Acoustofluidic Separation of Proteins Using Aptamer-Functionalized Microparticles.

We propose an acoustofluidic method for the triseparation of proteins conjugated with aptamer-coated microparticles inside a microchannel. Traveling surface acoustic waves (TSAWs) produced from a slanted-finger interdigital transducer (SFIT) are used to separate the protein-loaded microparticles of different sizes via the TSAW-driven acoustic radiation force (ARF). The acoustofluidic device consists of an SFIT deposited onto a piezoelectric lithium niobate substrate and a polydimethylsiloxane (PDMS) microfluidic channel on top of the substrate. The TSAWs propagating on the substrate penetrate into the sample fluid flow, where the human protein-conjugated microparticles are suspended, inside the PDMS microchannel. The microparticles are subjected to the TSAW-driven ARF with varying magnitude depending on their size and thus flow along different streamlines, leading to triseparation of the proteins. In this work, we used two different-sized streptavidin-functionalized polystyrene (PS) microparticles to capture two kinds of aptamers (apt15 and aptD17.4), which were labeled with a respective biotin molecule at one end. The biotin ends of the aptamers were attached to the microparticles through streptavidin-biotin linkage, whereas the free ends of the aptamers were used to capture their target proteins of thrombin (th) and immunoglobulin E (IgE). The resultant PS-apt15-th and PS-aptD17.4-IgE complexes, as well as mCardinal2, were used for experimental demonstration of acoustofluidic triseparation of the human proteins. We achieved simultaneous separation of proteins of three kinds (th, IgE, and mCardinal2) for the first time via the TSAW-driven ARF in the proposed acoustofluidic device.

Investigation of DPD transport properties in modeling bioparticle motion under the effect of external forces: Low Reynolds number and high Schmidt scenarios.

We have used a dissipative particle dynamics (DPD) model to study the movement of microparticles in a microfluidic device at extremely low Reynolds number (Re). The particles, immersed in a medium, are transported in the microchannel by a flow force and deflected transversely by an external force along the way. An in-house Fortran code is developed to simulate a two-dimensional fluid flow using DPD at Re ≥ 0.0005, which is two orders of magnitude less than the minimum Re value previously reported in the DPD literature. The DPD flow profile is verified by comparing it with the exact solution of Hagen-Poiseuille flow. A bioparticle based on a rigid spring-bead model is introduced in the DPD fluid, and the employed model is verified via comparing the velocity profile past a stationary infinite cylinder against the profile obtained via the finite element method. Moreover, the drag force and drag coefficient on the stationary cylinder are also computed and compared with the reported literature results. Dielectrophoresis (DEP) is investigated as a case study for the proposed DPD model to compute the trajectories of red blood cells in a microfluidic device. A mapping mechanism to scale the external deflecting force from the physical to DPD domain is performed. We designed and built our own experimental setup with the aim to compare the experimental trajectories of cells in a microfluidic device to validate our DPD model. These experimental results are used to investigate the dependence of the trajectory results on the Reynolds number and the Schmidt number. The numerical results agree well with the experiment results, and it is found that the Schmidt number is not a significant parameter for the current application; Reynolds numbers combined with the DEP-to-drag force ratio are the only important parameters influencing the behavior of particles inside the microchannel.

Sheathless Focusing and Separation of Microparticles Using Tilted-Angle Traveling Surface Acoustic Waves.

Sheathless focusing and separation of microparticles is an important preprocessing step in various biochemical assays in which enriched sample isolation is critical. Most of the previous microfluidic particle separation techniques have used sheath flows to achieve efficient sample focusing. The sheath flow dilutes the analyte and requires additional microchannels and accurate flow control. We demonstrated a tilted-angle traveling surface acoustic wave (taTSAW)-based sheathless focusing and separation of particles in a continuous flow. The proposed device consists of a piezoelectric substrate with a pair of interdigitated transducers (IDTs) deposited at two different angles relative to the flow direction. A Y-shaped polydimethylsiloxane (PDMS) microchannel having one inlet and two outlet ports was positioned on top of the IDTs such that the acoustic energy coupling into the fluid was maximized and wave attenuation by the PDMS walls was minimized. The two IDTs independently produced high-frequency taTSAWs, which propagated at ±30° with respect to the flow direction and imparted a direct acoustic radiation force onto the target particles. A sample mixture of 4.8 and 3.2 µm particles was focused and then separated by the actuation of the IDTs at 194 and 136 MHz frequencies, respectively, without using an additional sheath flow. The proposed taTSAW-based particle separation device offered a high purity >99% at the both outlets over a wide range of flow speeds (up to 83.3 mm/s).

On-Demand Droplet Capture and Release Using Microwell-Assisted Surface Acoustic Waves.

We demonstrate an acoustofluidic platform that uses surface acoustic waves (SAWs) for the facile capture of droplets inside microwells and their on-demand release. When the ac signal applied to the device is tuned to modulate the location of the SAW, the SAW-based acoustic radiation force retracts or pushes the droplets into or out of one of three microwells fabricated inside a microchannel to selectively capture or release the droplet.

Particle Separation inside a Sessile Droplet with Variable Contact Angle Using Surface Acoustic Waves.

A sessile droplet of water carrying polystyrene microparticles of different diameters was uniformly exposed to high frequency surface acoustic waves (SAWs) produced by an interdigitated transducer (IDT). We investigated the concentration behavior of the microparticles as the SAWs generated a strong acoustic streaming flow (ASF) inside the water droplet and exerted a direct acoustic radiation force (ARF) on the suspended particles, the magnitude of which depended upon the particle diameter. As a result of the ARF, the microparticles were concentrated according to their diameters at different positions inside the sessile droplet placed in the path of the SAW, right in front of the IDT. The microparticle concentration behavior changed as the sessile droplet contact angle with the substrate was varied by adding surfactant to the water or by gradually evaporating the water. The positions at which the smaller and larger microparticles were concentrated remained distinguishable, even at very different experimental conditions. The long-term exposure of the droplets to the SAWs was accompanied by the gradual evaporation of the carrier fluid, which dynamically changed the droplet contact angle as well as the concentration of particles. Complete evaporation of the fluid left behind several concentrated yet separated clusters of particles on the substrate surface. The effect of the droplet contact angle on particles' concentration behavior and consequent separation of particles has been uniquely studied in this SAW-based report.

A Pumpless Acoustofluidic Platform for Size-Selective Concentration and Separation of Microparticles.

We have designed a pumpless acoustofluidic device for the concentration and separation of different sized particles inside a single-layered straight polydimethylsiloxane (PDMS) microfluidic channel. The proposed device comprises two parallel interdigitated transducers (IDTs) positioned underneath the PDMS microchannel. The IDTs produce high-frequency surface acoustic waves that generate semipermeable virtual acoustic radiation force field walls that selectively trap and concentrate larger particles at different locations inside the microchannel and allow the smaller particles to pass through the acoustic filter. The performance of the acoustofluidic device was first characterized by injecting into the microchannel a uniform flow of suspended 9.9 µm diameter particles with various initial concentrations (as low as 10 particles/mL) using a syringe pump. The particles were trapped with ∼100% efficiency by a single IDT actuated at 73 MHz. The acoustofluidic platform was used to demonstrate the pumpless separation of 12.0, 4.8, and 2.1 µm microparticles by trapping the 12 and 4.8 µm particles using the two IDTs actuated at 73 and 140 MHz, respectively. However, most of the 2.1 µm particles flowed over the IDTs unaffected. The acoustofluidic device was capable of rapidly processing a large volume of sample fluid pumped through the microchannel using an external syringe pump. A small volume of the sample fluid was processed through the device using a capillary flow and a hydrodynamic pressure difference that did not require an external pumping device.

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

Lamb Wave-Based Acoustic Radiation Force-Driven Particle Ring Formation Inside a Sessile Droplet.

We demonstrate an acoustofluidic device using Lamb waves (LWs) to manipulate polystyrene (PS) microparticles suspended in a sessile droplet of water. The LW-based acoustofluidic platform used in this study is advantageous in that the device is actuated over a range of frequencies without changing the device structure or electrode pattern. In addition, the device is simple to operate and cheap to fabricate. The LWs, produced on a piezoelectric substrate, attenuate inside the fluid and create acoustic streaming flow (ASF) in the form of a poloidal flow with toroidal vortices. The PS particles experience direct acoustic radiation force (ARF) in addition to being influenced by the ASF, which drive the concentration of particles to form a ring. This phenomenon was previously attributed to the ASF alone, but the present experimental results confirm that the ARF plays an important role in forming the particle ring, which would not be possible in the presence of only the ASF. We used a range of actuation frequencies (45-280 MHz), PS particle diameters (1-10 µm), and droplet volumes (5, 7.5, and 10 µL) to experimentally demonstrate this phenomenon.

Transfer of Microparticles across Laminar Streams from Non-Newtonian to Newtonian Fluid.

Engineering inertial lift forces and elastic lift forces is explored to transfer microparticles across laminar streams from non-Newtonian to Newtonian fluid. A co-stream of non-Newtonian flow loaded with microparticles (9.9 and 2.0 µm in diameter) and a Newtonian carrier medium flow in a straight rectangular conduit is devised. The elastic lift forces present in the non-Newtonian fluid, undeterred by particle-particle interaction, successfully pass most of the larger (9.9 µm) particles over to the Newtonian fluid. The Newtonian fluid takes over the larger particles and focus them on the equilibrium position, separating the larger particles from the smaller particles. This mechanism enabled processing of densely suspended particle samples. The method offers dilution-free (for number densities up to 10,000 µL(-1)), high throughput (6700 beads/s), and highly efficient (>99% recovery rate, >97% purity) particle separation operated over a wide range of flow rate (2 orders of magnitude).

Generation of Dynamic Free-Form Temperature Gradients in a Disposable Microchip.

Temperature gradients (TGs) provide an effective approach to controlling solvated molecules and creating spatiotemporally varying thermal stimuli for biochemical research. Methods developed to date for generating TGs can only create a limited set of static temperature profiles. This article describes a method for establishing dynamic free-form TGs in polydimethylsiloxane (PDMS) as well as in gases and liquids in contact with the PDMS. The heating mechanism relies on the efficient acoustic absorption by the PDMS of high-frequency (5-200 MHz) surface acoustic waves (SAWs). MATLAB-aided actuation of a transducer enabled the generation and propagation of SAWs in a controlled fashion, which permitted spatiotemporal control over the temperature in the PDMS microstructures. This technique is exploited to perform one-shot high-resolution melting (HRM) analysis to detect single nucleotide polymorphisms (SNPs) in DNA. The experimental results displayed a 10-fold higher resolution and an enhanced signal-to-noise ratio compared to the results obtained using a conventional real-time PCR machine.

Microchannel anechoic corner for size-selective separation and medium exchange via traveling surface acoustic waves.

We demonstrate a miniaturized acoustofluidic device composed of a pair of slanted interdigitated transducers (SIDTs) and a polydimethylsiloxane microchannel for achieving size-selective separation and exchange of medium around polystyrene particles in a continuous, label-free, and contactless fashion. The SIDTs, deposited parallel to each other, produce tunable traveling surface acoustic waves (TSAWs) at desired locations, which, in turn, yield an anechoic corner inside the microchannel that is used to selectively deflect particles of choice from their streamlines. The TSAWs with frequency fR originating from the right SIDT and propagating left toward the microchannel normal to the fluid flow direction, laterally deflect larger particles with diameter d1 from the hydrodynamically focused sample fluid that carries other particles as well with diameters d2 and d3, such that d1 > d2 > d3. The deflected particles (d1) are pushed into the top-left corner of the microchannel. Downstream, the TSAWs with frequency fL, such that fL > fR, disseminating from the left SIDT, deflect the medium-sized particles (d2) rightward, leaving behind the larger particles (d1) unaffected in the top-left anechoic corner and the smaller particles (d3) in the middle of the microchannel, thereby achieving particle separation. A particle not present in the anechoic corner could be deflected rightward to realize twice the medium exchange. In this work, the three-way separation of polystyrene particles with diameters of 3, 4.2, and 5 µm and 3, 5, and 7 µm is achieved using two separate devices. Moreover, these devices are used to demonstrate multimedium exchange around polystyrene particles ∼5 µm and 7 µm in diameter.

Acoustofluidic lysis of cancer cells and Raman spectrum profiling.

The lysis of cancer cells inside a sessile droplet was performed using traveling surface acoustic waves (SAWs) without any chemical reagents. Raman spectrum profiling was then carried out to explore detailed cell-derived data. The Rayleigh waves formed by an interdigital transducer were made to propagate along the surface of an LiNbO3 substrate. Polystyrene microparticles (PSMPs) were used to establish mechanical cell lysis effectively, and gold nanoparticles (AuNPs) were added to enhance the Raman signals from the lysed cells by SAWs. The lysis efficiency was evaluated according to the size and concentration of the PSMPs in experiments where the frequency was varied. Lysis occurred mainly by mechanical collision using PSMPs in a high-frequency domain, and the lysis efficiency was improved by increasing the application time and the energy density of the SAWs. Raman signals from the lysed cells were greatly enhanced by nanogaps formed by the AuNPs, which were evenly distributed irrespective of the SAWs through the frequency-independent behavior of the AuNPs. Finally, detailed Raman spectra of MDA-MB-231, malignant breast cancer cells, were acquired, and various organic matter-derived peaks were observed. The 95% confidence region for cells subjected to lysis was more widely distributed than that of cells not subjected to lysis. The proposed SAW platform is expected to facilitate the detection of small quantities and to be applied in biomedical applications.

Optical levitation of a non-spherical particle in a loosely focused Gaussian beam.

The optical force on a non-spherical particle subjected to a loosely focused laser beam was calculated using the dynamic ray tracing method. Ellipsoidal particles with different aspect ratios, inclination angles, and positions were modeled, and the effects of these parameters on the optical force were examined. The vertical component of the optical force parallel to the laser beam axis decreased as the aspect ratio decreased, whereas the ellipsoid with a small aspect ratio and a large inclination angle experienced a large vertical optical force. The ellipsoids were pulled toward or repelled away from the laser beam axis, depending on the inclination angle, and they experienced a torque near the focal point. The behavior of the ellipsoids in a viscous fluid was examined by analyzing a dynamic simulation based on the penalty immersed boundary method. As the ellipsoids levitated along the direction of the laser beam propagation, they moved horizontally with rotation. Except for the ellipsoid with a small aspect ratio and a zero inclination angle near the focal point, the ellipsoids rotated until the major axis aligned with the laser beam axis.

Optofluidic particle manipulation in a liquid-core/liquid-cladding waveguide.

This paper describes a method for particle manipulation in a liquid-core/liquid-cladding optical waveguide system. Step-index and graded-index waveguides were modeled with consideration for, respectively, miscible and immiscible core and cladding fluids. The characteristic motions of four different particles with refractive indices of 1.59, 1.48, 1.37, and 1.22 were examined. The guided beam was assumed to be Gaussian in shape. Our results showed that high-refractive-index particles converged at the center of the core fluid due to a positive gradient force, whereas low-refractive-index particles converged at the flow periphery. The nonlinearity of the particle motion increased as the flow velocity and the guided beam waist decreased and the laser power and the particle size increased. The initial beam waist of the guided beam in the graded-index waveguide did not significantly affect the characteristics of the particle motion due to the effects of diffusion.

Behavior of double emulsions in a cross-type optical separation system.

The behavior of double emulsions in a cross-type optical particle separation system was studied for different combinations of refractive indices and different inner and outer layer radii. The radii and refractive indices of the double emulsions were easily adjusted by taking advantage of the coflowing geometry of a cross-type optical particle separation device. An analytical expression of the optical forces on a pair of concentric spheres was derived using the photon stream method in the ray optics regime. The predicted trajectories of the double emulsions by the optical force agreed well with the experimental data. This work has potential uses in cell separation by morphometry, drug delivery vehicle, and emulsion-based biomedical applications.

Nanoforest of hydrothermally grown hierarchical ZnO nanowires for a high efficiency dye-sensitized solar cell.

In this paper, in order to increase the power conversion efficiency we demonstrated the selective growth of "nanoforest" composed of high density, long branched "treelike" multigeneration hierarchical ZnO nanowire photoanodes. The overall light-conversion efficiency of the branched ZnO nanowire DSSCs was almost 5 times higher than the efficiency of DSSCs constructed by upstanding ZnO nanowires. The efficiency increase is due to greatly enhanced surface area for higher dye loading and light harvesting, and also due to reduced charge recombination by providing direct conduction pathways along the crystalline ZnO "nanotree" multi generation branches. We performed a parametric study to determine optimum hierarchical ZnO nanowire photoanodes through the combination of both length-wise growth and branched growth processes. The novel selective hierarchical growth approach represents a low cost, all solution processed hydrothermal method that yields complex hierarchical ZnO nanowire photoanodes by utilizing a simple engineering of seed particles and capping polymer.

Hydrodynamic interaction of dorsal fin and caudal fin in swimming tuna.

Tuna, which are known for high-performance swimming, possess a large crescent dorsal fin (DF) and a caudal fin (CF) that differ from those of other fishes. The hydrodynamic interaction between the DF and CF in tuna, which are represented by two tandem 3D flapping plates, is numerically explored in the present study. Hydrodynamic properties and wake structures of the models with and without a DF are compared to investigate the effects of the DF. The thrust on the CF is substantially enhanced by the DF, whereas the force on the DF is not affected by the CF. The constructive interaction between the leading-edge vortex (LEV) on the CF and the vortices shed from the dorsal fin (DFVs) is identified from 3D wake topology and 2D vorticity distributions. The circulation of spanwise vorticity quantitatively reveals that the LEV on the CF is strengthened by the same-signed DFV. The effect of the flapping phase of the CF is examined. The DF-CF interaction is sensitive to the flapping phase at a short spacing, whereas a long spacing between the two fins enables a robust constructive interaction in tuna swimming. A systematic study is carried out to explore the effects of the Strouhal number (St) and the Reynolds number (Re) on the interaction of the fins. The enhancement of thrust due to the DF is diminished at St = 0.63, whereas the Re does not substantially influence the constructive DF-CF interaction.

Acoustofluidic Stimulation of Functional Immune Cells in a Microreactor.

The cytotoxic response of natural killer (NK) cells in a microreactor to surface acoustic waves (SAWs) is investigated, where the SAWs produce an acoustic streaming flow. The Rayleigh-type SAWs form by an interdigital transducer propagated along the surface of a piezoelectric substrate in order to allow the dynamic stimulation of functional immune cells in a noncontact and rotor-free manner. The developed acoustofluidic microreactor enables a dynamic cell culture to be set up in a miniaturized system while maintaining the performance of agitating media. The present SAW system creates acoustic streaming flow in the cylindrical microreactor and applies flow-induced shear stress to the cells. The suspended NK cells are found to not be damaged by the SAW operation of the adjusted experimental setup. Suspended NK cell aggregates subjected to an SAW treatment show increased intracellular Ca2+ concentrations. Simultaneously treating the NK cells with SAWs and protein kinase C activator enhances the lysosomal protein expressions of the cells and the cell-mediated cytotoxicity against target tumor cells. These have important implications by showing that acoustically actuated system allows dynamic cell culture without cell damages and further alters cytotoxicity-related cellular activities.

Manipulation of cancer cells in a sessile droplet via travelling surface acoustic waves.

The behaviours of microparticles inside a sessile droplet actuated by surface acoustic waves (SAWs) were investigated, where the SAWs produced an acoustic streaming flow and imparted an acoustic radiation force on the microparticles. The Rayleigh waves formed by a comb-like interdigital transducer were made to propagate along the surface of a LiNbO3 substrate in order to allow the manipulation of microparticles in a label-free and non-contact manner. Polystyrene microparticles were first employed to describe the behaviours inside a sessile droplet. The influence of the volume of the sessile droplet on the behaviours of the microparticles was examined by changing the contact angle of the droplet. Next, cancer cells were suspended in a sessile droplet, and the influence of contact angle on the behaviours of the cancer cells was investigated. A long gelation time was afforded by using a PEGylated fibrin gel. A primary tumour was mimicked by patterning the cancer cells to be concentrated in the middle of the sessile droplet. The non-contact manipulation property of acoustic waves was indicated to be biocompatible and enabled a structure-free platform configuration. Three-dimensional aggregated culture models were observed to make the cancer cells display an elevated expression of E-cadherin. The efficacy of the anticancer drug tirapazamine increased in the aggregated cancer cells, attributed to the low levels of oxygen in this formation of cancer cells.

Antibiotic susceptibility test under a linear concentration gradient using travelling surface acoustic waves.

An efficient and accurate antibiotic susceptibility test (AST) is indispensable for measuring the antimicrobial resistance of pathogenic bacteria. A minimal inhibitory concentration (MIC) can be obtained without performing repeated dilutions of the antibiotic by forming a linear antibiotic concentration gradient in a microfluidic channel. We demonstrated a device designed to use travelling surface acoustic waves (TSAWs) to enable a rapid formation of an antibiotic gradient in a few seconds. The TSAWs produced by a focused interdigital transducer deposited on the surface of a piezoelectric (LiNbO3) substrate generated an acoustic streaming flow inside a microfluidic channel, which mixed confluent streams of antibiotics in a controlled fashion. The growth of bacteria exposed to the antibiotic gradient was determined by measuring the MIC, which was used as an indicator of the effectiveness of the AST. The concentration gradient produced using our device was linear, a feature that enhanced the reliability of measurements throughout the microchannel. Two ASTs, namely Pseudomonas aeruginosa against gentamicin and levofloxacin were chosen for the case of slowly proliferating bacteria, and one AST, namely Escherichia coli against gentamicin, were chosen for the rapidly proliferating case. Appropriate antibiotic doses for Pseudomonas aeruginosa and Escherichia coli were each obtained in an efficient manner.