Investigating the effect of phospholipids on droplet formation and surface property evolution in microfluidic devices for droplet interface bilayer (DIB) formation

Determining the validity of the in silico model

We have designed an in silico (computational) model and, together with experimental (in situ) data, used it to better understand the effect that lipids have on the formation of droplets for the creation of DIBs at a microfluidic T-junction. The graphical representation of the in silico model and the key dimensions of the model and of the microfluidic device used for in silico and in situ data acquisition, respectively, can be found in Fig. 2. In both cases, aqueous (buffer) droplets were formed in an oil (hexadecane) phase at a microfluidic T-junction, with the lipid (DPhPC) dosed in either of the two phases. Control data without lipids were also gathered.

When developing an in silico model, it is important to determine how well it mimics, and hence predicts, real-world situations. The validity of our in silico model was verified according to whether it accurately predicted droplet length under control (no-lipid) conditions in situ. If the model satisfied this criterion, we could then attempt to use it to predict and explain the droplet lengths which would be observed for the other in situ (lipid-out, lipid-in) conditions. First, we determined whether the in situ and in silico droplet lengths were significantly different (two-level ANOVA) for the no-lipid condition. Then, to check whether the addition of lipid caused a change in droplet length in silico, we carried out a three-way ANOVA between no-lipid, lipid-out, and lipid-in conditions.

Our data show that the in silico model we developed satisfies our proposed criterion for validity, as shown in Fig. 4. We measured the length (as a proxy for volume) of a droplet following its formation at a T-junction both in silico and in situ with no lipid present in the system. The first column in Fig. 3(a) shows representative images under the no-lipid condition. We analyzed the droplet length formed with no lipid in the system, and two-level ANOVA between the in silico and in situ droplet lengths showed no statistically significant (p = 0.38) difference between them. Figure 3(b) shows these in silico (purple) and in situ (green) data for the no-lipid condition.We then changed the contact angle and interfacial tension values in our in silico model to match those of the conditions with lipids (i.e., lipid-in and lipid-out) with the aim of determining whether it was able to show the effect that lipids have on the droplet length. These simulations ignore mass transfer and adsorption effects to focus on modeling the effect of the surfactant on the interfacial tension and contact angle only. These data are shown in purple in the graph shown in Fig. 3(b). Three-level ANOVA between the three in silico conditions revealed a statistically significant difference between the no-lipid, lipid-out, and lipid-in conditions (p=7.4−6). Therefore, we conclude that the model does not include all of the physics required to predict the droplet length when lipids are added.To understand the lack of change in droplet length upon addition of lipid, we remind the reader that this comparison was done using only the first five droplets. This means that it is possible that at early times the channels may still be equilibrating in one or more ways. For example, we see in Fig. 3(a) that the contact angle in situ does not completely match the contact angle in silico. This is likely due to mass transfer effects, but could also be due in part to complexities related to defining a contact angle since we have not attempted to include contact angle hysteresis. However, because of this, we will now compare our results with measurements of thousands of droplets at later times.

Determining the effect of phospholipids on droplet formation in situ

The reduced droplet size observed for lipid containing solutions in silico is expected due to their reduced interfacial tensions: 21.04 mN m−1 for lipid-out and 10.89 mN m−1 for lipid-in, compared to 55.2 mN m−1 for no lipid.23,3923. J. D. Wehking, M. Gabany, L. Chew, and R. Kumar, “Effects of viscosity, interfacial tension, and flow geometry on droplet formation in a microfluidic T-junction,” Microfluid. Nanofluid. 16, 441–453 (2014). https://doi.org/10.1007/s10404-013-1239-039. A. Goebel and K. Lunkenheimer, “Interfacial tension of the water/n-alkane interface,” Langmuir 13, 369–372 (1997). https://doi.org/10.1021/la960800g Another factor that is known to affect droplet length is the contact angle, with higher contact angles resulting in smaller droplets.4343. H. Liu and Y. Zhang, “Droplet formation in a T-shaped microfluidic junction,” J. Appl. Phys. 106, 034906 (2009). https://doi.org/10.1063/1.3187831 This contact angle effect is consistent with the decrease in droplet length observed in the lipid-out conditions, where θdevice increased from 157° for no lipid to 180° for lipid-out. However, lipid-in conditions exhibited the smallest droplet length, despite the contact angle being smaller at 121°. Hence, the greatest factor affecting droplet length in silico appears to be the interfacial tension, as otherwise the contact angle decrease seen with the lipid-in conditions would very likely lead to droplets being larger than observed, as the effects of contact angle and interfacial tension on droplet size are working in opposition to each other.23,4323. J. D. Wehking, M. Gabany, L. Chew, and R. Kumar, “Effects of viscosity, interfacial tension, and flow geometry on droplet formation in a microfluidic T-junction,” Microfluid. Nanofluid. 16, 441–453 (2014). https://doi.org/10.1007/s10404-013-1239-043. H. Liu and Y. Zhang, “Droplet formation in a T-shaped microfluidic junction,” J. Appl. Phys. 106, 034906 (2009). https://doi.org/10.1063/1.3187831Carrying out two-level statistical comparison between the in situ, lipid containing conditions, and the in silico, no-lipid condition, there was no statistically significant difference observed for either lipid-out (p = 0.35) or lipid-in (p = 0.48). This demonstrates that, at least for the first ffive droplets, both lipid-out and lipid-in systems in situ behave indistinguishably from the predicted behavior of a system containing no lipid. These findings are surprising for a microfluidic system, as it is known that stable DIBs can be formed on a microfluidic device orders of magnitude faster than in bulk solution,4444. S. Thutupalli, J.-B. Fleury, A. Steinberger, S. Herminghaus, and R. Seemann, “Why can artificial membranes be fabricated so rapidly in microfluidics?,” Chem. Commun. 49, 1443–1445 (2013). https://doi.org/10.1039/c2cc38867g suggesting that the assembly of lipid at the fluid interface should also be considerably faster than those determined by the pendant drop method.1717. E. Hildebrandt, H. Nirschl, R. J. Kok, and G. Leneweit, “Adsorption of phospholipids at oil/water interfaces during emulsification is controlled by stress relaxation and diffusion,” Soft Matter 14, 3730–3737 (2018). https://doi.org/10.1039/C8SM00005K However, it is important to note that we are studying how droplet formation occurs when phospholipids are present. DIBs will only form if the droplets come together slowly enough once they are properly covered with a phospholipid monolayer, otherwise droplets will merge. This also highlights the possible pitfalls in extrapolating computational data to the real world, as in silico determination of the relationship between interfacial tension and droplet size may be misleading when predicting a corresponding droplet size upon the addition of surfactants.However, we can also look beyond the first five droplets to see what happens at longer times. To gain more insight into the dynamics of equilibration, we also measured the average droplet size for thousands of droplets during continuous operation of the microfluidic device for three different flow rates at each of the three lipid conditions. The droplet lengths measured for each case are plotted in Fig. 4 as a function of the Capillary number, Ca, defined asHere, η is the viscosity and Q is the volumetric flow rate of the continuous phase (oil), A is the cross-sectional area of the channel where the droplets are formed, and γ is the interfacial tension. In each case, the equilibrium value of γ was used to generate the plot.Figure 4 shows several interesting results. First, we see that the average droplet lengths for the three lipid conditions are still approximately the same at the lowest flow rate (the lowest Capillary number for each data set), though they are larger than the first five droplets. The increased size at later times shows that indeed there is some kind of equilibration that is not yet complete for the first five droplets. However, it also highlights the importance of using appropriate non-dimensionalization when comparing conditions. Specifically, if one only compared the droplet length for each of the three lipid conditions at the lowest flow rate, one might conclude that adding lipid makes no difference. However, the systems can only be called hydrodynamically equivalent when they are operated at the same Capillary number4545. G. F. Christopher, N. N. Noharuddin, J. A. Taylor, and S. L. Anna, “Experimental observations of the squeezing-to-dripping transition in T-shaped microfluidic junctions,” Phys. Rev. E 78, 036317 (2008). https://doi.org/10.1103/PhysRevE.78.036317 and so should only be expected to have the same droplet length if the Capillary number (and other relevant dimensionless quantities like viscosity ratio, flow rate ratio, geometric aspect ratios, etc.) are the same.In line with the previous point, another result that we see in Fig. 4 is that at the same Capillary number, the mean droplet size for each condition is quite different. This proves that adding lipid changes the droplet formation process and that adding lipid to the droplet phase (lipid-in) is different from adding it to the continuous phase (lipid-out). The reasons for this difference are discussed in more detail below. A final result that we wish to point out in Fig. 4 is that the standard deviation in the droplet length for the no-lipid case is much larger than for other conditions. This is not unexpected since droplet formation is known to be disordered for a system without surfactant, and the formation process is irregular.4646. R. Dreyfus, P. Tabeling, and H. Willaime, “Ordered and disordered patterns in two-phase flows in microchannels,” Phys. Rev. Lett. 90, 144505 (2003). https://doi.org/10.1103/PhysRevLett.90.144505

Determining the effect of phospholipids on long-term microfluidic device operation

In addition to playing a role during droplet formation, we have previously shown that surfactants in microfluidic devices also change the surface properties of the rest of the device.88. A. P. Debon, R. C. R. Wootton, and K. S. Elvira, “Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies,” Biomicrofluidics 9, 024119 (2015). https://doi.org/10.1063/1.4917343 Having established that lipids alter the droplet formation to affect droplet length, we then examined the effect of lipids on the wetting properties of the microfluidic device. In order to examine whether there are any changes to the surface properties of the microfluidic device during operation in situ, a relationship between contact angle, as a means to quantify wetting characteristics, and a parameter that can be seen visually on a microscope needed to be established. One such variable is the recession distance (drecession), which is the distance that the aqueous fluid recedes into the inlet channel after the formation of a droplet [Fig. 2(b)]. Note that there is no backflow in the device and this recession distance is simply a reflection of the location where the droplet broke away from the upstream fluid.We have observed experimentally that as devices develop poor wetting properties, the distance that the fluid interface recedes following droplet formation grows shorter. Moreover, we have observed that shortly after drecession reaches zero, the aqueous fluid interface begins to move further into and down the channel, a critical step in the transition to the dripping droplet failure mode (Fig. 1).88. A. P. Debon, R. C. R. Wootton, and K. S. Elvira, “Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies,” Biomicrofluidics 9, 024119 (2015). https://doi.org/10.1063/1.4917343 To test whether drecession was a suitable visual reporter parameter for the change in device surface properties, we used our in silico model to perform a single-parameter, parametric sweep of device contact angles (θdevice) over the range of 120°–180°, and ran each of the models over the window of time needed for three droplets to form, to confirm whether a clear relationship between the θdevice and drecession exists. This contact angle range was chosen to accommodate the entire range of contact angles seen in silico during the initial model validation.While we readily acknowledge the limitations that we have already identified in the in silico model, precedent exists for using such an idealized model of droplet formation, as previous computational work relating droplet size to interfacial tension typically assumes that interfacial tension is a fundamental property of the fluid system, rather than a time-dependent process induced by a surfactant.3,24,32,433. X.-B. Li, F.-C. Li, J.-C. Yang, H. Kinoshita, M. Oishi, and M. Oshima, “Study on the mechanism of droplet formation in T-junction microchannel,” Chem. Eng. Sci. 69, 340–351 (2012). https://doi.org/10.1016/j.ces.2011.10.04824. X. Li, L. He, Y. He, H. Gu, and M. Liu, “Numerical study of droplet formation in the ordinary and modified T-junctions,” Phys. Fluids 31, 082101 (2019). https://doi.org/10.1063/1.510742532. M. Y. A. Jamalabadi, M. DaqiqShirazi, A. Kosar, and M. S. Shadloo, “Effect of injection angle, density ratio, and viscosity on droplet formation in a microfluidic T-junction,” Theor. Appl. Mech. Lett. 7, 243–251 (2017). https://doi.org/10.1016/j.taml.2017.06.00243. H. Liu and Y. Zhang, “Droplet formation in a T-shaped microfluidic junction,” J. Appl. Phys. 106, 034906 (2009). https://doi.org/10.1063/1.3187831 More specifically, prior work with the natural phospholipids found in soy lecithin shows the inability of solutions containing soy lecithin to form droplets lipid-out at a Y-junction, while Span 80-containing solutions did, which was explained by the poor mobility of lecithin in hexadecane, and therefore a slow drop in interfacial tension relative to Span 80.2121. F. Y. Ushikubo, F. S. Birribilli, D. R. B. Oliveira, and R. L. Cunha, “Y- and T-junction microfluidic devices: Effect of fluids and interface properties and operating conditions,” Microfluid. Nanofluid. 17, 711–720 (2014). https://doi.org/10.1007/s10404-014-1348-4 Therefore, the in silico model was used as a qualitative check of our hypothesis that drecession is correlated to θdevice.As shown in Fig. 5, our in silico model shows a clear dependence of drecession on θdevice. This figure shows both the raw data for drecession measured at each θdevice, as well as an empirical fitted curve. A non-linear, positive relationship exists between θdevice and drecession over this range, showing that even under simplified conditions, drecession is a suitable means of indicating changes in contact angle visually on microscopy images for in situ experiments. With this quantitative relationship between drecession and θdevice established, we have a means of characterizing the surface properties of a microfluidic device during its operation and we can hence examine the effect of lipids on surface property evolution over time.To determine the effect of DPhPC on device surface properties, video footage of the microfluidic T-junction was collected from the point when water first reached the T-junction until device failure. Device failure was defined to occur when when droplet formation ceased due to co-flow or the formation of plugs, whichever occurred first. The results of measuring drecession for the first 100 s (or until device failure) are shown in Fig. 6. This figure shows quantitatively that drecession decreases over time in situ for droplets created with no lipids (black squares), lipid-out (red circles) or lipid-in (blue triangles). This, in turn, implies a decrease in the contact angle, and an increase in the ability of the water phase to wet the channel as time progresses.In Fig. 6, we also show images just after a droplet pinch-off event at the start of the flow (first row of images) and just after the point when drecession is no longer visible (second row of images). This is clear visual confirmation that the wetting properties of the channel are changing over time and in each of the images an arrow shows the approximate location of the contact line where the droplet phase is still adhering to the wall. The effect is even more pronounced at later times as shown in the third row of images in Fig. 6. At much later times, we see that the contact line, where the wetted region ends, has progressed farther down into the channel, forcing droplet breakup to occur further down the channel and resulting in transition to the dripping regime and eventually the co-flow regime.

When comparing the three different lipid conditions at later times, we see that in the lipid-out case the wetted region has propagated only a short distance into the channel even at very long times. The lipid-in case, however, has allowed the wetted region to propagate about halfway down the visible portion of the channel in approximately the same amount of time, resulting in the dripping failure mode. Finally, in the no lipid case, the wetted region has quickly propagated beyond the visible portion of the channel resulting in complete device failure.

Surfactants are known to coat the walls of PDMS microfluidic devices during their operation, contributing to their equilibrium surface properties.88. A. P. Debon, R. C. R. Wootton, and K. S. Elvira, “Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies,” Biomicrofluidics 9, 024119 (2015). https://doi.org/10.1063/1.4917343 It is also generally assumed that a surfactant dosed in the aqueous phase of water-in-oil droplets has no effect on the surface properties of the microfluidic device. However, it is common in the field of microfluidics to “prime” the channels in the microfluidic device by flowing only the oil phase (for water-in-oil droplet formation) through the device prior to droplet formation, though it is not clear whether it is the oil or the surfactant (or both) that is affecting the surface characteristics during this process. Here, we wanted to study the effect that the presence of lipids has on the longevity of the device. Our data provide the first in situ quantification of the different rates of surface property changes when phospholipids are used in a PDMS microfluidic device.The data in Fig. 6 show that the addition of lipid clearly affects the device longevity, whether it is dosed in the oil phase or in the aqueous phase. Where no lipid is present, droplet formation ceases to be possible within 1 s of device usage. When lipid is added lipid-out, there is a gradual decrease in device performance over time, as signified by the drop in the recession distance. This means that droplet formation ceases to occur cleanly at the T-junction and begins to occur further into the channel over the course of 100 s. When the lipid is added lipid-in, this process occurs much more suddenly at around 1 s after initiation of droplet formation. These data show that the surfactant-like behavior of lipids in microfluidic devices is complicated.

We measured the contact angle of oil on PDMS to be 39°, which decreases to 23° upon the addition of lipid. Likewise, we measured the contact angle of the aqueous phase on PDMS to be 114°, which decreases to 70° upon the addition of lipid. This suggests that the lipid behavior is changing based on its preference for interaction with the oil phase or with the surface of the PDMS. The decrease in contact angle upon addition of the lipid to the oil phase suggests that the lipid enhances the wetting of the oil to the PDMS surface by making the surface more lipophilic, and hence that the lipid molecules arrange themselves tail out on the channel surface. Likewise, the decrease in contact angle upon addition of the lipid to the aqueous phase suggests that when droplets are in contact with the channel walls, the lipid makes the surface of the PDMS more hydrophilic, and hence that the presence of droplets encourages the lipid molecules to arrange themselves headgroup out on the channel surface.

We therefore rationalize our findings as follows. In the lipid-out case, the gradual decrease in the recession distance indicates that the surface of the channel is becoming more hydrophilic over time as more droplets are created, which agrees with our prior work showing that droplets effectively strip the surfactant from the channel surface causing wetting of the aqueous phase.88. A. P. Debon, R. C. R. Wootton, and K. S. Elvira, “Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies,” Biomicrofluidics 9, 024119 (2015). https://doi.org/10.1063/1.4917343 After 100 s of device operation, we can still form droplets but these are created further down the channel. In the lipid-in case, initially the device behavior follows the same trend as with the no-lipid case, but after around 10 s mass transfer of the lipid from the droplet to the channel wall makes the walls even more hydrophilic than in the lipid-out case.

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