We would like to acknowledge the support given and facilities provided by the Department of Mechanical Science and Engineering Rapid Prototyping Lab at the University of Illinois to enable this work.

1. Rapid prototype mold design and fabrication
<ol>
	<li>Open AutoCAD on a PC. Select File on the taskbar, then select Open and browse to and click on a three-dimensional (3D) model file of the channel mold having .dxf or .dwg extension.</li>
	<li>Select the entire model by clicking and dragging a box around it. Export the design as a .stl file by selecting File |&#160;Export,&#160;then Other formats and choosing .stl from the dropdown box.</li>
	<li>Upload the file to a high precision resin stereolithographic (SLA) printer such as Formlabs FORM3. Pour the resin into the resin chamber and initiate printing and produce the mold with the smallest z-axis steps (25 micron for Formlabs CLEAR resin).</li>
	<li>Wait for the automatic part printing to be completed. 
	NOTE: Molds with features as small as 0.1 mm can be fabricated in this way.</li>
	<li>After removing the part from the resin, agitate it in isopropanol for 5 min to remove any remaining resin.</li>
	<li>Dry the mold with air or nitrogen gas for 2 min. 
	NOTE: Conventional microfluidic mold fabrications with silicon wafers and photolithography with any SU8 or KMPR photoresists can also be used to produce a mold with smaller features.</li>
	<li>Cure the dried mold at 60 &#176;C in UV light for a maximum of 1 h.</li>
</ol>
2. PDMS microchannel fabrication
<ol>
	<li>Place the mold on a sheet of aluminum foil. To ease the delamination of PDMS, spray coat the mold with silicone mold release in 1 or 2 passes.</li>
	<li>Pour PDMS resin and cross-linker into a disposable cup in the ratio of 10:1 by weight and mix with a disposable spoon.</li>
	<li>Pour the resulting mixture onto the mold to produce a film of required thickness. To prevent large channel wall deformation, maintain PDMS thickness of more than 5 mm or 3-4 times the maximum feature thickness.</li>
	<li>Place the mold with poured PDMS into the degas chamber and close the lid. Ensure that the O-ring hermetically seals the chamber.</li>
	<li>Close the exhaust valve and turn on the vacuum rough pump to initiate degassing.</li>
	<li>Degas the poured mixture in a vacuum pump for over 4-6 cycles with each cycle lasting approximately 5 min. Manually remove any remaining bubbles (in corners and trenches) using a fine wire.</li>
	<li>Set oven temperature to 80 &#176;C and allow it to preheat. Place the mixture in the oven at 80 &#176;C for 2 h to cure.</li>
	<li>Remove the cured mold from the oven and leave it at room temperature for 10 min to cool.</li>
	<li>Using a scalpel, carefully cut out the edges of the mold. For optimal delamination, use a syringe to inject isopropanol in between the mold and the cured PDMS.</li>
	<li>Peel the cured PDMS off from the mold and cut it into individual devices with a razor blade. The size of each device must range between 10 mm x 10 mm to 30 mm x 70 mm to be bonded with the glass slide.</li>
	<li>Make a hole of 1.0-3.0 mm diameter at the inlet and outlet using a biopsy punch.</li>
	<li>Turn on the handheld radio frequency (RF) plasma generator. To activate the glass slide, steadily pass the wire electrode over a clean dry glass slide multiple times for 2 min. Maintain a wire to glass gap of approximately 5 mm. Place the device side of the cured PDMS in contact with the activated glass slide and then place in an 80 &#176;C oven for 2 h.</li>
	<li>Cut polyethylene inlet and outlet tubing to the required length and insert them into the inlet and outlet holes.</li>
	<li>To prevent tube detachment during operation, apply silicone sealant on the contact surface and let cure for 2 h to secure the tubing.</li>
</ol>
3. Oscillatory driver assembly
<ol>
	<li>Clamp the alligator clip ends of a pair of alligator-to-pin wires to the terminals of a speaker. Here a 15 W speaker with an 8 cm cone was used although other speakers can also be used.</li>
	<li>Place the aux controller chip on an insulating container. Insert the pin ends into the screw sockets of the aux controller chip and firmly tighten with a screwdriver to ensure connectivity.</li>
	<li>Connect one end of an aux cable to the controller chip and the other end to an aux port on a computer or smartphone.</li>
	<li>Connect a 12 V direct current (DC) adapter to the power supply. Power the controller chip on by connecting the coaxial end of the DC adapter to the power socket.</li>
	<li>Using an internet browser, navigate to an online tone generator website (e.g., https://www.szynalski.com/tone-generator/ ).</li>
	<li>Type in the desired frequency (5-1200 Hz) in the online application. Scroll the volume bar to the required amount (e.g., 100%).</li>
	<li>Click on the Wave-Type Generator symbol and select the desired waveform (sine, square, triangle, sawtooth). Note the default is a sine waveform. Press Play to actuate the speaker.</li>
</ol>
4. Adapter assembly
NOTE: The complete speaker-to-tube adapter assembly is illustrated by the schematic in Figure 1.
<ol>
	<li>Fix the speaker&#160;(Figure 1(I)) onto the 3D printed speaker mount (Figure 1(II)) (see speakermount.stl in Supplementary File 1) by sticking a tape over the curved surface and either side of the mount.</li>
	<li>Orient the speaker vertically with the speaker cone surface facing up. Place the 3D printed adapter&#160; (Figure 1(III)) (see speakertubeadapter.stl in Supplementary File 2) concentrically on the speaker cone.</li>
	<li>Apply silicone sealant generously along the edges of the adapter and let cure for 2 h.</li>
	<li>Position the speaker and speaker mount on the microscope stage and tape down to prevent movement during operation.</li>
	<li>Cut a 200 &#956;L micro-pipette tip approximately 2 cm from its narrow end and dispose the wider half of the tip. The narrow conical end will serve as a wedge seal for reversible attachment.</li>
	<li>Connect the polyethylene tubing (Figure 1(V)) to the microchannel (Figure 1(VI)) outlet by first threading through the micro-pipette tip (Figure 1(IV)), and then through the adapter&#39;s coaxial end and finally out through the side.</li>
	<li>Firmly wedge the narrow end of the pipette tip into the adapter&#39;s coaxial end to create a detachable tight seal.</li>
</ol>
5. Operation of the experimental setup for oscillatory flows in microchannels
<ol>
	<li>Add tracer particles into a vial of 22% weight/weight (w/w) glycerol solution to produce a neutrally buoyant suspension with a volume fraction of 0.01%-0.1% polystyrene in liquid at 20 &#176;C. Mix vigorously by shaking to produce a homogenous suspension.</li>
	<li>Load a 1 mL inlet syringe with 1 mL of sample. Mount and fasten the loaded syringe onto an automatic syringe pump. Insert the syringe needle into the inlet tubing of the device to create a watertight seal.</li>
	<li>Ensure the outlet tube is routed through the adapter assembly and into a reservoir (see previous section on adapter assembly).</li>
	<li>Turn on the syringe pump. Using the touch screen, select the syringe type as Becton-Dickinson 1 mL. Then, select Infuse. Then select the required flow rate (0-1 mL/min) or flow volume (&#60; 1 mL).</li>
	<li>Initiate the steady flow using the syringe pump. Wait until sufficient volume of fluid has flowed and the outlet tube is filled with liquid up to the speaker. 
	NOTE: The oscillatory amplitude for a given setting will not vary with steady transport flow if the outlet tube is primed.</li>
	<li>Select a required frequency, amplitude, and waveform in the tone generator application as described in step 3.5 and press Play to generate oscillatory flow inside the microchannel.</li>
</ol>
6. Observation and amplitude measurement
<ol>
	<li>Mount the device on the microscope. Set up the optical configuration by selecting an objective lens with a magnification between 10x and 40x adjusting the focal plane and positioning the stage.</li>
	<li>To obtain measurements in a well-defined focal plane, ensure that the depth of field of the objective lens is smaller than the channel depth by a factor of 5 or more.</li>
	<li>To observe the oscillatory flow, use a high-speed camera with a frame rate of at least twice the oscillation frequency as calculated using the Nyquist sampling theorem. For a practically useful resolution of the waveform, measure at least 10 points per time period using a framerate &#62; 10 times that of the oscillation frequency.</li>
	<li>Alternatively, to observe only the rectified or long-time effects of pulsatile flows, perform stroboscopic imaging by setting the observation frequency to any perfect divisor of the oscillation frequency.</li>
	<li>For both direct and stroboscopic imaging, use a camera equipped with a global shutter to avoid the jello-effect. In either case, keep the exposure time considerably smaller than the oscillation time period (by a factor of 10 or more) to prevent streaking.</li>
	<li>To measure the oscillation amplitude without a high-speed camera, record at a framerate maintained close to but not equal to the stroboscopic frame rate (e.g., 49 frames/s for a 50 Hz signal). This results in a highly slowed-down oscillation from which the amplitude can be accurately measured.</li>
	<li>Observe and record the amplitude measurements.</li>
</ol>

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</ol>

The authors have nothing to disclose.

We have demonstrated the assembly (see protocol critical steps 3 and 4) and operation (see protocol critical steps 5 and 6) of an external speaker-based apparatus for the generation of oscillatory flow with frequencies in the range of 10 to 1000 Hz in microfluidic devices. Particle tracking of suspended tracer particles is required to determine the fidelity of the harmonic motion as well as for calibrating the range of oscillation amplitudes achievable over the range of operating frequencies. The amplitude-frequency curv...

The precise control of liquid flow rate in microchannels is crucial for lab-on-a-chip applications such as droplet production and encapsulation1, mixing2,3, and the sorting and manipulation of suspended particles4,5,6,7. The predominantly used method for flow control is a syringe pump that produces highly controlled steady flows dispensing either a fixed volume of liquid or a fixed volumetric flow rate, often limited to entirely unidirectional flow. Alternative strategies for producing unidirectional flow include using gravitational head8, capillary forces9, or electro-osmotic flow10. Programmable syringe pumps allow for a time-dependent bidirectional control of flow rates and dispensed volumes but are limited to response times greater than 1 s due to the mechanical inertia of the syringe pump.
Flow control at shorter time scales unlocks a plethora6,11,12,13,14,15 of otherwise inaccessible possibilities due to qualitative changes in flow physics. The most practical means of harnessing this varied flow physics is through acoustic waves or oscillatory flows with time periods ranging from 10-1- 10-9 s or 101&#160;-109 Hz. The higher end of this frequency range is accessed using bulk acoustic wave (BAW; 100 kHz-10 MHz) and surface acoustic wave (SAW; 10 MHz-1 GHz) devices. In a typical BAW device, the entire substrate and the fluid column are vibrated by applying a voltage signal across a bonded piezoelectric. This enables relatively high throughputs but also results in heating at higher amplitudes. In SAW devices, however, the solid-liquid interface is oscillated by applying voltage to a pair of interdigitated electrodes patterned on a piezoelectric substrate. Due to the very short wavelengths (1 &#181;m-100 &#181;m) particles as small as 300 nm can be precisely manipulated by the pressure wave generated in SAW devices. Despite the ability to manipulate small particles, SAW methods are limited to local particle manipulation since the wave rapidly attenuates with distance from the source.
At the 1-100 kHz frequency range, oscillatory flows are usually generated using piezo-elements that are bonded to a polydimethylsiloxane (PDMS) microchannel above a designed cavity16,17. The PDMS membrane above the patterned cavity behaves like a vibrating membrane or drum that pressurizes the fluid within the channel. At this frequency range, the wavelength is larger than the channel size, but the oscillation velocity amplitudes are small. The most useful phenomenon in this frequency regime is the generation of acoustic/viscous streaming flows, which are rectified steady flows caused due to non-linearity inherent in the flow of liquids with inertia18. The steady streaming flows typically manifest as high-speed counter-rotating vortices in the vicinity of obstacles, sharp corners, or micro-bubbles. These vortices are useful for mixing19,20 and separating 10 &#181;m sized particles from the flow stream21.
For frequencies in the range of 10-1000 Hz, both the velocity of the oscillatory component and its associated steady viscous streaming are considerable in magnitude and useful. Strong oscillatory flows in this frequency range can be used for inertial focusing22, facilitate droplet generation23, and can generate flow conditions (Womersley numbers) that mimic blood flow for in vitro studies. On the other hand, streaming flows are useful for mixing, particle trapping, and manipulation. Oscillatory flow in this range of frequencies can also be accomplished using a piezo-element bonded to the device as described above23. A significant hurdle to implementing oscillatory flows through a bonded piezo element is that it requires features to be designed beforehand. Furthermore, the bonded speaker elements are not detachable, and a new element must be bonded to each device24. However, such devices present the advantage of being compact. An alternative method is using an electromechanical relay valve20. These valves require pneumatic pressure sources and custom control software for operation and therefore increase the technical barrier to testing and implementation. Nevertheless, such devices enable the application of set pressure amplitude and frequency.
In this article, the construction, operation, and characterization of a user-friendly method to generate oscillatory flows in the frequency range of 10-1000 Hz in microchannels is described. The method offers numerous advantages such as cost-effective assembly, ease of operation, and ready to interface with standard microfluidic channels and accessories such as syringe pumps and tubing. Additionally, compared to previous similar approaches25, the method offers the user selective and independent control of oscillation frequencies and amplitudes, including the modulation between sinusoidal and non-sinusoidal waveforms. These features allow users to easily deploy oscillatory flows and, therefore, facilitate widespread adoption into a broad range of currently existing microfluidic technologies and applications in the fields of biology and chemistry.

<table><tbody><tr><td>&lt;strong&gt;Oscillatory Driver Assembly&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alligator-to-pin wire</td><td>Adafruit</td><td>3255</td><td>Small alligator clip to male jumper wire (12)</td></tr><tr><td>Aux cable</td><td>Adafruit</td><td>2698</td><td>3.5 mm Male/Male stereo cable 1 m</td></tr><tr><td>Controller chip</td><td>Damgoo</td><td>TPA3116</td><td>50w+50w 2 channel audio amplifier (bluetooth and AUX)</td></tr><tr><td>DC adapter</td><td>Adafruit</td><td>798</td><td>12 V DC 1A regulated switching power adapter</td></tr><tr><td>Micro-pipette tip</td><td>VWR Signature</td><td>37001-532</td><td>200 ul micropipette tip</td></tr><tr><td>Silicone sealant</td><td>Loctite</td><td>908570</td><td>Clear silicone waterproof sealant (80 ml)</td></tr><tr><td>Speaker</td><td>Drok</td><td>6843996</td><td>4.5 inch 4 Ohm 40 W speaker</td></tr><tr><td>Speaker mount</td><td></td><td></td><td>3D printed from &#039;speakermount.stl&#039; in supplementary files</td></tr><tr><td>Speaker-to-tube adapter</td><td></td><td></td><td>3D printed from &#039;speaketubeadapter.stl&#039; in supplementary files</td></tr><tr><td>&lt;strong&gt;Microchannel Manufacture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biopsy punch</td><td>Miltex</td><td>15110</td><td>Biopsy punch with plunger (1 - 4 mm)</td></tr><tr><td>Degasser</td><td></td><td></td><td></td></tr><tr><td>Disposable cup</td><td></td><td></td><td></td></tr><tr><td>Disposable spoon</td><td></td><td></td><td></td></tr><tr><td>Glass Slides</td><td>VWR Signature</td><td>16004-430</td><td>3&quot; x 1&quot; pre clean 1 mm thick</td></tr><tr><td>Mold</td><td></td><td></td><td>Si - SU-8 or 3D printed</td></tr><tr><td>Oven</td><td>Fischer Scientific</td><td></td><td>Isotemp</td></tr><tr><td>PDMS resin and cross-linker</td><td>Dow Chemical</td><td>4019862</td><td>Sylgard 184 PDMS resin and crosslinker (500 g)</td></tr><tr><td>Polyethylene tubing</td><td>Becton Dickinson Intramedic</td><td>427440</td><td>Polyethylene tubing (PE 60 - PE 200)</td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td>Single edge industrial razor blades</td></tr><tr><td>RF plasma generator</td><td>Electro-Technic Products</td><td>BD - 20</td><td>High frequency generator</td></tr><tr><td>Silicone Mold Release</td><td>CRC</td><td>03301</td><td>Food Grade Silicon Mold release (16 oz)</td></tr><tr><td>&lt;strong&gt;Observation and Characterization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td>Edgertronic</td><td></td><td>SC2+</td></tr><tr><td>Lens</td><td>Nikon</td><td></td><td>Plan Fluor 10x</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Ti Eclipse manual stage</td></tr><tr><td>Needles</td><td>Becton Dickinson</td><td>305175</td><td>&amp;nbsp;PrecisionGlide 20G</td></tr><tr><td>Syringe</td><td>Becton Dickinson</td><td>1180100555</td><td>Monoject 1 ml</td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td></td><td>Dual syringe programmable syringe pump</td></tr><tr><td>Tracer Particles</td><td>Spherotech</td><td>PP-10-10</td><td>Polystyrene tracer particles 1 um</td></tr></tbody></table>

assembly and characterization of an external driver for the generation of sub-kilohertz oscillatory flow in microchannels

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid samples, such as chemical reagents and cell cultures, is predominantly accomplished through steady flows that are typically driven by syringe pumps, gravity, or capillary forces. The use of complementary oscillatory flows is seldom considered in applications despite its numerous advantages as recently demonstrated in the literature. The significant technical barrier to the implementation of oscillatory flows in microchannels is likely responsible for the lack of its widespread adoption. Advanced commercial syringe pumps that can produce oscillatory flow, are often more expensive and only work for frequencies less than 1 Hz. Here, the assembly and operation of a low-cost, plug-and-play type speaker-based apparatus that generates oscillatory flow in microchannels is demonstrated. High-fidelity harmonic oscillatory flows with frequencies ranging from 10-1000 Hz can be achieved along with independent amplitude control. Amplitudes ranging from 10-600 &#181;m can be achieved throughout the entire range of operation, including amplitudes &#62; 1 mm at the resonant frequency, in a typical microchannel. Although the oscillation frequency is determined by the speaker, we illustrate that the oscillation amplitude is sensitive to fluid properties and channel geometry. Specifically, the oscillation amplitude decreases with increasing channel circuit length and liquid viscosity, and in contrast, the amplitude increases with increasing speaker tube thickness and length. Additionally, the apparatus requires no prior features to be designed on the microchannel and is easily detachable. It can be used simultaneously with a steady flow created by a syringe pump to generate pulsatile flows.

To illustrate the capability and performance of the above setup, representative results of oscillatory flow in a simple linear microchannel with a square cross-section are presented. The width and height of the channel are 110 &#181;m and its length is 5 cm. First, we describe the motion of spherical polystyrene tracer particles and how these can be used to check the fidelity of the oscillatory signal as well as the range of oscillation amplitudes achievable. We then discuss the effect of specific fluid properties or mic...

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Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

The protocol demonstrates a convenient method to produce harmonic oscillatory flow from 10-1000 Hz in microchannels. This is performed by interfacing a computer-controlled speaker diaphragm to the microchannel in a modular manner.

Microfluidic technology has become a standard tool in chemical and biological laboratories for both analysis and synthesis. The injection of liquid ...

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Research

JoVE Journal

Engineering

2.3K Views.  University of Illinois at Urbana-Champaign. Onze techniek brengt oscillerende stromen naar biologen en chemici met behulp van microfluïdica. Dit apparaat is snel te monteren, gemakkelijk te gebruiken en is een plug-and-play-methode voor het produceren van hifi-oscillerende stromen. De procedure wordt gedemonstreerd door Giridar Vishwanathan, een promovendus uit mijn laboratorium.Klem om te beginnen de alligatorclipuiteinden van een paar alligators vast om draden vast te pinnen aan de aansluitingen van een 15 watt luidspreker me...

Video: Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Biology

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Bioengineering

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Induction of Microstreaming by Nonspherical Bubble Oscillations in an Acoustic Levitation System

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. Experimental observations suggest that the temporary permeabilization of these barriers may be due to shear stress that is exerted on cell tissues by cavitation microstreaming. Cavitation microstreaming is the generation of vortex flows which arise around oscillating ultrasound microbubbles. To produce such liquid flows, bubble oscillations must deviate from purely spherical oscillations and include either a translational instability or shape modes. Experimental studies of bubble-induced flows and shear stress on nearby surfaces are often restricted in their scope due to the difficulty of capturing shape deformations of microbubbles in a stable and controllable manner. We describe the design of an acoustic levitation chamber for the study of symmetry-controlled nonspherical oscillations. Such control is performed by using a coalescence technique between two approaching bubbles in a sufficiently intense ultrasound field. The control of nonspherical oscillations opens the way to a controlled cavitation microstreaming of a free surface-oscillating microbubble. High-frame rate cameras allow investigating quasi-simultaneously the nonspherical bubble dynamics at the acoustic timescale and the liquid flow at a lower timescale. It is shown that a large variety of fluid patterns may be obtained and that they are correlated to the modal content of the bubble interface. We demonstrate that even the high-order shape modes can create large-distance fluid patterns if the interface dynamics contain several modes, highlighting the potential of nonspherical oscillations for targeted and localized drug delivery.

A fast and reliable technique is proposed to control the shape oscillations of a single, trapped acoustic bubble that is based on coalescence technique between two bubbles. The steady-state, symmetry-controlled bubble shape oscillations allow analysis of the fluid flow generated in the vicinity of the bubble interface.

When located near biological barriers, oscillating microbubbles may increase cell membrane permeability, allowing for drug and gene internalization. ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...

Developmental Biology

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Assembly and Characterization of an External Driver for the Generation of Sub-Kilohertz Oscillatory Flow in Microchannels

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