Objective:
The goal of this experiment is to study droplet generation behavior of a flow focusing device, understand the pressure level effect on droplet radius and period via video processing. Students will use a pressure pump, a 3D digital microscope and a flow focusing device to generate droplets at varying sizes. Also, they will learn fabrication of microfluidic devices.
Theory Overview :
Microfluidics is the key science and technology that enables fluid manipulation and control in channels that have dimensions on the order of micrometers (Fig.1). The change in fluid physics in microscale ensures novel use of microfluidic systems. Although the first microfluidic device was a gas chromatograph invented in 1975, these systems have not used for biological or chemical applications since 1990s [1]. Microfluidic device fabrication technology was derived from microelectromechanical system (MEMS) technology.
During the last decades, microfluidic systems became a widespread research area and gave birth to easy-to-use and miniaturized platforms that can be applied to biological and chemical analysis. Microfluidics can be divided into three groups as continuous microfluidics, discrete (multi-phase flow, droplet-based, segmented flow) microfluidics and digital microfluidics (Fig.2.).
Continuous microfluidics is based on continuous liquid flow manipulation. These systems are usually used for simple and well-defined functions such as chemical separation and biochemical applications. Since the surface property of the entire system affects fluid flow at any location in the system, these systems are not suitable for integration and scalability. Discrete microfluidics compartmentalizes and manipulates small volumes of liquid with two immiscible phases. Microdroplets are suitable for very small amount of liquid handling. Since droplet is isolated from its surrounding, any material inside droplet (reagent, cell, protein etc.) is preserved throughout the system. Droplet loading, mixing, sorting, merging, break-up enables high-throughput chemical and biological experimentation due to kHz level droplet generation. Both continuous and discrete microfluidics operate in microchannels. However, digital microfluidics is manipulation of small liquid volumes on open structures using electrowetting method. Electrowetting is changing surface properties of a material by applying electric field. On independently addressed electrodes, a small volume of liquid is moved from one electrode to another. These systems enable merging of different material loaded droplets. Electrowetting on-dielectric (EWOD) is a common method in digital microfluidics.
Paper-based microfluidic systems are considered Microfluidics 2.0. This method is inexpensive and easy-to-use. Changing hydrophobicity of the paper at different zones using a printer and wax, channels are fabricated [4]. Printing reagents and other materials to the test zones makes microfluidic paper-based analytical devices (µPADs) cheaper point-of-care diagnostic devices. Being user-friendly and cheap, paper-based microfluidic systems are developed for disease diagnostics in third world countries [5].
Glass, Si and polymers are the three main materials for fabricating microchannels. According to application, any of these three materials can be used. Si wafer is not a suitable material for optical measurement techniques due to its opacity. Glass is a fragile material and etching it rather difficult than etching a Si wafer. The bonding process requires high voltages or temperatures. Also, a clean room was needed for the fabrication process [6]. The dimensions of polymers change when they interact with some chemicals such as alcohols. Silicon can be used for applications requiring high temperatures such as quantum dot synthesis [7]. Glass microchannels are resistant to chemicals and reusable. A polymer type, polymethylmethacrylate (PMMA), is suitable for high volume production of cartridges. On the other hand, polydimethylsiloxane (PDMS), is the "working horse" of scientists, since it is a non-toxic, opaque material and can be cured at low temperatures. PDMS is a rather cheap material and fabrication of a microfluidic device becomes quite easy with soft lithography techniques. Microfluidic systems are integrated with different components to operate properly. Pumps, valves, mixers, pressure and flow sensors are some of the fluidic components that are integrated with microfluidic systems.
Microfluidic systems or lab-on-a-chip technologies generate, manipulate and process small liquid volumes. Due to their miniaturized nature, microfluidic systems especially microdroplet based microfluidic systems provide several advantages such as, enhanced analytical performance with respect to macroscale techniques, low cost and ability to process large libraries of samples in a short amount of time (high throughput). Nowadays, these platforms are used for DNA sequencing, chemical and biochemical screening, PCR (polymerase chain reaction), protein crystallization, directed evolution of proteins, detection of rare diseases, cell-cell interactions, single cell analysis [8]–[14].
Droplet generation
In the literature, there are two main passive droplet formation generators, T-junction and flow-focusing device. Two immiscible fluids are driven from two separate channels and meet at a junction that is determined by the specific geometry of the channels. In 2001, Thorsen et al. published an article titled "Dynamic pattern formation in a Vesicle-Generating Microfluidic Device" [15]. For the first time, they accomplished droplet generation with two immiscible fluids using a T-junction. Both water and oil were continuously driven to the microchannel (Fig.3). The water obstructs the main channel at the junction, while oil flows through the channel. At this moment, high shear forces occur. The flow is not linear and static due to interactions between the boundary of two liquids. This instability arises from the competition between surface tension and shear forces. The competition generates droplets. The size and speed of droplets are finely tuned by adjusting water and oil flow rates or pressures.
Anna et al. used flow-focusing technique in a planar microchannel to form droplets [16]. In this technique, two immiscible phases are driven to the one orifice, where the outer channel carries oil, and the inner channel carries water. These three channels form a cross at the intersection. The oil comes from two sides of the water and applies pressure to it so that water breaks into droplets. Using this technique, varying size of droplets can be generated at very high speeds. Three regimes occur during droplet formation depending on physical properties of fluids and external variables. These regimes are categorized as squeezing, dripping and jetting [17]. The physical properties of liquids such as interfacial tensions, viscosities, and external variables as flow rates of fluids, channel dimensions and geometry are used to categorize droplet formations [18]. The dimensionless numbers originated from aforementioned variables determine these regimes. Capillary (Ca) number is the most important dimensionless number for droplet formation and its value varies between 10-3 and 10 (Eq. 1). In Eq. 1, µ is the dynamic viscosity of the fluid, V is velocity of the fluid and is interfacial tension between two liquids. Capillary number relates viscous forces with interfacial tension.
Ca=(μ*V)/γ Eq.1
Abate et al. stated that monodisperse droplets are generated at low capillary numbers for T-junctions and at high capillary numbers for flow-focusing devices [18]. In a T-junction two regimes occur: squeezing and dripping. When dispersed phase (water) completely blocks the mainstream channel and there is pressure drop along the droplet due to channel blockage, the regime is called squeezing regime. In dripping regime, droplets do not completely block the main channel and are smaller than the dimension of the main channel. In a flow-focusing device dripping and jetting are two regimes that occur during droplet formation. In dripping regime, the dispersed phase breaks now it enters the junction and turns into droplets. These droplets are immediately carried away by continuous phase. In jetting regime, dispersed phase goes into continuous phase and for a while they co-exist in the main channel [17].
How do we use Navier-Stokes eqn for microfluidics?
The Navier-Stokes equations are a set of partial differential equations that describe the motion of viscous fluids (Fig.4). These equations are crucial in understanding fluid dynamics, including the behavior of fluids in microfluidic devices.
In microfluidics, the flow regime is often laminar, meaning the fluid particles move in parallel layers without significant mixing. This is due to the small scale of microfluidic channels, where viscous forces dominate over inertial forces. The Navier-Stokes equations, when simplified for laminar flow, provide a powerful tool for analyzing and predicting the behavior of fluids in these devices.
Key aspects of how the Navier-Stokes equations define microfluidics include:
Laminar flow: The equations can be simplified for laminar flow, making them easier to solve and analyze.
Viscous forces: The equations account for viscous forces, which are particularly significant in microfluidic channels due to their small size.
Pressure gradients: The equations can be used to calculate pressure gradients, which drive fluid flow in microfluidic devices.
Velocity profiles: The equations can be used to determine the velocity profile of the fluid within the channel, which is important for understanding flow behavior.
Design and optimization: The equations can be used to design and optimize microfluidic devices, ensuring that the desired flow characteristics are achieved.
In summary, the Navier-Stokes equations are fundamental to understanding and designing microfluidic systems. By providing a mathematical framework for describing fluid flow, these equations enable researchers and engineers to analyze and optimize the performance of microfluidic devices for various applications.
Fabrication of microfluidic devices
Photolithography is a cornerstone technique in microfluidic device fabrication. It involves transferring a pattern from a photomask onto a substrate using light.
Si wafer Preparation: A Si wafer is cleaned and prepared for the lithography process.
Photoresist Application: A light-sensitive negative SU-8 photoresist is spin-coated onto the substrate, forming a thin, uniform layer.
Photomask Exposure: The photomask, containing the desired pattern, is placed over the photoresist. UV light is then shone through the mask, exposing the photoresist in the patterned areas.
Development: The exposed photoresist is developed in a chemical solution. The exposed areas are either removed (positive photoresist) or remain (negative photoresist), depending on the type of photoresist used.
Channel Formation with soft lithography : Soft lithography is a method that refers to replicating mold structure using polymeric, "soft", materials by stamping. For fabrication of microchannels, PDMS (polydimethylsiloxane) is poured on Si wafer and heated on a hot plate. Cured PDMS is peeled off from the Si wafer, cut and punched.
Bonding :The cut PDMS substrate that contains 3 edges of the microfluidic channel is then bonded to a glass slide to close the microchannels via oxygen plasma.
Other Fabrication Techniques: While photolithography is a widely used method, other techniques can also be employed for microfluidic device fabrication, such as:
Laser Ablation: A laser beam is used to remove material from a substrate, creating microchannels or features.
3D Printing: Additive manufacturing techniques can be used to directly fabricate microfluidic devices.
The choice of fabrication technique depends on factors such as the desired device complexity,material properties, and cost. Careful optimization of the fabrication process is essential to ensure the desired performance and reproducibility of microfluidic devices.
Process flow is given in Fig.5.
Pressure Pump and 3D digital microscope
The experimental system comprised of Elveflow pressure pump, Microqubic 3D digital microscope and microfluidic flow focusing device (Fig.6).
2. Pre-Lab Quiz
Quiz Date: xxxxxx
The quiz will cover the microfluidics, droplet generation devices, Navier Stokes equations, fabrication of microfluidic devices.
Please watch all the videos. You will be responsible from them as well.
3. Experimental Procedure
Step-by-Step Instructions:
Switch on Elveflow pump. Make sure inlet ports are closed.
Open vacuum source.
Open ESI software. Click on Calibration button. Let the system calibrate itself. Calibrate the system twice.
Switch on Microqubic 3D digital microscope.
Open Microqubic software. Choose See3Cam camera.
Under camera options adjust Exposure, set it to Auto.
Put your microfluidic chip on the stage.
Using joystick adjust focus until you observe microfluidic channels.
Connect DI and silicone oil vial to the inlets of pump.
Never let fluid to go inside pressure line.
On ESI software, click Stop All button.
Give 10 mbar pressure to the DI line. Wait until fluid comes at the edge of the precision tip.
Insert DI precision tip to the DI inlet on the microfluidic chip.
Close pressure.
Give 50 mbar pressure to the silicone oil line. Wait until fluid comes at the edge of the precision tip.
Insert DI precision tip to the DI inlet on the microfluidic chip.
Close pressure.
Go to DI inlet. Increase pressure to 20 mbar. Watch for the fluid while it is going through the microchannel. At this point you can lower the pressure, because DI should not go into the silicone oil channel. The reverse is also valid.
Go to silicone oil inlet. Increase pressure to 40 mbar. Watch for the fluid while it is going through the microchannel. At this point you can lower the pressure, because silicone oil should not go into the DI channel.
Using microscope joystick, go to the junction where droplet will be generated.
Adjust pressures accordingly until you observe droplet generation.
After droplet generation is observed, increase pressure of DI and silicone oil proportionally to observe varying size and speed droplets.
4. Data Collection
During the lab, collect the following:
Take a note of pressure values of both DI and silicone oil.
Take videos using Microqubic software.
Use these videos for image processing to determine droplet sizes (Code will be provided).
5. Post-Lab Report
Due Date: xxxxxx
Submit a report plotting :
DI pressure value vs. Droplet size for 10 different DI pressure values while fixing silicone oil pressure. Do not forget to include units.
Silicone pressure value vs. Droplet size for 10 different silicone oil pressure values while fixing DI pressure. Do not forget to include units.
Comparison of experimental data with theoretical predictions.
Additional resources :
EWOD device [ https://www.youtube.com/watch?v=srezdlbTQnU&ab_channel=Baebies%2CInc. ]
Paper-based microfluidic system [https://www.youtube.com/watch?v=7OPe-KcdKMM&ab_channel=LemelsonMIT ]
T-junction device droplet generation 1 : [https://www.youtube.com/watch?v=Gpt1vkVAZJo]
T-junction device droplet generation 2 : [ https://www.youtube.com/watch?v=aysInwrMyeM&ab_channel=WickedMicrofluidics ]
Flow focusing device droplet generation 1: [ https://www.youtube.com/watch?v=V_Vs81pLYoQ&ab_channel=CIDARLAB]
Flow focusing device droplet generation 2 : [ https://www.youtube.com/watch?v=T8uzesK2Aiw&ab_channel=ElveflowMicrofluidics ]
Fabrication of microfluidic devices : [ https://www.youtube.com/watch?v=RxoZhAevZVo&ab_channel=MicrofluidicsandMultiphaseFlowLaboratory-SUNYBinghamton ]
Pressure driven flow controller : [ https://www.youtube.com/watch?v=niWfINDUub4&t=220s ]
3D digital microscope : [ https://www.youtube.com/watch?v=IPmvcTY6J9M ]
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Responsible TA : Pelin Kübra İşgör, pisgor21@ku.edu.tr