Modelling at a Microscale

Multi Lamination is a principle that relies on splitting the fluid stream into multiple smaller streams which are then recombined, increasing the interfacial area and promoting diffusion. This method is particularly effective in laminar flow conditions where molecular diffusion is the primary mixing mechanism.

Splitting and recombining structures (SAR) based designs mix fluids by dividing the main inlet stream into two or more subchannels and then recombining them. This process increases the interfacial area between different fluid streams (by causing the interface between streams to become distorted), promoting chaotic advection rather than relying solely on molecular diffusion.

During our research, we realized SAR micromixers are simple to construct, cost-effective, and very effective at inducing chaotic mixing. This makes them ideal for other iGEM teams hoping to incorporate microfluidic mixing into their projects.

Baffles and obstacles are used in microfluidic devices to enhance mixing by changing the flow direction and producing transverse flows. Changes in flow direction cause the fluid to fold and stretch continuously, while the transverse flows create separation vortices and flow dead zones that inhibit substance transport.

While these were very effective at mixing laminar flow, due to the subtractive nature of our manufacturing process, we weren’t sure we would be able to implement protruding obstacles in our channels.

This mixing strategy utilizes variations in channel cross-sections to enhance fluid mixing through the generation of vortices and flow velocity changes. It leverages reverse pressure gradients that form when fluid flows from a narrow channel into a wider one, creating expansion vortices. These vortices disrupt the flow and promote chaotic advection, creating eddy currents that increase the mixing of fluids.

These are also very easy to fabricate with laser ablation, and can be efficient micromixers. However, one potential drawback is the formation of flow dead zones, since expansion vortices can be separated from the main stream. This can be mitigated by ensuring a high enough fluid velocity within the channels to maintain flow in all regions.

These mixers utilize a helical or spiral channel design to enhance mixing through secondary flow generation, which is crucial in low Reynolds number regimes where turbulent mixing is not feasible. The helical structure induces rotational flow components, increasing the interaction between fluid layers and promoting efficient mixing within a compact device footprint. This design is particularly effective in achieving rapid and thorough mixing, making it ideal for various lab-on-a-chip applications.

While having many of the benefits of CAE designs, they lack the potential drawback of dead flow zones, making it very attractive with our team.

Dean vortices are pairs of counter rotating vortices generated in curved microchannels due to the interaction between centrifugal forces and viscous forces. They are one of the most effective ways to mix fluids, typically induced by spiral or serpentine microchannels. The working principle is that the smaller the radius, the stronger the centrifugal forces, which results in better mixing. When fluids flow in a curved microchannel, central fluids flow faster than the peripheral fluids, causing the outside fluids to be pushed to the top and bottom, generating counter-rotating vortices. These structures have no risk of flow dead zones and are easy to manufacture.

For a detailed description of our simulation process, please refer to this page: 📈Notes on CFD simulation

Description:
The Herringbone Helix Flow Chip integrates two powerful passive mixing strategies—herringbone ridges and helical flow—to enhance the efficiency of fluid mixing within microchannels (Channon et al., 2021). The
herringbone ridges, patterned along the channel walls, create chaotic advection by generating transverse flows, which disrupt laminar streams and increase the interaction between fluid layers. Combined with the helical flow design, which induces secondary rotational flows, this chip ensures thorough mixing even at low Reynolds numbers. This dual-flow design makes it ideal for applications requiring precise control over reaction conditions, such as DNA synthesis.

Why it was chosen:
The Herringbone Helix Flow geometry was chosen due to its
ability to effectively mix fluids even in low-flow conditions, making it highly suitable for precise DNA synthesis where maintaining controlled conditions is critical. Its design efficiently mitigates dead zones, ensuring complete mixing within a compact footprint. Moreover, the robustness of its helical flow design makes it ideal for processes requiring high reliability and consistency, while still being energy efficient and easy to fabricate.

Description:

The Multi Lamination Chip is designed to split fluid streams into multiple smaller laminar flows, increasing the interfacial surface area between different fluid layers. As the fluid passes through the microchannels, these streams are recombined, promoting molecular diffusion at the interfaces (Suh & Kang, 2010). This lamination effect enhances mixing efficiency without the need for external agitation, making it ideal for processes that rely on consistent and uniform fluid distribution, such as DNA synthesis or chemical reactions in synthetic biology.

Why it was chosen:
This Multi Lamination Chip geometry was selected for its
high precision in creating uniform fluid layers, which enhances the accuracy of reagent mixing during DNA synthesis. This chip provides fine control over mixing processes without the need for complex external controls. Its straightforward design ensures rapid fabrication and easy scalability, making it an excellent choice for high-throughput applications that prioritize precision over turbulence-driven mixing.

Description:

The “Pinball” Contraction and Expansion Chip leverages a series of alternating narrow and wide sections along the microfluidic channel (Viktorov et al., 2016). As fluid passes through these constricted and expanded regions, vortices are generated in the expansion zones due to reverse pressure gradients. This “pinball” effect disrupts the flow and enhances chaotic advection, ensuring that fluid streams mix effectively. The geometry of the chip is carefully designed to induce vortex formation at strategic points, allowing for efficient mixing with minimal energy input.

Why it was chosen:
The “Pinball” Contraction and Expansion Chip was chosen for its
unique ability to generate vortices through simple geometric features, making it highly efficient in inducing chaotic mixing without requiring external forces. This chip offers a balance between effective mixing and minimal material use, allowing for both energy and material conservation. It’s also easily fabricated with laser ablation, which aligns with our project’s goal of using sustainable, low-cost methods for scalable biomanufacturing.

Description:

The “Countered Spirals” Dean Vortices Chip utilizes curved microchannels to induce secondary flows known as dean vortices (Hong & Yeom, 2022). When fluids pass through these curved paths, centrifugal forces act on the fluid, creating pairs of counter-rotating vortices that significantly enhance mixing. The spiral design of this chip maximizes the interaction between fluid layers, promoting efficient mixing in systems with laminar flow regimes. The geometry ensures that as the fluid flows through the alternating spiral channels, the mixing is amplified by the continuous formation of dean vortices.

Why it was chosen:
The “Countered Spirals” Dean Vortices Chip was selected for its
ability to generate strong secondary flows in curved channels, making it highly effective at mixing even at low Reynolds numbers. This chip provides a unique advantage by maintaining laminar flow while inducing sufficient turbulence to ensure uniform mixing. It is particularly well-suited for applications that require delicate handling of fluids, such as DNA synthesis, where turbulent mixing might cause shear stress. Additionally, the curved design is easily fabricated using laser ablation techniques, ensuring that it meets both the sustainability and scalability goals of the project.

Description:

The “F Channel” Split and Recombination Chip operates by dividing fluid streams into smaller substreams that flow through branched channels (Kim et al., 2005). These substreams are later recombined, increasing the interfacial surface area between different fluid layers, which promotes diffusion and enhances molecular mixing. The “F” shape of the channels ensures that fluids are split multiple times, generating a higher degree of interaction between the streams to promote effective mixing.

Why it was chosen:
The “F Channel” Split and Recombination Chip was chosen for its
ability to achieve thorough mixing via simple geometric manipulation, making it a robust and efficient solution for fluid mixing in DNA synthesis processes. This chip excels in applications where the goal is to maximize the interaction between fluid streams in a controlled, laminar flow environment. Its design is easy to fabricate using laser ablation, ensuring quick production, and its scalability ensures that it can handle higher-throughput operations while still minimizing reagent use, aligning with our sustainability goals.

Description:

The Criss-Cross Split and Recombination Chip employs a series of alternating cross-channel segments to split the fluid flow into multiple streams, which are then recombined (Xia et al., 2005). The criss-cross pattern maximizes the interaction between streams, effectively disrupting laminar flow and enhancing the diffusion of molecules between the layers. This geometry ensures that as fluids pass through the chip, they undergo multiple splitting and merging cycles, which promotes efficient mixing through chaotic advection.

Why it was Chosen:

The Criss-Cross Split and Recombination Chip was selected for its high efficiency in achieving chaotic advection through repeated splitting and recombination. This chip is particularly effective at maintaining uniform mixing without introducing turbulent flow, making it ideal for DNA synthesis applications where precise fluid handling is critical. Its design supports high-throughput mixing while remaining simple to fabricate using laser ablation techniques, aligning with the project’s objectives of scalability, cost-efficiency, and minimal environmental impact. Additionally, the criss-cross geometry allows for highly reproducible results, making it a versatile option for a wide range of microfluidic processes.

Description:
The Fin Shaped Baffles Chip utilizes strategically placed fin-shaped obstacles within the microfluidic channel to alter the flow direction and generate transverse flows (Cortes-Quiroz et al’s, 2010). As fluids pass through the fins, the flow is continuously folded and stretched, creating vortices and enhancing chaotic advection. The unique fin geometry disrupts laminar flow in a controlled manner, ensuring that fluids mix efficiently even at low Reynolds numbers. The design minimizes dead zones and improves mixing efficiency by taking advantage of flow separation and reattachment around the fins.

Why was it chosen:
The Fin Shaped Baffles Chip was selected for its ability to induce chaotic mixing through simple yet effective geometric features, making it highly reliable for situations where precise and consistent fluid mixing is required. The fin-shaped baffles significantly enhance transverse flow without introducing excessive turbulence, ensuring that delicate reactants, such as those used in DNA synthesis, are mixed uniformly. This design is easily manufactured using laser ablation, making it both cost-effective and scalable. The chip’s ability to maintain high mixing efficiency while minimizing energy and reagent use aligns perfectly with the sustainability and scalability goals of our project.

Description:
The Serpentine Dean Vortices Chip features a serpentine microchannel layout that forces fluid to follow curved paths, generating dean vortices through the interaction of centrifugal and viscous forces (Yin et al., 2021). As the fluid flows through the alternating turns of the serpentine design, secondary vortices are formed, increasing mixing efficiency by promoting cross-stream diffusion. The continuous curves within the chip generate strong vortices, ensuring that even laminar flows experience effective mixing.

Why was it chosen:

The Serpentine Dean Vortices Chip was chosen for its exceptional ability to create strong Dean vortices within a compact, curved design, making it highly effective for applications that require efficient mixing in confined spaces. The serpentine geometry allows for increased mixing without requiring high fluid velocities, making it energy efficient and suitable for delicate biological processes, such as DNA synthesis. Its fabrication is straightforward using laser ablation, and its design minimizes reagent use, aligning with the project’s sustainability goals. The chip’s ability to consistently generate vortices ensures reliable mixing performance, making it a versatile tool for scalable biomanufacturing.

Description: The ZigZag Contraction and Expansion Chip employs a zigzag microchannel design that alternates between narrow and wide sections. This repeated contraction and expansion generate significant flow disturbances, creating vortices and enhancing chaotic advection. As the fluid passes through the narrow sections, it accelerates, and upon reaching the expanded sections, it decelerates, promoting mixing through turbulence. The zigzag geometry increases the interaction between fluid layers and prevents the formation of dead zones, leading to more efficient mixing even at low Reynolds numbers. (Natsuhara et al.’s, 2022).

Why Was It Chosen?
The ZigZag Contraction and Expansion Chip was selected for its ability to generate consistent and efficient mixing through simple geometric manipulation. The contraction-expansion pattern in the zigzag layout allows for effective mixing without requiring external energy sources, making it a sustainable solution for fluid mixing in DNA synthesis applications. Its ease of fabrication using laser ablation techniques ensures that it can be reproduced at a low cost while maintaining high performance. Additionally, the chip’s design promotes scalability, as its predictable and controlled mixing behavior can be easily adapted for larger-scale biomanufacturing processes.

Description:
The Planar Split and Recombination Chip uses a series of planar microchannels to split the fluid into smaller streams, which are then recombined downstream (Husain et al., 2018). The planar configuration allows for efficient manipulation of the fluid in a two-dimensional space, promoting increased interaction between the split streams. This layout generates chaotic advection through the constant splitting and recombining of fluid streams, enhancing diffusion and mixing efficiency without requiring complex geometries or external energy inputs.

Why Was It Chosen?
The Planar Split and Recombination Chip was chosen for its ability to achieve effective mixing within a simple, planar structure, making it ideal for applications where spatial constraints are a consideration. Its planar design minimizes the complexity of fabrication while still promoting strong mixing through passive means. The chip is straightforward to manufacture using laser ablation techniques, which aligns with our project’s goal of low-cost, scalable production. Additionally, its simplicity reduces the potential for fabrication errors, ensuring reliable performance in DNA synthesis and other biomanufacturing processes. The planar structure also allows for easy integration into multi-layer microfluidic systems, making it a versatile and efficient solution.

Description:
The Cantor Baffles Chip leverages a fractal-inspired baffle design based on Cantor sets, where baffles are arranged in a self-similar, repeating pattern (Wu & Chen, 2019). This fractal geometry enhances the mixing of fluids by creating multiple points of flow disruption at different scales, generating small and large vortices. As fluids pass through the channels, they are subjected to multiple changes in direction, leading to chaotic advection and highly efficient mixing. The Cantor baffles are designed to increase the surface area for fluid interaction without significantly increasing the complexity of the channel layout.

Why was It chosen:
The Cantor Baffles Chip was chosen for its ability to induce efficient mixing through a fractal pattern that optimizes flow disruption across multiple scales. This chip’s unique baffle arrangement ensures thorough mixing with minimal energy input, making it suitable for high-throughput processes such as DNA synthesis. The fractal design promotes mixing at both micro and macro levels, which enhances fluid interaction and reduces dead zones. Additionally, the chip is easy to fabricate using laser ablation, which ensures that the design can be reproduced at low cost and with minimal environmental impact. The fractal baffle structure also minimizes material use while maximizing performance, aligning with the project’s goals of sustainability and scalability.

For usability and reproducibility, we produced design specifications for all microfluidic chips that are to be designed. These specifications are modified from Dr. Albert Folch’s BIOEN 455 course at the University of Washington.

• Length of the chip has to be 50.00 mm
• Width of the chip has to be 60.00 mm
• There must be four holes in the corner of every layer, with a diameter of 5.00 mm
• Distance between the center of the four holes to the edge of the chip has to be 4.00 mm
• All inlet and outlet fluidic channel holes have to be 2.00 mm
• The distance between any inlet or outlet hole has to be greater than 10.00 mm
• Layers must be printed in 1mm thick acrylic PMMA plastic

• Sheets are to be bonded together by 3M 9495MP double sided adhesive tape

  

Once the simulation is run and results are calculated, we can then see the flow trajectory and select point parameters to analyze the mass fraction of water at the outlet channel. Intuitively, the outlet channel should be the point at which the fluids should be maximally mixed.

Previously, methods for quantifying mixing between two or more coloured liquids involved a measure of standard deviation through mixing indices (Hashmi & Xu, 2014). Thus, we want to communicate the spread of concentrations that are present at our outlet valve, which would also involve calculating the standard deviation of the mass fraction. The point parameters function provides the mass fraction at 100 points (can be altered) on the outlet channel. The values were then exported into excel to calculate the standard deviation. This measure will tend to 0 as the fluid is mixed more fully.

We therefore produced two evaluation criteria related to the results obtained from these fluid simulations.

Evaluation Criteria #1: Standard Deviation at Outlet Chip

The first criteria will score each design based on the standard deviation value we obtained at the outlet channels. The closer the standard deviation is to 0, the greater the satisfaction as the fluid is mixed more fully closer to 0. This is a linear relationship as satisfaction levels decrease the same when there is an increase in standard deviation.

Evaluation Criteria #2: Simulation Run Time

The second criteria will score each design based on the time it took for the simulation to run and calculate results. The runtime would represent how easy and quickly the mixing and experiment will occur in the lab. A piecewise optimum curve was used to take into consideration the accuracy of our simulation model. The mesh density must be decreased for a very fast runtime, which would decrease the accuracy of the analysis results. Thus, to optimize both accuracy and runtime, max satisfaction would occur at 1.5 minutes, the standard time required to run a flow simulation at a medium-mesh density of 4.

Following laser printing all our chip designs, we thought of a simple titration experiment to physically test the mixing efficiency of each chip. We used two syringe pumps, which would pump NaOH through one inlet and HCl through another inlet. The base and acid would have an indicator, bromothymol blue, added to them, which would indicate blue for base and yellow for acid. As the strong base and acid mixes throughout the chip channels, it would neutralize to a green color at the outlet hole.

Evaluation Criteria #3: Titration Colour Value

Using a digital color meter, the hexadecimal # and RGB values of the liquid color at the outlet channel were measured. The RGB values of the neutralized green, most basic blue, and most acidic yellow were used as references to compare against to produce the satisfaction curve. A piecewise optimum curve was used as anything above the neutralized green color is considered too yellow / too acidic and anything below is considered too blue / too basic. The minimum values were obtained from the most basic blue color and the most acidic yellow color. Anything above or below the neutralized green value would lower the satisfaction.

Evaluation Criteria #4: Plastic Waste

While implementing microfluidic chips in our workflow encourages a more sustainable use of reagents and reactions, we need to ensure the manufacturing process is as sustainable as possible. Some designs have differing complexity and differing amounts of layers. Each layer is made out of PMMA plastic; thus, to evaluate our plastic use, satisfaction decreases with more plastic layers. It is discrete since layers are either 3, 4, 5, or 6. Maximum satisfaction is at 3 layers since all LPS designs have the same base and face chip, with a minimum of one specially designed chip in between (SPS can have 2 layers). The maximum layer of 8 is decided upon design requirement #11.

Evaluation Criteria #5: Assembly Difficulty

Following the laser ablation of each chip, plastic bits must be manually removed and each layer has to be assembled. Depending on the intricacy and complexity of the channel design, some layers may require a microscope to carefully remove microscopic bits of plastic. The placement of adhesive also affects how easy it is to assemble each layer. Based on these factors, the difficulty in assembling each design can differ. Thus, individuals that contributed to assembling each chip are asked to qualitatively determine the difficulty on a scale of 1 to 10. More satisfaction is associated with decreased difficulty, thus, a decreasing linear plot is used.

Here, we included the results from our SolidWorks Flow Simulations and Titration Experiments for each of the LPS chips. We can observe how our computational simulations are actually reflected in realistic, in-lab experiments with the chips.

”Herringbone” Helix Flow Chip

We can see that the two inlet liquids are being mixed at the intersection of the Y. Molecules are rocking back and forth at the chevrons, allowing mixing to occur.

“Binary Tree” Multilamination Chip

We can see that the liquid was not able to flow through the outer channels, which indicates a flow dead zone, leading to no mixing in those areas. The amount of molecules flowing through the left and right channels are also disproportionate. This simulation result was consistent with in-lab results as we observed the fluid flowing much slower through the left channel than the right, and no fluid flowing through the outer channels. Possible explanations for the flow dead zone formations could be that the sharp corners leading to multiple channels could have significantly decreased the flow velocity. While assembling the layers on SolidWorks, the layers may have been slightly uneven or asymmetric, leading to imperfect splitting / combining of the channels.

“Pinball” Contraction and Expansion Chip

The molecules are flowing around the pinball obstacles through the two layers, promoting mixing. By observing the colours, the liquid is pretty well mixed right when the two liquids meet, at the intersection.

“Countered Spirals” Dean Vortices Chip

The two liquids are mixed well at the intersection of the inlet channels as it enters the spirals. The molecules flows through the spirals smoothly, as it alternates flow directions, become increasingly well mixed as it goes down the channels.

“F Channel” Split and Recombination Chip

The two liquids are mixed well after flowing through the first “F”. The molecules are divided relatively evenly at the F arms, which are then combined before moving onto the next “F”.

“Criss-Cross” Split and Recombination Chip

The two layers come together to form criss-cross shaped channels, where the molecules overlap in alternating directions at each criss-cross. The molecules are then recombined and mixed before reaching the next criss-cross.

Titration Experiment Results

Footage from our Titration Experiments was compiled into the video below: