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

Summary

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.

Abstract

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 µm can be achieved throughout the entire range of operation, including amplitudes > 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.

Introduction

The precise control of liquid flow rate in microchannels is crucial for lab-on-a-chip applications such as droplet production and encapsulation¹, mixing^2,3, and the sorting and manipulation of suspended particles^4,5,6,7. 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 plethora^{6,11,12,13,14,15} 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 -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 µm-100 µm) 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 cavity^16,17. 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 mixing^19,20 and separating 10 µm 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.

Protocol

1. Rapid prototype mold design and fabrication

1. 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.
2. Select the entire model by clicking and dragging a box around it. Export the design as a .stl file by selecting File | Export, then Other formats and choosing .stl from the dropdown box.
3. 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).
4. 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.
5. After removing the part from the resin, agitate it in isopropanol for 5 min to remove any remaining resin.
6. 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.
7. Cure the dried mold at 60 °C in UV light for a maximum of 1 h.

2. PDMS microchannel fabrication

1. 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.
2. Pour PDMS resin and cross-linker into a disposable cup in the ratio of 10:1 by weight and mix with a disposable spoon.
3. 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.
4. Place the mold with poured PDMS into the degas chamber and close the lid. Ensure that the O-ring hermetically seals the chamber.
5. Close the exhaust valve and turn on the vacuum rough pump to initiate degassing.
6. 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.
7. Set oven temperature to 80 °C and allow it to preheat. Place the mixture in the oven at 80 °C for 2 h to cure.
8. Remove the cured mold from the oven and leave it at room temperature for 10 min to cool.
9. 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.
10. 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.
11. Make a hole of 1.0-3.0 mm diameter at the inlet and outlet using a biopsy punch.
12. 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 °C oven for 2 h.
13. Cut polyethylene inlet and outlet tubing to the required length and insert them into the inlet and outlet holes.
14. To prevent tube detachment during operation, apply silicone sealant on the contact surface and let cure for 2 h to secure the tubing.

3. Oscillatory driver assembly

1. 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.
2. 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.
3. Connect one end of an aux cable to the controller chip and the other end to an aux port on a computer or smartphone.
4. 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.
5. Using an internet browser, navigate to an online tone generator website (e.g., https://www.szynalski.com/tone-generator/ ).
6. Type in the desired frequency (5-1200 Hz) in the online application. Scroll the volume bar to the required amount (e.g., 100%).
7. 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.

4. Adapter assembly

NOTE: The complete speaker-to-tube adapter assembly is illustrated by the schematic in Figure 1.

1. Fix the speaker (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.
2. Orient the speaker vertically with the speaker cone surface facing up. Place the 3D printed adapter  (Figure 1(III)) (see speakertubeadapter.stl in Supplementary File 2) concentrically on the speaker cone.
3. Apply silicone sealant generously along the edges of the adapter and let cure for 2 h.
4. Position the speaker and speaker mount on the microscope stage and tape down to prevent movement during operation.
5. Cut a 200 μL 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.
6. 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's coaxial end and finally out through the side.
7. Firmly wedge the narrow end of the pipette tip into the adapter's coaxial end to create a detachable tight seal.

5. Operation of the experimental setup for oscillatory flows in microchannels

1. 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 °C. Mix vigorously by shaking to produce a homogenous suspension.
2. 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.
3. Ensure the outlet tube is routed through the adapter assembly and into a reservoir (see previous section on adapter assembly).
4. 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 (< 1 mL).
5. 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.
6. 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.

6. Observation and amplitude measurement

1. 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.
2. 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.
3. 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 > 10 times that of the oscillation frequency.
4. 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.
5. 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.
6. 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.
7. Observe and record the amplitude measurements.

Results

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 µm 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 microflu...

Discussion

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...

Materials

List of materials used in this article

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