMS substrate with the acoustic system
(Fig. 6A). The spot size is 110 μm and separated by 40 μm.
The coefficient of variation (CV) of the 10 ×20 microarray is
2.93% which is likely due to the flexible nature of the sub-
strate. In addition, we printed TJUcharacters on this flexi-
ble substrate using writing ink as another sample (Fig. 6B).
The result indicates that our system is robust for large-scale
fabrication and proves the ability of the system to print dif-
ferent patterns on different substrates.
Conclusion
In this work, we developed a novel droplet dispensing system
based on a MEMS acoustic resonator with a resonant fre-
quency as high as 2.45 GHz. A controlled liquid needle is gen-
erated at the liquidair interface through strong body force in-
duced by the acoustic device. By controlling the time duration
of the liquid needle which is in contact with the target sub-
strate, different size high-quality DNA and protein arrays are
successfully patterned on different substrates. Due to its dis-
tinct working manner, the system avoids some drawbacks of
conventional methods (e.g. clogging or satellite spots). The
size of the printed spot as well as the volume dispensed can
be well controlled down to the micrometer scale (femtoliter
in volume). Considering that the acoustic devices are CMOS
compatible, they can be further minimized and integrated,
enabling dispensing liquid samples with ultra-small volume,
as well as fabricating multiplexed and large-scale patterns.
Since this acoustic-based approach is non-invasive, such a
technique can be applied to a variety of different materials in-
cluding inorganic, organic, and biological inks for generating
analytical bio-chips and has great potential in developing
other interesting droplet-based applications.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
The authors gratefully acknowledge financial support from the
National Natural Science Foundation of China (NSFC No.
61674114, 91743110, 21861132001), the National Key R&D Pro-
gram of China (2017YFF0204600), and the 111 Project (B07014).
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Lab on a Chip Paper
Published on 18 July 2018. Downloaded by Tianjin University on 7/26/2018 3:13:36 AM.
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... He et al. proposed a drop-on-demand droplet preparation technique based on a gigahertz acoustic resonator. The liquid was pushed upward, and produced droplets which were further transported onto the hydrophobic substrate under the highly strong force induced by the acoustic field at the center of piezoelectric device (Fig. 2a) [22]. Foresti et al. achieved the precise control of droplets in microliter-to-nanoliter by utilizing the highly localized acoustic field generated by a subwavelength Fabry-Perot resonator [23]. ...
... Highly localized acoustic field [22,23] Bioprinting-based microfluidics platform ...
... Droplets preparation technique induced by a gigahertz acoustic resonator. Reproduced from Ref.[22]. With permission from Royal Society of Chemistry, Copyright 2018; b Integrated system of inkjet printing and PESI-MS. ...
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