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Trapping of Sub-100 nm Nanoparticles Using Gigahertz Acoustofluidic Tweezers for Biosensing Applications

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Trapping of Sub-100 nm Nanoparticles Using Gigahertz Acoustofluidic Tweezers for Biosensing Applications

Abstract and Figures

We present a nanoscale acoustofluidic trap (AFT) which manipulates nanoparticles in a microfluidic system actuated by a gigahertz acoustic resonator. The AFT generates independent standing closed vortices with high-speed rotation. By carefully designing and optimizing the geometric confinements, the AFT is able to effectively capture and enrich sub-100 nm nanoparticles with low power consumption (0.25~5 μW/μm2) and rapid trapping (within 30 s), showing greatly enhanced particle operating ability towards its acoustic and optical counterparts. Using specifically functionalized nanoparticles (SFNPs) to selectively capture target molecules from the sample, the AFT produces a molecular concentration enhancement of ~200 times. We investigated the feasibility of the SFNPs-assisted AFT preconcentration method for biosensing applications, and successfully demonstrated its capability for serum prostate specific antigen (PSA) detection. The AFT is prepared with a fully CMOS-compatible process, and thus can be conveniently integrated on a single chip, with potential for “lab-on-a-chip” or point-of-care (POC) nanoparticle-based biosensing applications.
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Nanoscale
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ISSN 2040-3364
PAPER
Qian Wang et al.
TiC2: a new two-dimensional sheet beyond MXenes
Volume 8 Number 1 7 January 2016 Pages 1–660
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Duan, W. Pang and M. Reed, Nanoscale, 2019, DOI: 10.1039/C9NR03529J.
Trapping of Sub-100 nm Nanoparticles Using Gigahertz Acoustofluidic
Tweezers for Biosensing Applications
Weiwei Cui a,b)#, Luye Mu b), Xuexin Duan a)* , Wei Pang a), and Mark A. Reed a,b)*
a) State Key Laboratory of Precision Measuring Technology & Instruments, College of Precision
Instrument and Optoelectronics Engineering, Tianjin University, Tianjin 300072, China.
b) Department of Electrical Engineering and Yale University, New Haven, Connecticut 06520, United
States
* Corresponding authors: xduan@tju.edu.cn; mark.reed@yale.edu.
Abstract
We present a nanoscale acoustofluidic trap (AFT) which manipulates nanoparticles in a microfluidic
system actuated by a gigahertz acoustic resonator. The AFT generates independent standing closed
vortices with high-speed rotation. By carefully designing and optimizing the geometric confinements,
the AFT is able to effectively capture and enrich sub-100 nm nanoparticles with low power
consumption (0.25~5 μW/μm2) and rapid trapping (within 30 s), showing greatly enhanced particle
operating ability towards its acoustic and optical counterparts. Using specifically functionalized
nanoparticles (SFNPs) to selectively capture target molecules from the sample, the AFT produces a
molecular concentration enhancement of ~200 times. We investigated the feasibility of the
SFNPs-assisted AFT preconcentration method for biosensing applications, and successfully
demonstrated its capability for serum prostate specific antigen (PSA) detection. The AFT is prepared
with a fully CMOS-compatible process, and thus can be conveniently integrated on a single chip, with
potential for “lab-on-a-chip” or point-of-care (POC) nanoparticle-based biosensing applications.
Introduction
Nanoscale manipulation is essential in many fundamental biology studies and biomedical applications.
For example, the ability to trap and confine nanoparticles would enable techniques for the local
enrichment of targeted analytes, for the development of ultra-sensitive biosensors1 and novel
drug-delivery approaches2, 3. Magnetic nanoparticles have been largely applied to biosensing
applications including target separation and concentration4. However, external magnetic field is
inevitable for these applications, which has been concerned for their negative influence on the
biological activities of targeted proteins or cells. Optical techniques including optical tweezers5,
plasmonic tweezers6, and opto-thermal tweezers7 have been explored for micro- and nanoscale
manipulations as well. However, they generally require giant equipment which is not compatible to
point-of-care (POC) applications. On the other hand, materials in nanoscale size range have unique
structural and functional properties with wide applications in biosensing and medical diagnosis, which
have been explored for decades.8,9 Thus, nano-trapping techniques owning properties of
simple-operation, low-power, convenient integration, and universal operation on any type objects are
desired for developing high-performance biosensors.
By merging acoustics with micro/nanofluidic systems, acoustofluidics has emerged as a versatile and
non-invasive technique for particle and cell manipulation.10,11 Generally, acoustofluidic approaches
utilize either acoustic radiation forces or acoustic streaming effects, that can operate particles in an
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invasive way without any requirement on their chemical properties. For the past decades,
multifunctional manipulations of microscale particles have been demonstrated using acoustic radiation
forces generated by acoustic bubbles12, bulk acoustic waves (BAWs) 13,14, and surface acoustic waves
(SAWs) 15. In these applications, the acoustics work at frequencies ranging from kHz to several MHz,
corresponding to wavelengths from millimeters to hundreds of micrometers. To manipulate
nanoparticles, the acoustic wavelength is required to be as the same scale as the nanoparticles16,17. Thus,
to get a wavelength of several micrometers or even sub-micron, the acoustic frequency should be in the
GHz range.18 However, such high frequency sound waves would dramatically increase acoustic
dissipation into the liquid and effectively trigger acoustic streaming on solid-liquid interface, which
would defocus the particles confined in the nodes or anti-nodes.19 As far as we know, there have been
no reports of standing wave (including those arising from SAWs) based strategies that are able to
directly manipulate particles less than 500 nm.20 An alternative is to utilize acoustic streaming, where
localized acoustic fields with large gradients10, 19, 21 or acoustic-driven vibrating microstructure (such as
bubbles22 and sharp edges23) create micro-vortices which can provide particle trapping, separation, and
other types of manipulations. Previous work used focused SAWs to transfer acoustic radiation forces
into fluids to trigger a pair of vortices across the microchannel, which have been demonstrated with
potential for continuous nanoparticle sorting.24, 25 Another strategy is to guide the vertical component of
the acoustic radiation to rotate fluids within a square glass capillary,26 where a single vortex was
successfully formed in the center of the tube to concentrate nanoparticles as small as ~100 nm.
Different from the direct trapping methods, microparticle-seeded acoustic trapping chip has been
developed for micro/nanoparticles enrichment using the scattering radiation forces inside the gaps of
pre-trapped microbeads.27, 28 Up to now, effective trapping of sub-100 nm nanoparticles using acoustic
methods still remains as a challenge.
Working at the GHz frequency, thin film piezoelectric bulk acoustic resonators prepared with standard
microfabrication processes have been shown to produce very efficient fluid mixing due to vortex
generation.29-31 The vortices created by the resonators have demonstrated excellent performance within
microfluidic systems due to the following: (1) the resonator has a pure perpendicular (to the film)
vibration mode, which directly couples almost all the acoustic energy into the fluid; (2) the GHz
working frequency (ω) contributes to an enhanced body force, which scales with ω4;30 (3) the resonator
creates an acoustic field confined into a small region (with areas from ~100 µm2 to 20,000 µm2). By
tuning the microfluidic confinement, rotating vortices are formed around the resonator, which has been
used to develop an acoustofluidic trap (AFT) that is able to effectively manipulate micro- and
nanoscale particles. Here, we show that the acoustofluidic vortex strategy can be developed into a
bioassay platform by introducing into the AFT specifically functionalized nanoparticles (SFNPs) for
selective capture and concentration of biomarkers from complex samples. The selective capture of
target molecules by the SFNPs was conducted in a centrifugal tube with only several microliters of
sample required. After the incubation, the reacted solution was introduced into the AFT for
concentration. A molecular concentration increase of at least two orders has been demonstrated with
the biotin-Streptavidin (biotin-SAv) model. The advantage of SFNPs (high surface to volume ratio and
selective capture of targets) combined with the advantage of the AFT (for concentration) enables a
universal biosensor strategy for biomolecule concentration and quantification. Accurate, rapid and
portable prostate specific antigens detection in serum was realized, demonstrating its capability in
point-of-care clinical diagnosis.
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Results and Discussions
Figure 1 shows the essentials of the AFT biosensor. Figure 1a is a schematic illustration of the AFT
chip that consists of a nano-electromechanical resonator with a pentagon on the top as the resonator
region,30 a polydimethylsiloxane (PDMS) microchannel which is bounded to the substrate, and
functionalized nanoparticles trapped in the vortices. A completed AFT biosensor is shown in Figure 1b,
with two input/output tubes connected to the PDMS to introduce nanoparticles, target sample solution,
or wash buffer. Working in the GHz regime, the acoustic resonator produces a large acoustic pressure
field32 to generate highly focused forces in the fluid, which induces 3D vortices in a microfluidic
system.
To construct a molecule capturing and enriching chip via the AFT, specific binding nanoparticles
(SFNPs) are as seeds or capturer entities for target molecules (Figure 1a) and finally trapped and
assembled in the vortex array for molecular enrichment. As the body force activated by the GHz
acoustic pressure (AP) is enormous, the vortex rotates in an ultra-fast speed and stimulates a large
volume in the flowing sample solution. Figure 1c schematically illustrates a cross section of the AFT,
indicating the principles of the vortex array generation and how the vortex drives fluid to sample the
SFNPs.
To further study the trapping ability of the AFT, force analysis of the particles in the vortex is carried
out, revealing a linear relationship between streaming forces (viscous and inertial force) and particle
size (Figure 1d). Therefore, correctly chosen nanoparticles can be effectively trapped, whereas small
molecules flow through the trap. Traditional acoustic traps utilize radiation force which scales with the
third power of particle radius, making it powerful to confine larger microparticles but too weak to trap
smaller particles with diameter less than 1 μm.33,34 Trapping forces induced by the optical method are
reported at the order of pN35 for particles with a diameter of 100 nm, and that of the negative
dielectrophoresis (nDEP) traps is rather weak by which the trapping force is of order pN even for
microparticles.36 However, the AFT generates a trapping force (determined by the inertial force) of
several nN for 100 nm particles, showing an obvious advantage over the above-mentioned techniques.
Additionally, it is worth mentioning that the AFT uses the action of fluidic motion, and thus the
trapping forces acting on particles are independent on their properties (such as charge, magnetic,
dielectric constant, etc.). Furthermore, because the vortex streaming regions are not spatially
overlapping the acoustic pressure region (i.e. the AP in Figure 1c), the vortex trapping in the AFT is
dominated by streaming forces that extracts particles from the flow and inertial forces that confine
them in the vortex.
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Figure 1. (a) Schematics of the AFT chip with vortices (blue lines) generated by the acoustic resonator
(pentagonal region), wherein nanoparticles are stably trapped inside the region. The black arrow denotes the fluid
flow direction. (b) Fabricated chip with overlaying PDMS microchannel. The PDMS is partially cut away to show
some resonator structures (gold dots). The active channel is filled with red ink for visualization. (c) Force and
motion schematic of the SFNPs trapped in the vortex. “AP” corresponds to the region of acoustic pressure. The
dashed red curves represent the streamlines of the vortex. The arrows in (c) represent viscous force (blue), inertial
force (pink), and acoustic radiation force (black), respectively. The AFT selectively captures and enriches the
target analyte (green) assisted with the SFNPs. (d) Inertial and viscous forces on particles in the AFT as a function
of particle size.
Optimization of AFT chip
As streaming forces (mainly stokes force and inertial force) dominate the particle trapping,
optimization of the vortex to get improved velocity field is significant for the AFT. We first used
numerical simulation to study the vortex inside the AFT chip. Details of the simulation are presented in
the Supporting Information, Figure S1, S2. As revealed by previous work,30,37,38 increasing the applied
power can directly lead to higher rotating velocity. However, for biosensor applications, the power is
limited due to the unwanted heating effect when higher power is applied37, 39, which would influence
the bioactivity of the proteins. To achieve a stronger vortex, another strategy is tuning the dimension of
the fluidic channel. As previously reported, the vortex could be enhanced by increasing the channel
height18, 40. In the acosutofludic system, the geometric parameters of the acoustic-fluidic boundaries
play dominant roles in the manipulation performance.11,18,41 Here, we analyze the geometric influence
by carefully studying the match between resonator size and channel height. As Figure 2a shows, a
pentagon-shaped resonator is confined inside a microchannel filled with aqueous solution. The
vibrating region distributed within the pentagon region generates a huge body force in the interface
when the GHz acoustic waves propagate from the solid substrate into the liquid. The analysis of the
resonator in air and its’ coupling in liquid are presented in the supporting information. To avoid the
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extra difficulty of a 3D calculation, we choose a cross-section in the x-z direction (shown in Figure 2a)
to conduct a 2D simulation of the GHz acoustic stimulated Navier-Stokes fluidic field. Figure 2b
presents one example of simulation results with the width of resonator set to 150 μm (the width of the
fabricated resonator), and the channel height as 105 μm. By plotting the velocity field under different
geometric confinements characterized by the channel height to resonator width ratio (H/W), we can
determine the optimized parameters of the AFT chip. As shown in Figure 2c, when the H/W ration
equals one, the velocity is the highest. For the resonator used inside the AFT, the pentagon area is
20,000 μm2, and the width varies from 120 μm to 180 μm. Thus, the channel height in a range around
150 μm is reasonable. Additionally, for a fixed channel height, the vortex can be tuned by increasing
the resonator area. As Figure 2d shows, there is a linear relationship between velocity and resonator
width under 105 μm fluidic confinement. Thus, for the resonator in position 1 with a characterization
width of w1 (170 μm) and position 2 with width of w2 (180 μm), there are two different vortices
wherein vortex 2 (Vtx2) is stronger than vortex 1 (Vtx1). Under the streaming forces, particles are
trapped from vortex 1 to vortex 2, and finally most of trapped particles are concentrated inside the
vortex 2. As a result, each edge of the resonator has two concentration regions, and there are totally ten
individual vortices observed around the pentagon region (as the insert picture in d shows).
Figure 2. Scaling of the maximum velocity in the AFT trap as a function of fluidic height and resonator size. (a)
Schematic of the acoustofluidic simulation confinement, with simulated velocity field in the labeled cross section
in x-z plane (green plane) presented in (b). (c) The maximum velocities are plotted as a function of the ratio of
fluidic height to resonator width. (d) The velocity versus resonator width, showing that wider resonator region
generates stronger vortex field. The below insert in (d) presents the different width in different positions along the
resonator edge. The upper insert shows the ten-individual fluorescent vortices formation generated by one
pentagon resonator. All the simulations in this paper use with same parameters, except for the variation of fluid
height.
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Trapping dynamics of nanoparticles in the AFT vortex
To characterize the particles trapping dynamics by the AFT, fluorescein isothiocyanate (FITC)-labeled
particles were used to trace the particle motions. First, a solution with 1.71 µm polystyrene particles
was introduced into the AFT. As Figure 3a shows, FTIC-labeled particles are uniformly suspended in
the channel when the device is off. When turning on the device, vortex streaming was immediately
triggered as shown in Figure 3b. There are ten individual vortices distributed around the resonator,
looking like a flower which fits well with the theoretical analysis in Figure 2d. Particles moved towards
and then entered the vortex regions through two stable positions near the channel boundaries. At the
same time, a depletion region was formed around the vortex, and the area became larger with the
particles being trapped. Finally, particles were depleted and the vortex became saturated by 50 s
(Figure 3c). To investigate the role of acoustic power and further characterize the trapping dynamics of
the AFT, the fluorescence intensity of the focused particles under different powers was recorded
real-in-time as Figure 3c shows. The final fluorescence intensity and time at the 90% saturated
intensity point were plotted versus powers in Figure 3d. Higher power leads to faster and more
nanoparticle concentration because a stronger vortex traps particles from a bigger area at a higher
speed. In most of the cases, particles could be trapped within 50 s, and only about 15 s for 100 mW
(Figure 3e).
Figure 3. Trapping motion trace of 1.71 μm FITC-labeled polystyrene particles by the AFT. (a-c) Frames from a
video segment. (a) Device off. (b)When the device is turned on, suspended FITC-labeled particles move into the
vortex (trapping paths are labeled as the pink arrows in (b)), and almost all particles nearby are trapped in the
focused regions, leaving a depletion region around (as red dash line defended in (b)). (c) Saturation at 50 s. The
channel has a height is 105 μm and a width of 1000 μm, with white dash lines representing the channel boundaries.
(d) Real-in-time fluorescence intensity of the concentrated particles in the AFT chip under different applied
acoustic power. (e) Saturated fluorescence intensity (green) and time (red) versus applied acoustic power with data
acquired at the 90% saturation point.
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The theoretical analysis of Figure 1c-d reveals the potential of the AFT for nanoparticle trapping and
enrichment. To demonstrate this, we conducted experiments using an AFT with a specific channel size
(105 µm (height)×1000 µm (width)). Figure 4a-b present the trapping of polystyrene nanoparticles
ranging from 87 nm to 390 nm. All the nanoparticles were labeled with FITC, and the fluorescence
intensity of the vortex was monitored to characterize the enriching process. Figure 4c shows the
trapping results of gold colloids, demonstrating successful enrichments of sub-100 nm particles (down
to 10 nm). The trapping principle of the AFT shows that faster-rotating vortices contributes to
increased trapping forces, which lead to more effective trapping for smaller particles. The vortex is
driven by the acoustic-induced body force above the AP region (shown in Figure 1c), with the body
force scaling with the fourth powder of the acoustic frequency.19 Therefore, further increasing the
applied frequency can make the AFT much more effective for smaller nanoparticles and potentially
even molecules in the future.
Figure 4. Demonstration of the trapping ability of the AFT. (a) Dynamic process of the trapping of FITC-labeled
polystyrene nanoparticles of different size. (b) Fluorescence image of the enriched nanoparticles with diameters of
87 nm, 210 nm, and 390 nm, showing an effective trapping and enriching of sub-100 nm particles. The
corresponding saturated time respectively are 4 min, 1 min, and 30 s, when the pictures were taken. (c)
Microscopy image of the enriched gold colloids with diameter ranging from 10 nm to 100 nm, demonstrating a
trapping limit of gold nanoparticle size ~ 10 nm. The scale bars in (b) and (c) are all 200 μm.
Molecule enrichment with nanoparticle-assisted strategy
Preconcentration of target molecules from complexed sample is a general strategy to enhance the limit
of detection (LOD) of biosensors. Current methods to concentrate analyte prior to sensing include field
amplified stacking (FAS)42,43, isotachophoresis (ITP),44,45 electrokinetic trapping (EKT),46,47
conductivity gradient focusing (CGF)48, 49, and electric field gradient focusing (EFGF)50. These
technologies require careful consideration of the electrical behavior of the sample matrix, and
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background electrolyte composition and ion concentration. Non-electrical extraction techniques can
take the form of on-chip vapor impinging51, chips designed for liquid/liquid extraction52, or the
inclusion of packed beads for solid-phase extraction53. The design of the platform is only limited to the
physical and electrical boundaries of the separation desired. These reported technologies can be
operated on microfluidic devices, showing good capability for sample preconcentration. However, a
universal microfluidic molecular concentration method with high throughput, non-contact,
bio-compatibility, and easy integration with biosensors is still required. Specific functionalized
microbeads and nanoparticles have been widely used for blood sample purification, molecular
quantification, drug delivery, and related studies 54-56. Meanwhile, interactions between nanoparticles
and biological objects such as protein, DNA, RNA, exosomes, and cells have been well studied57-60,
offering many available strategies for particle-assisted fundamental research and applications. The
developed AFT has demonstrated an excellent ability to manipulate particles ranging from microbeads
to sub-100 nm nanoparticles, providing a powerful tool for particle-assisted biological sample process.
Here, our approach to molecule enrichment using the AFT is based on specifically functionalized
nanoparticles (SFNPs). We use the SFNPs to capture the targets first and then focus and concentrate
them in the AFT, which facilitates the optical quantification of the SFNPs. The process can be operated
in an “offline” (external to the trap) way, such that protein-NPs interaction can occur in a large volume
to overcome diffusion limitations, followed by injecting the reacted solution into the AFT for
concentration. As shown in Figure 1d, the trapping of molecules are ineffective because of their rather
small size. By specific labeling the targeted molecules with SFNPs, the AFT can capture and focus the
molecules effectively with greatly enhanced trapping forces.
As biotin-SAv complex has been widely used in biosensing 61, 62 or molecule interaction studies 63, we
first applied biotin-coated nanoparticles to capture and enrich SAv molecules. The approach is
schematically shown in Figure 5a. Biotin-labeled 270 nm SFNPs are mixed with FITC-labeled SAv
and incubated at room temperature in a tube (0.5 mL) for 15 min. If the SAv molecules are present,
they will be captured by the biotin-modified nanoparticles to form a SFNP-SAv complex. As shown in
Figure 5b, when this mixture is introduced into the AFT and the acoustic resonator is turned on, the
SFNPs-SAv will be concentrated in the vortex, resulting in significantly enhanced fluorescence
intensity signals. The top panels of Figure 5b are for SFNPs that have not been exposed to SAv; and
the bottom panels are SFNPs which has been incubated with SAv. Notice that the bottom left panel has
an unobservable fluorescence intensity, versus the concentrated signal when the AFT is activated.
Under the same acoustic power and applied flow rate, the number of focused NPs stays the same, as
well as the shape of the vortex. Therefore, the final signal is determined by the amount of SAv captured
by each SFNP, which can be used for molecule quantification. To verify the potential of the
nanoparticles-assisted AFT for enhanced molecule detection, SAv with concentrations ranging from 1
ng/mL (corresponding to 18.5 pM) to 2.5 µg/mL (corresponding to 46.3 nM) were respectively mixed
with biotin-labeled SFNPs and concentrated in the AFT, followed by measuring the fluorescence
intensity of the vortex (Figure 5c). The fluorescence intensity is averaged over a number of vortex
positions (circled in red in Figure 5d). A Sigmoidal fitting curve of the responses to SAv concentrations
demonstrates the quantitative potential of the NPs-assisted AFT for bioassay. We note that the
incubation time will determine the shape of this calibration curve, depending on whether the system is
approaching a saturation condition.
Figure 5c also shows two fluorescence intensity values (dashed lines) of SAv solution in PBS buffer
measured without a trap, allowing one to determine the AFT enhancement. Notice that these values (in
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the 10s to 100s of µg/mL range) intersect the experimental calibration curve at much lower
concentration (in the 10s to 100s of ng/mL), giving an improved concentration of at least 200 times in
the AFT. For optical detection, the fluorescence signal is enhanced by ~100 times for different SAv
concentrations (Figure 5d). Because of the signal enhancement factor, no wash step is required to get
the optimized signal to noise ratio (SNR). Furthermore, the AFT concentrates and holds the
nanoparticles in a non-contact way, thus the enriched samples are convenient to transfer to other
downstream sensing modules such as ELISA platform and mass spectrometer analysis. The AFT also
allows for locally in-situ sample preparation and detection by integrating microfabricated biosensors
close to the resonator. The gold surface of the resonator could also work as the working electrode of
electrochemical sensor 63, impedance sensor, 64 or other biosensors interface 65.
Figure 5. Scheme of the acoustofluidic-enhanced biomolecule detection, and results of signal enhancement via the
AFT platform. (a) An illustration of the bioassay procedure, where fluorescently-tagged analytes are bound to
nanoparticles. (b) Experimental demonstration of SAv trapping and concentration via the NPs-assisted AFT. The
top panels are for SFNPs that have not been exposed to SAv, with the AFT off and on; the bottom panels are for
SFNPs that have been exposed to SAv, Notice that the bottom left panel has an unobservable fluorescence
intensity, versus the concentrated signal when the AFT is activated. (c) The fluorescence intensity as a function of
the SAv concentration, fit with a sigmoidal curve. The black dash lines represent the calibrated fluorescence
intensity of FITC-SAv solution within the AFT, with a concentration of 100 µg/mL and 12.5 µg/mL. The mean
value of the ten-individual vortex fluorescence intensity was plotted. (d) Signal enhancement as a function of SAv
concentrations. The method to calculate signal enhancement is to take the fluorescence intensity in the vortex,
outside the channel as the background value, and only FITC-SAv away from the AFT. For more details of the
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signal enhancement calculation, please see Supporting Information, Figure S3. The scale bar in (b) is 200 μm.
Serum acoustofluidic PSA detection
To demonstrate the application of the AFT biosensor in real disease diagnosis, immunosensing
experiments in serum sample have been performed to detect PSA with clinically relevant
concentrations from 0.1, 0.25, 1, 2.5, to 25 ng/mL. As shown in Figure 6a, capture antibody
(anti-PSA-antibody-1) labeled nanoparticles in 1X PBS, PSA serum solution (spiked sample), and
detection antibody (anti-PSA-antibody-2) were well mixed and incubated in a (500 μL) tube for 40
min. The detection antibody was labeled with FITC and used with a fixed concentration of 7.5 µg/mL.
After incubation at room temperature, the reacted solution was introduced into the AFT chip. When
turning on the acoustic device, the AFT drags and concentrates PSA captured nanoparticles into the
vortices. ELISA assays were formed in presence of PSA molecules, resulting in selectively bound PSA
and FITC on the nanoparticle surface. In this case, we observe fluorescent vortices formed in the AFT
as Figure 6b shows. Without PSA, no obvious fluorescence is observed in the vortex (Fig. 6(b), 0
ng/mL). Upon introduction of PSA, clear vortices are observed (Fig. 6(b), 1 ng/mL). Due to the
difference of vortex in different positions, six of the ten which has the strongest and even fluorescent
pixel during the experiments are selected as the signal regions. The intensity of the vortex fluorescence
is linearly dependent on the PSA concentration as shown in Figure 6c (R2 = 0.9922). The linear
response indicates that the AFT chip is capable of conducting quantitative detection of the target
molecules with a generic immunoassay procedure. In clinical applications, the clinically relevant PSA
concentration is in the range of 1~10 ng/mL, which is clearly covered by the AFT sensor, and well
above the limit of detection (~0.05 ng/mL) denoted by the dashed line in Figure 6c. Benefitting from
the molecular enrichment effect, the AFT biosensor does not require a wash step, which makes the
operation very simple. In addition to this, the AFT platform is convenient and compact, with easy
integration with other detectors, low sample consumption, and fast response. In addition, the offline
test can be performed without pumps which largely benefits Point-of-care (POCT) applications. The
reacted protein-NPs solution direct injection into the AFT with a pipette is shown in Figure S4 of
Supporting Information. Besides, the microfabricated acoustic resonator can be integrated with an
electrowetting-driven digital microfluidic chip,66, 67 wherein the bioassay can be conducted in a small
droplet (see Supporting Information, Figure S5). All these features show the promise of the AFT
biosensor as a versatile tool for POCT medical diagnosis strategies and biomedical instrumentation.
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Figure 6. Acoustofluidic serum PSA detection. (a) Illustration of PSA detection using SFNPs-assisted procedures.
(b) Fluorescence images of AFT biosensor without and with PSA in serum (290 nm NPs + PSA in serum + 7.5
μg/mL detection antibody). (c) Fluorescence intensity of the concentrated SFNPs as a function of PSA
concentration.
Conclusions
In summary, a microfabricated acoustofluidic trap has been experimentally demonstrated with the
ability to manipulate sub-100 nm nanoparticles. Polystyrene particle as small as 87 nm and gold
colloids of 10 nm have been successfully trapped and enriched in the AFT. These operations are
realized under rather low power consumption (0.25~5 μW/μm2) and within a short time (generally
within 30 s), showing its excellent capability in small nanoparticle manipulation. Compared with
magnetic methods which require complex systems to operate, magnetized materials, and sophisticated
feedback actuations,68-70 the AFT uses acoustic-induced hydrodynamic force, making it compatible
with any kind of object, independent of materials or medium. Thus, the AFT provides a bio-compatible
nano-manipulating platform that can integrate nanoparticle-protein interactions and has great potential
for versatile biological or clinical applications. As far as we know, it is the first time to use acoustic to
directly capture and manipulate sub-100 nm objects, promoting the capability of acoustic trap or
tweezer from sub-micron to sub-100 nm size range. Benefiting from the selective enrichment of
molecules by ~200 times, the performance of the AFT biosensor is greatly improved, without a wash
step. This is demonstrated here with a serum PSA quantification bioassay, which satisfies the demand
of clinical diagnosis featured with high accuracy, low limit of detection (below 0.1 ng/mL), and
ultralow sample consumption (~0.5 μL). The AFT provides a simple, high throughput, no wash, and a
practical approach for nanoscale biosensing which will contribute to the development of point-of-care
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biosensors.
Methods
Device fabrication
The acoustic resonator devices were fabricated on 100 mm undoped silicon (Si) wafers, starting
from deposition of the Bragg reflector, made of aluminum nitride (AlN) and silicon dioxide (SiO2)
layers alternatively deposited through PVD and CVD, respectively. Then a sandwiched structure
comprising of bottom electrode (BE), piezoelectric layer (AlN), and top electrode (TE) was
deposited and patterned layer by layer. BE of the acoustic device was made of 600 nm thick Moly
and deposited via physical vapor deposition (PVD) on top of the Bragg reflector, and the film was
then patterned by photolithography and plasma etching into a pentagon. PVD was used again to
deposit the piezoelectric layer, a 1000 nm thick AlN film on top of the BE, with a crystal
orientation along c-axis. In the final step, the resonator was capped with a gold TE film deposited
using electron beam evaporation followed by wet etch. Thicknesses of the Au electrodes and the
underlying Cr adhesive lr were 300 and 50 nm, respectively. The electrode area was configured to
be 20,000 μm2 so that the resonator has a characteristic impedance of 50 Ω in order to match the
impedance of external circuits. Microchannels made of polydimethylsiloxane (PDMS) were
prepared by soft lithography. Finally, the acoustic device was integrated with the PDMS
microchannel chip permanent bonding, placing the acoustic device in the center of the channel.
Schematic illustration of the fabrication process is presented in the Supporting Information, Figure
S6.
Nanoparticle and molecule samples
The FITC-labeled polystyrene particles with diameter of 87 nm and 1.71 µm were purchased from
Polysciences; the FITC-labeled 210 nm polystyrene nanoparticles were purchased from Brangs
laboratories, Inc.; gold colloids with diameters of 10, 40, and 100 nm were all bought from BBI
international; Biotin-labeled 270 nm polystyrene nanoparticles were bought from Spherotech; SAv
and full serum were ordered from Sigma; SAv labeled 290 nm nanoparticles were purchased from
Spherotech; PSA, capture antibody (biotin labeled anti-PSA-antibody-1), and detection antigen
(FITC labeled anti-PSA-antibody-2) were purchased from Linc-bio (Shanghai). The PSA and
anti-PSA antibodies are monoclonal. PBS (10×, pH 7.0) buffer containing 0.2% v/v Tween
(Sigma) as the surfactant was used as the solvent. The capture antibody was modified on the
nanoparticle surface by mixing SAv-labeled nanoparticles suspensions with 10 μg/mL
biotin-labeled anti-PSA antibody solution and reacting for 30 min. The modified nanoparticles
were washed twice with PBS buffer and then resuspended in PBS buffer.
Experimental setup
All experiments were recorded by a microscope (Olympus, BX53) integrated with a CCD camera
(DP73) and captured at 25 frames per second. The AFT is excited by a radio frequency (RF) signal
generator (MXG Analog Signal Generator, Agilent, N5181A 100 kHz-3GHz) RF source
instrument; To introduce samples into the microchip and accurately control the flow rate, syringe
pumps (New Era Pump Systems, Inc., NE-1000) are connected with the AFT system via Teflon
tubing (0.75 mm inner diameter). Fluorescence measurements were conducted by the microscope
with a maximum excitation wavelength of 495 nm, and maximum emission wavelength of 530 nm.
Meanwhile, the optical parameters were kept constant, with exposure time of 400 ms for SAv
detection and 1.5 s for serum PSA detection, objective lens, and fixed picture-taking software
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sets. All the experiments were conducted at room temperature.
Acknowledgements
X. Duan gratefully acknowledges financial support from the Natural Science Foundation of China
(NSFC No. 91743110, 61674114, 21861132001), National Key R&D Program of China
(2017YFF0204600), Tianjin Applied Basic Research and Advanced Technology
(17JCJQJC43600), the Foundation for Talent Scientists of Nanchang Institute for
Microtechnology of Tianjin University, and the 111 Project (B07014).
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Supplementary resource (1)

... Acoustical devices, such as surface-acoustic-wave (SAW) chips [1][2][3] and micro-electrode-dot-array (MEDA) chips [4,5], are now being widely utilized in various microfluidic applications in fields such as biochemistry, biomedical and clinical diagnose [6][7][8] because of their great ability on manipulating particles. GHz bulk-acoustic-wave (BAW) resonator, as a newly proposed method, is also proved to have good performances on manipulating particles, especially to particles in sub-micron sizes [9,10]. The good property to properly excited GHz BAW resonator on trapping smaller particles is greatly benefited from the ultra-high speed vortex field that it generates [9]. ...
... GHz bulk-acoustic-wave (BAW) resonator, as a newly proposed method, is also proved to have good performances on manipulating particles, especially to particles in sub-micron sizes [9,10]. The good property to properly excited GHz BAW resonator on trapping smaller particles is greatly benefited from the ultra-high speed vortex field that it generates [9]. ...
... It can be observed that the maximum value of velocity in vortex can touch several meters per second. Based on the fact that minimum trapping diameter of particles is quasi-reciprocal to vortex velocity, the GHz BAW resonator provides great ability on trapping particles with very small diameter if particles are suspending in the microfluidic channel [9]. ...
Conference Paper
Full-text available
Ultra-high-speed vortex field generated by GHz bulk-acoustic-wave (BAW) resonator can bring well ability on particle trapping and manipulation in microfluid, but the ability is weakened when high throughput of sample solution is injected. This paper investigates mechanism of this throughput induced performance deterioration to GHz BAW resonator both theoretically and experimentally, and Region of Effective (RoE) is termed to evaluate the actual performance. Then, RoEs are measured to resonators with different excitation power in different throughput, and prevention method to the weakening is derived. This paper provides explanation to deterioration mechanism and guides to future development for GHz BAW-resonator based applications.
... Through integration of this device into microfluidic channels, confined high-speed microvortices were induced by the GHz acoustic streaming, and this approach was successfully applied to ultrafast fluid mixing 36 and particle trapping in a microfluidic system. 37 Although different-sized particles (from several micrometers to less than 100 nm) can be trapped by these GHz acoustic streaming tweezers (AST), the particle behavior in such 3D microvortex systems remains largely unknown. In this work, we study GHz AST and the associated particle manipulation in an open solution chamber without any physical confinement. ...
Article
Contactless acoustic manipulation of micro/nanoscale particles has attracted considerable attention owing to its near independence of the physical and chemical properties of the targets, making it universally applicable to almost all biological systems. Thin-film bulk acoustic wave (BAW) resonators operating at gigahertz (GHz) frequencies have been demonstrated to generate localized high-speed microvortices through acoustic streaming effects. Benefitting from the strong drag forces of the high-speed vortices, BAW-enabled GHz acoustic streaming tweezers (AST) have been applied to the trapping and enrichment of particles ranging in size from micrometers to less than 100 nm. However, the behavior of particles in such 3D microvortex systems is still largely unknown. In this work, the particle behavior (trapping, enrichment, and separation) in GHz AST is studied by theoretical analyses, 3D simulations, and microparticle tracking experiments. It is found that the particle motion in the vortices is determined mainly by the balance between the acoustic streaming drag force and the acoustic radiation force. This work can provide basic design principles for AST-based lab-on-a-chip systems for a variety of applications.
... Thus implementation of such devices in biological studies that involve single-cell manipulation [1] and non-organic tissues have grown in the past decade. Examples range from nanometer-scale [2] to micro-scale acoustic traps for solid particles [3] to the manipulation of fluids using ultrasonic transducers [4]. Biomedical in-vivo applications have been recently experimentally proven, with the manipulation of glass spheres inside the bladder of a living pig [5]. ...
Preprint
Full-text available
In this work, we will describe an experimental setup for a standing--wave ultrasound trap for air microbubbles in oil. We develop a model for the finite acoustic beam using the angular spectrum technique, and reconstruct the pressure field using the General Lorenz-Mie Theory framework, which was validated using a finite elements method (FEM) simulation. Using Stokes' drag law, we were able to obtain the radius of the trapped bubbles and estimate the minimum acoustic force necessary to trap them, which ranged from 3 nN to 780 nN. We also present the force profile as a function of distance for different bubble that were trapped experimentally, and show that a standing wave formed by interfering infinite plane waves cannot explain the observed acoustic trapping of bubbles in 3-D.
... When placing such membranes in the bottom wall of a cavity containing a liquid, we show that specific higher harmonics of the order n 10 in the membrane induce acoustic pressure fields in the liquid with interference patterns that result in the formation of a single, strong trapping region located 50 -100 µm above the membrane, where a single suspended cell can be trapped in all three spatial directions. The choice of this model system is motivated by the increasing use of disk-shaped membranes in acoustofluidic applications, such as capacitive micromachined ultrasonic transducers (CMUT) for imaging, inkjet printing, and testing [4], thin-film resonators for mixing and biosensing [5,6], and silicon-membrane devices for particle manipulation [7]. In the field of acoustofluidics, electrode shaping is of course used extensively when dealing with surface acoustic waves (SAW), as the electrodes directly define these waves [8]. ...
Preprint
Full-text available
Excitations of MHz acoustic modes are studied numerically in 10-um-thick silicon disk membranes with a radius of 100 and 500 um actuated by an attached 1-um-thick (AlSc)N thin-film transducer. It is shown how higher-harmonic membrane modes can be excited selectively and efficiently by appropriate patterning of the transducer electrodes. When filling the half-space above the membrane with a liquid, the higher-harmonic modes induce acoustic pressure fields in the liquid with interference patterns that result in the formation of a single, strong trapping region located 50 - 100 um above the membrane, where a single suspended cell can be trapped in all three spatial directions. The trapping strength depends on the acoustic contrast between the cell and the liquid, and as a specific example it is shown by numerical simulation that by using a 60% iodixanol solution, a cancer cell can be held in the trap.
... We previously reported an acoustic tweezering technique using a single gigahertz bulk-acoustic-wave (GHz BAW) resonator that has been proven with good trapping ability to particles down to sub-100 nm [30], [31]. It is reasonable to outlook a novel addressable sub-micron bioparticle trapping application by composing multiple GHz BAW into one array. ...
Article
Addressable trapping and manipulations of micro/nano-scale bioparticles is often necessary and critically important in microfluidic devices for biological and medical applications. While GHz bulk-acoustic-wave (BAW) resonators have shown their excellent performance in trapping, rotating, and focusing particles down to nanometers, arraying them for addressable manipulation is a great challenge due to the crosstalk among resonators. This paper presents the electrical and mechanical crosstalk analysis to GHz BAW array with proposed circuit-level models. Crosstalk mechanisms are thoroughly revealed, which contribute to clarify the design rules of GHz BAW array with low crosstalk. A 4 x 4 BAW array chip is designed and experimental results show the chip can achieve addressable control with negligible crosstalk, demonstrating the rationality of the analysis. The crosstalk analysis, as well as the derived design rules, will lead to the design of a high-performance GHz BAW array with great potential for applications that require massive, addressable, and precise particle manipulation. [2021-0236]
Article
Acoustic micro-manipulation has emerged as a promising tool for biomedical studies and clinical diagnostics. We briefly introduce the underlying mechanisms for acoustic micro-manipulation, and highlight recent advances and its potential for biomedical applications. Perspectives on the future developments and new opportunities are also included.
Article
Acoustofluidics-assisted colorimetric method, a promising point-of-care test (POCT) strategy, has been applied in convenient detection of target molecules using the sensing nanomaterials, but remains a formidable challenge to design fluorescent probe with profuse color evolution, realizing sensitive, simple and accurate determination of analyte. Here, a novel triple-emission fluorescent probe is constructed through a simple blend of two ratiometric fluorescent probes (blue-organosilane-polymerized carbon [email protected]2 [email protected]/CdS quantum dots ([email protected]2@r) and blue-organosilane-polymerized carbon [email protected]2 [email protected]/CdS quantum dots ([email protected]2@g)) at the optimized volume ratio of 11:8. As signal reporters of the triple-emission probe, outer modified “r” and “g” will be quenched in sequence by target via two-step response strategy while the inner “b” as internal standard remains constant. When acoustic field is applied, the effect of acoustofluidics-based nanoparticles concentration makes triple-emission probe aggregate completely, accompanying fluorescence color signal being enhanced, sensitivity of colorimetric assay being improved and multicolor-variation (from orange to dark-goldenrod to dark-olive-green to sea-green to dark-cyan to final steel-blue) with the increase of analyte amount. For POCT demonstration, hemoglobin (Hb) is used as a model target and the captured fluorescent photos of triple-emission probe aggregates are analyzed by a chromaticity analysis application on the smartphone to calculate the red, green and blue values, which are used for accurate Hb quantification with the limit of detection of 1.99 mg/L. Whole test is finished within ∼12 min. The acoustofluidics-manipulated triple-emission probe biosensing strategy has also been applied to the rapid and visual quantitative detection of Hb in human blood.
Article
Up-to-date particle sieving schemes face formidable challenges for sieving label-free submicron molecules with similar sizes and dielectric constants but diverse shapes. Herein, optical sorting of polystyrene particles with various shapes is illustrated in optofluidic nanophotonic paired waveguide (ONPW) composed of chalcogenide semiconductor Sb2Se3. The Sb2Se3-ONPW creates the coupling length (CL) between the neighboring hot spots that can be actively modulated via the transition of Sb2Se3 between amorphous (AM) and crystalline (CR) phases. Submicron particles interfere with the coupled hotspots, which can exert various optical torques on the particles according to their profiles. In the model system, spherical (diameter of 0.5 μm) and rod-shaped (diameter of 0.5 μm, length of 1.5 μm) polystyrene particles were employed to mimic two types of bacteria, namely, Staphylococcus aureus and rod-shaped Escherichia coli, respectively. For the AM state, the CL value is ∼7.0 μm, enabling the structure to trap the sphere stably in the hot spots. For the CR state, the CL value becomes ∼25 μm, leading to stable trapping of the rod-shaped particle. In this work, the working wavelength was fixed at 1.55 μm at which both AM- and CR-Sb2Se3 are transparent. Our scheme may offer a paradigm shift in shape-selective sieving of biomolecules and fulfill the requirements of the new-generation lab-on-chip techniques, where the integrated manipulation system must be much more multifunctional and flexible.
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