What is Microfluidics?

Microfluidics is the flow and mixing of microlitre volumes of fluids via capillary action, where the fluids are contained within wells and channels made of glass and polydimethylsiloxane gel (PDMS). Figure 1 shows how the cured gel creates an enclosure between itself and the glass slide beneath. Essentially, this forms what you call a “lab-on-a-chip” device.

Figure 2: Photo of a microfluidics chip showing 3 channels.

A schematic diagram of the potential chip is shown in figure 3. The chip would require two inlets: one where the blood sample is deposited (Start chamber) and one where the stock solution is deposited (Middle chamber). The stock solution contains the siderophore-iron complexes that lipocalin in the blood sample would bind to. The two solutions would mix completely in the main channel before entering the end chamber where the bacteria will be contained. Once the mixed solution flows into the end chamber, it can stay there indefinitely until the bacteria takes up the siderophore-iron complexes and produces fluorescence. As an extension of this project, an albumin chelator could be added just before the Middle chamber in order to chelate albumin before it could come into contact with the siderophore-iron complexes.

protocol of the manufacturing process. Food colouring is used in place of blood and siderophore-iron solution to test how well the solutions mixed.

In order to determine the best microfluidics design for our project, we tested out different mixing designs as well as the fluid flow rate in different channel lengths.

The Theory

Mixing in microfluidics occurs in two ways: via diffusion or the introduction of turbulence. In order to encourage mixing in a microfluidics chip, one must induce it in either or both ways.

Firstly, diffusion is the movement of particles from a region of higher concentration to a region of lower concentration. For instance, when the blood and stock solution are flowing side by side, lipocalin would move from the blood into the stock solution since there is higher concentration of lipocalin in the blood as compared to the stock solution. This also holds true for the siderophore-iron complexes, which diffuse from the stock solution into the blood. Over time, there is an equal concentration of reagents on both sides and thus the solutions are mixed. In order to increase the rate of diffusion and hence mixing, the distance that the particles need to diffuse across needs to be shortened by minimising the width of the channel. For the protocol used, the minimum width that is feasible is 0.5 mm.

Introducing turbulence encourages mixing much like how stirring liquid in a mug helps to mix the contents.There are two types of flow: laminar and turbulent. Laminar flow is when the liquid particles travel in an orderly manner parallel to the channel length and in a single direction. The velocity of the liquid can be represented well using equations. In turbulent flow, the direction and speed at which each particle moves is unpredictable. The Reynolds number quantifies whether a flow is laminar or turbulent (Falkovich, 2011). It is defined as follows, where a higher Reynolds number indicates a more turbulent regime.

With respect to the lengths of a microfluidics chip (maximum 10 cm) , viscosity (3.5 × 10⁻³ Pa.s) and density (1060 kg/M³) of blood, the Reynolds number is around 10 (Bruus, 2007), which indicates laminar flow with minimal turbulence. The viscosity and density of blood remains constant throughout the test and the velocity cannot be easily controlled. However, the flow can be made more turbulent by increasing the length of the channel.

The Design Ideas

Design 1: The Capillary Stop Valve Idea

The initial design for mixing is shown in figure 4. Due to the surface tension of the liquid, the stock solution contained within the middle chamber would not flow out into the main channel. This design is called a capillary stop valve and has been shown to work in literature (Mastrangelo et al, 1998). The flow of liquid from the middle channel can only be initiated when the blood sample flows past it.

Figure 4: Diagram of the mixing idea 1 containing a capillary stop valve. Surface tension holds the blue liquid in place like a valve until the red liquid flows past, pulling the blue liquid into the main channel where they mix.

This design allows the stock solution to be contained in a chamber prior to a blood sample being taken. The length of the main channel between the middle chamber and the end chamber can be specified so that the binding of siderophores to lipocalin would have reached equilibrium by the time the solution reaches the end chamber. A potential pitfall of this design is that the surface tension is inadequate to contain the stock solution.

Design 2: The Well Idea

The second design consists of two layers of PDMS (figure 5). The bottom layer has a well holding the stock solution (blue). The middle well needs to be filled to the brim but should not flow into the channels when the top layer is placed on it. This can be done by calculating the volume that the well can hold prior to adding the stock solution. The blood sample (here shown in yellow) will flow across the well, mix with the blue solution and flow out of the well as the mixed green solution.

This design is harder to manufacture, as two layers of PDMS have to be made and aligned with each other and this often incorporates considerable amount of human error and poor reproducibility. It has also not been seen in literature before, thus the effectiveness of this design is unknown. The advantage to this design is that it is a simple idea that does not depend on the quality of the manufactured PDMS, unlike capillary stop valves (design 1). The timing could also be easily controlled by varying the lengths of the channels.

Figure 5: Diagram of how mixing design 2 should work. The yellow fluid flows over the bottom well and mixes with the blue fluid contained there before flowing to the end chamber.

Design 3: The Mixing Chamber Idea

The third design consists of a large mixing chamber where the two fluids will spread out over a larger area and thus flow over a longer distance, which would give them a higher chance of mixing. A longer distance to flow over also means we can extend the time for which the reagents are in contact, further promoting adequate mixing. The final channel is deliberately made to be long so that if the fluids are not mixed by the time they enter the final channel, they could mix via diffusion within the smaller width of the channel. When the fluid flows from the mixing chamber to the final chamber, the reduction in width of the channel promotes the fluid to reach the end chamber. This is important as one of the problems we faced in the lab was the fluid stopping in the final chamber mid-way before reaching the end.

Figure 6: Diagram (top view) of mixing design 3 containing a large mixing chamber. The yellow and blue fluids should mix within the mixing chamber.

This design has already been shown to work in the lab before and thus should be more foolproof. However, there might be difficulty controlling when the 2 solutions mix, thus potentially resulting in one solution entering the end chamber before the mixed solution does. Since the blood can enter the last chamber without any adverse effects on the bacterial cells and the results obtained, this disadvantage can be mitigated by adding the blood before adding the siderophore-iron solution into their respective inlet chambers.

The Results

Design 1: The Capillary Stop Valve Idea

Capillary stop valves have been shown to work in literature (Mastrangelo et al, 1998) but did not seem to work in our lab. We assume this is due to the quality of the PDMS chip produced when using our equipment and protocol (Boldock, 2013).

Figure 7 contains a photo of our result. The yellow solution, which represented the stock solution, did not stay in the chamber and flowed out into the main channel instead. Thus, it had to be quickly added after the red solution, representing the blood sample. The addition of the yellow solution had to be timed perfectly, as the red solution had the tendency to flow into both chambers. As can be seen from the photo, there is some leakage of red solution into the inlet chamber containing the yellow solution.

Figure 7: Photo showing the result of mixing design 1. Red food colouring representing the blood is added in the leftmost chamber, while yellow food colouring representing the stock solution is added in the middle chamber. Both fluids flowed towards rightmost end chamber.

The two solutions are not fully mixed by the time they reach the end chamber. The channel between the end chamber and the yellow chamber should have been longer and/or narrower to allow mixing via diffusion to take place.

Design 2: The Well Idea

Overall, the results indicate that the design did not work as expected.

In the first test, with a well of 4 mm diameter, blue dye from the inlet chamber entered the well and sank to the bottom. When the well was eventually filled, the yellow dye at the top of the well was pushed out into the channel (figure 8). Thus, while mixing did occur at the bottom of the well, the liquid going out of the well was completely yellow. Furthermore, when the well was covered by the top layer of PDMS, an air bubble was formed on top of the well, preventing the blue dye from entering the well. This was mitigated by punching a hole in the top layer to release the air.

Figure 8: Results of mixing design 2 with a 4mm yellow well. The blue dye sank to the bottom of the well and pushed the yellow dye out into the channel. The top two photos show the results from the top and side views; the bottom image is a diagram that represents the results.

Since the blue dye seemed to be denser than the yellow dye, as indicated by how it sank to the bottom of the well, we tested what would happen if the dye in the well was denser instead. Figure 8 shows the results. The two dyes were able to mix as shown by the green solution flowing out of the well. The green solution also managed to flow back towards the start chamber. However, this would not pose a problem as what matters is that a green solution reaches the end chamber.

Figure 8: Results of mixing design 2 with a 4 mm blue well. As the yellow dye was less dense than the blue dye, it was able to mix with the blue dye.

As blood is denser than the stock solution, it would be better to place the blood in the well rather than in the start chamber. Punching a hole in the top PDMS layer above the well to prevent an air bubble from forming has also helped in allowing blood to be deposited in the well.

It would be preferable for the well to hold the stock solution instead, as that would allow the stock solution to be transported with the chip instead of separately. Hence, we tested what would happen if we reduced the diameter of the well while using the same design (top view of figure 4). As predicted from earlier experiments, the blue dye sank to the bottom of the well due to its higher density. The blue dye also managed to flow in the channel around the well so that both the yellow and blue dye were in the main channel. Mixing did occur, as shown by the green solution in between the yellow and blue dyes. However, the blue dye reached the end chamber before the green solution and there is no way of knowing how much blue dye will reach the end chamber before the green solution arrives.

Figure 10: Results of mixing design 3. The blue and yellow dyes mixed well to give a green solution.

Figure 11: A close up photo of the PDMS of mixing design 3.

The Theory

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The Results

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