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Mixing enhancement induced by viscoelastic micromotors in microfluidic platforms

Fine manipulation of fluid flows at the microscale has a tremendous impact on mass transport phenomena of chemical and biological processes inside microfluidic platforms. Fluid mixing in the laminar flow regime at low Reynolds is poorly effective due to the inherently slow diffusive mechanism. As a strategy to enhance mixing and prompt mass transport, here, we focus on polyelectrolyte multilayer capsules (PMCs) embodying a catalytic polyoxometalate as microobjects to create elastic turbulence and as micromotors to generate chaotic flows by fuel-fed propulsions. The effects of the elastic turbolence and of the artificial propulsion on some basic flow parameters, such as pressure and volumetric flow rate are studied by a microfluidic set-up including pressure and flow sensors. Numerical-handling and physical models of the experimental data are presented and discussed to explain the measured dependence of the pressure drop on the flow rate in presence of the PMCs. As a practical outcome of the study, a strong decrease of the mixing time in a serpentine microreactor is demonstrated. Unlike our previous reports dealing with capillarity flow studies, the present paper relies on hydrodynamic pumping experiments, that allows us to both develop a theoretical model for the understanding of the involved phenomena and demonstrate a successfully microfluidic mixing application. All of this is relevant in the perspective of developing microobject based methods to overcome microscale processes purely dominated by diffusion with potential improvements of mass trasport in microfluidic platforms.


Introduction
Integrated microfluidic platforms for lab-on-a-chip (LOC) and organ-on-a-chip (OOC) applications are of fast growing applicative interest with huge potential for the development of laboratory scale and industrial products. Compared to bulky and traditional platforms, LOCs and OOCs possess many potentials and benefits (small consumption of reagents and samples, short reaction times, highly parallel and multiplexed analysis and portability) which make them useful for biochemical analyses, chemical processing applications [1,2], environment and sanitation safety [3], drug delivery, cell culture manipulation [4][5][6]. Nowadays, due to the important advances in fabrication technologies and miniaturization, LOCs and OOCs can be realized with sub-micrometer level of precision and reproducibility. Moreover, significant improvements in terms of device performance are due to the are able to move in the fluid like swimming micro-organisms and produce gas bubbles; 3) gas bubbles motion can be further enhanced by applying ultrasonic waves.
In our previous works, the H2O2 dismutation by means of Ru4POM-PMCs was studied in a capillary flow regime with a focus on kinematics quantities (velocity and acceleration of the fluid) [36,37] and the pressure-driven turbulence effects (corresponding to an applied pressure of tens of Pa) were demonstrated to blow up flexible thin membranes for gas sensing [38] or liquids pumping applications [39]. As further step, stimulated by the considerable development of applications of micromotors in the pharmaceutical, biomedical and chemical fields, in this work we evaluated the impact of activated PMCs on the fluid mixing under hydrodynamic pumping conditions, which, unlike capillary flow regime, are operative common working flow conditions. Compared to viscoelastic polymers, that are miscible with the working liquid and hence difficult to remove from the mixture, soft PMCs can be easily removed from the reaction environment via centrifugation or external magnetic fields whenever decorated with magnetic nanoparticles. Moreover, when stimulated by a proper chemical, PMCs act as self-propelled micromotors due to an oxygen bubble-driven mechanism where the propulsion is due to pressure changes and recoil effects favored by bubble generation in the presence of H2O2 fuel source. Hence, capsules moving in the background fluid speed up the diffusion-limited mass transport through detaching or bursting bubbles from their surface. level for LOC and OOC platforms to improve mass transport in experimental conditions purely dominated by diffusion.

Materials
For the fabrication of microchannels, commercially available B-270 glasses, cut in 2.5 cm x 2.5 cm x 1.1 mm (width x depth x height) slides and covered with 450 nm-thick Chromium (Cr) layer (Telic, USA), were used as solid substrates. Hydrochloric acid (HCl), ammonium fluoride (NH4F), and hydrofluoric acid (HF) were purchased from Sigma-Aldrich (Taufkirchen, Germany For the mixing experiments, hydrogen peroxide (H2O2, 30%) and eosin were purchased from Sigma-Aldrich (Milan, Italy).
Briefly, 20 mg of calcium carbonate (CaCO3) microparticles were sequentially resuspended for 12 min in 1 mL of 0.5 M NaCl solution containing the polyanion PSS (2 mg mL-1, pH = 6.5) or the polycation PAH (2 mg mL-1, pH = 6.5). The excess polyelectrolytes were removed by three washing with 1 mL of milli-Q H2O and three centrifugation steps (4500 rpm for 5 s). For preparing PMCs of batch B1, the microparticles coated with two bi-layers of (PSS/PAH) were incubated in 1 mL of Ru4POM solution (H2O, pH = 5.0, 2 mg mL-1, 0.35 mM). Then, three additional bi-layers of (PSS/PAH) were deposited to obtain the following multilayer shell composition (PSS/PAH) (Ru4POM/PAH)2(PSS/PAH)3 (batch B1, Figure 1a). Instead, for obtaining PMCs of batch B2, the microparticles carrying five bi-layers of (PSS/PAH) were incubated in 1 mL of Ru4POM solution (H2O, pH = 5.0, 2 mg mL-1, 0.35 mM) to obtain the following multilayer shell (PSS/PAH)5(Ru4POM) (batch B2, Figure 1b). PMCs without Ru4POM (batch termed B3) were also prepared and used as control system (Figure 1c). The sacrificial CaCO3 cores were removed by complexation with EDTA buffer. Finally, the PMCs were stored as suspension in 500 μL of milli-Q H 2 O at 4 °C. All obtained PMCs showed a spherical shape, with average diameter of 4.00 + 0.04 m (Figure S1a-c). The PMCs number per volume was determined by direct counting by a haemocytometer under a microscope by using a 20X objective in phase contrast channel. The number of PMCs was estimated to be around 9.05x108 per mL in batch B1, 1.12x109 PMCs per mL in batch B2, and 7.8x108 PMCs per mL in batch B3. The uptake of the Ru4POM in batches B1 and B2 was estimated by UV-Vis analysis (Cary 300 UV-Vis, Varian). It was calculated from the absorbance difference (=270 nm,  = 74846 M -1 cm -1 ) between initial feeding solution (total Ru 4 POM) and the supernatants recovered after the incubation step of microparticles with the feeding solution of Ru4POM (free Ru 4 POM) ( Figure S1d).
A total amount of 203 M and 189 M of Ru4POM was estimated for batch B1(9.05x108 per mL) and for batch B2 (1.12x109 PMCs per mL).

Flow sensor -based measurements in a linear microchannel
In perspective of mass transport in pure laminar regime, we analyzed the effects on flow dynamics of all the three PMC batches (B1, B2 and B3) inside a glass linear microchannel under hydrodynamic pumping conditions. In particular, we monitored pressure drop and volumetric flow rate based on a set-up including microfluidic flow sensors (Elveflow®, ELVESYS S.A.S, France, see Figure S3) connected to a 1.5 cm long glass microchannel patterned with a nominal width of 300 m by using photolithography on a B-270 glass substrate [44]. After the geometry transfer, the glass substrate was etched with buffered oxide etch (BOE) solution by using the microwave reactor system (Anton Paar Multiwave 3000, Labservice Analytica s.r.l., Italy) as reported elsewhere [45] for obtaining a channel depth of 60 μm. Then, two holes were processed by using microdriller (MICRO miller MF70, Proxxon, Germany), in order to create inlet/outlet ports to be used as connections to the Elveflow® sensors. The channel was then thermally bonded to a glass top plate [46,47]. Based on the same fabrication procedure of the linear glass channel, a 270 mm long, 400 µm wide and 100 µm deep serpentine glass microreactor was prepared for mixing experiments.
For each experiment, an aqueous solution containing 5M H2O2 and 1.9 x 103cps L-1 (with ca 0.43 and 0.32 μM Ru4POM adsorbed in the case of B1 and B2) was prepared. The Ru4POM act as a H2O2 dismutation catalytic trigger, thus providing localized oxygen gas bursts to generate turbulence [34]. Under such dynamic conditions, the inlet and outlet sensors monitored the inlet pressure (Pin), the outlet pressure (Pout) and the volumetric output flow rate (Qout) during a designed time of two minutes (long enough to observe multiple events of peaked pressure). The evolution of the inlet volumetric flow rate was also monitored to assess its temporal stability and consistency with the nominal value set in our experiments. The sensors were characterized by sensitivity in the range 2 -60 L min-1.
The pressure drop along the microchannel, as well as Qout of the reactive batches (B1, B2), the nonreactive batch (B3) and the transport fluid (H2O), were compared and analyzed using OriginPro 8.0 software to handle and plot the measured data.

Microfluidic experiments for mixing applications
The two reactive mixtures (containing B1, B2) and the non-reactive one (with B3) were also used to test the PMCs effects on mass transport during diffusive mixing of fluids in a serpentine glass microreactor (270 mm long and with internal volume of 4 L). Solutions of PMCs (B1, B2 and B3) and H2O2, prepared as above reported, were red colored using eosin for an optimal visualization of the laminar flow and diffusive mixing process. Each sample was injected into one inlet port of the serpentine microreactor at a flow rate of 50 L min-1 while pure water has been flowing at the same rate through the other inlet port.
The performance of each PMC solution, in terms of mixing efficiency, was evaluated by following the flows along the serpentine channel with an optical microscope (NIKON mod. DS-5MC camera).
The mixing length was estimated by monitoring the color change of an eosin solution in the microreactor in comparison with the one observed when eosin/H2O2 without PMCs and aqueous solutions flow. The images were acquired at a residence time (Rt) 0 s, 0.5 s and 3.24 s for each solution of PMCs and compared with images acquired in absence of PMCs. These images were converted to grayscale images and analyzed by using ImageJ software to accurately estimate the efficiency and length-scale of the mixing. In particular, for each image the average change in intensity profile was examined over several lines perpendicular to the channel axis drawn along the channel. All the average values were normalized to the intensity of the colored stream before mixing and plotted versus off-center distances (representing the distance off the longitudinal centerline of the microchannel) [21]. The data reproducibility was checked in different chips. Moreover, a coefficient of variation (CoV) ≡ σ/μ [21], defined as the ratio of the standard deviation of the concentration to the mean value of the various concentrations at each datum point, was determined. This ratio, related to the mixing efficiency, was calculated at the inlet region, first meander and middle of the serpentine.
It is exactly zero for complete mixing and 1 in absence of mixing. We calculated CoV as average and standard deviation of the values obtained from three experiments performed on three different devices for each mixture of PMCs and for the solution without PMCs used as reference.

Experimental data analysis
In order to rationalize our experiments and their aim we remark a few procedure points.
Since the motion inertia typical of the capillary-flow regime stops the flow inside the microchannels over short distances from the inlet section, such working conditions are interesting for a limited number of LOCs [37] in which an efficient mixing of reagents is not needed.
Hence, differently from our kinematic studies under capillary-flow regime [35,36], in this work we analyze the effects of the catalytic activity of PMCs inside a glass linear microchannel under hydrodynamic pumping conditions. This issue is of most applicative interest for LOCs and OOCs relying on mass transport processes in continuous flow.
Moreover, in addition to the PMCs loaded with the Ru4POM as inner layer (B1), we also explored the behaviour of the capsules with the catalyst adsorbed on the external layer (B2).
This comparison would let evaluate the influence of the spatial confinement of the catalyst between the polyelectrolyte multilayers on the mixing efficiency.
To investigate physics underlying our experimental findings, our results are presented and discussed according to the following guidelines and interpretative analysis steps.
i) As a preliminary investigation, the behavior of the flowing pure water (i.e., the carrier fluid of PMCs) as control system was modeled to investigate the hydrodynamics of milli-Q water (H2O) flowing inside the microchannel in laminar regime steady-state conditions ( Figure S2 in SI). The results were compared with the corresponding measured quantities to confirm the quality and well-working of the etched linear microchannel ( Figure S2 and S4 in SI and corresponding discussion).
ii) The effect of non-reactive capsules (i.e., B3) in presence of H2O2 on the carrier fluid (H2O) was tested by defining the pressure drop of B3 normalized to the pressure drop of the transport fluid, that is where ΔP stands for the pressure difference between inlet and outlet pressure values measured by the microfluidic sensors connected with the inlet and outlet of the microchannel (experimental set-up in Figure S3 in SI).
(iii) The effect of the dismutation reaction resulting from vigorous O2 production was tested by defining the pressure drop of the reactive samples (i.e., B1 and B2) normalized to the above defined pressure drop of B3, that is In regard to the normalization protocol reported in equation 1, it is worth noticing that it is not a limit and doesn't affect the predictions and results of our study.
Indeed, as the inset of Figure 2(a) shows, the experimental pressure-drop curves of the not reactive batch B3 and pure water are nearly overlapping over the whole sampled range of Qin, with slight differences/fluctuations for increasing flow rate due to capsule-induced viscoelastic effects (as it will be detailed in Paragraph 3.2). Hence, our experiments indicate that the pressure drops associated with H2O and H2O2 are experimentally comparable/undistinguishable. As a confirmation, viscoelastic perturbation induced by PMCs in pure water have been observed in absence of H2O2 (see Figure 2C plot of ref. [37]). Additionally, microbubbles dispersed in aqueous solutions [54] exhibits a pressure drop consistent with our experiments involving B3 in H2O2.
Therefore, in practice, there is no difference in using H2O rather than H2O2 as control fluid for measuring the pressure drop versus Qin of unreactive capsules through formula (1). Conceptually, since i) capsules rather than H2O2 determine the behavior of B3, ii) the concentration of H2O2 changes over time in the case of B1 and B2, and iii) H2O is the common steady background of all batches under study, we preferred to introduce normalization with respect to H2O rather than to the fuel.

Pressure drop distribution: interpretative model of the experimental data
The microfluidic sensors connected with the inlet and outlet of the microchannel allowed us to measure the temporal evolution of inlet and outlet pressure (Pin and Pout) and flow rate (Qin and Qout) during the experiments involving pure water, non-reactive capsules and the dismutation reaction driven by H2O2.
In regard of the carrier fluid, it is worth noticing that the observed consistency between the the increase of resistance due to the capsules can be exploited to mix the continuous fluid [48].
Differently, in the presence of bubbles generated through reactions between the motor material (Ru4POM) and the solution fueled by H2O2, mixing is enhanced and prompted by a random recoil effect caused be the continuous growth and ejection of bubbles from the reactive capsules.
This chaotic mechanism is inherently more effective for mixing than capsules stretching and folding the continuous liquid.
Turning back to the ∆P profile versus Qin for B3 as plotted in Figure 3a, corresponding to the green curve of Figure 2b, it clearly shows the occurrence of two different regimes: decreasing ∆P with a minimum value at 15 μL min -1 and then increasing ∆P with a non-linear trend. The Qout versus Qin curve (Figure 3b) has a similar profile but with a minimum at 10 μL min -1 . This not linear dependence of Qout versus Qin is consistent with non-linear increase of the system velocity in presence of PMCs described elsewhere [37].
In order to explain the ∆P vs Qin curves on a physical basis, the best fitting curve based on the least To explain such behavior that significantly differs from that of water (shown as black curve in Figure   2b), theories about motion of capsules along a pipe have to be taken into consideration. The motion of capsules in a pipe can be classified into different regimes which depend on the flow speed and the capsule density [49]. Based on the reported regimes, once Qin is defined as the bulk fluid velocity, our results suggest that at injection-flow below 15 μL min-1, Qin is too low, meaning that an insufficient drag is developed on the capsules to overcome the contact friction between the capsules and the pipe and allows the capsules to slide and move away from the channel inlet. Consequently, capsules rest on the pipe floor and hinder the flow, hence decreasing the Qout and determining a pressure drop ∆P. This behavior is confirmed by the excellent parabolic fit shown in Figure 3c; the quadratic term is physically related to the damping effect due to the drag force. At 15 μL min-1 ≤ Qin ≤ 25 μL min-1, another regime is observed that can be ascribed to velocity of the fluid high enough to cause the capsules to slide along the pipe (Qin > Qi, where Qi is the incipient flow at which the capsule starts to slide). Once the motion of the capsules onsets, depending on the value of Qin, it is expected that the liquid bypasses or transports the capsules. If the fluid velocity is still relatively low, the contact friction between the capsules and the pipe is high, and the capsule velocity is less than the fluid velocity as well as the capsules reduce the volumetric flow rate. Consistently, in our experiments, Qout is lower than Qin and ∆P starts to increase linearly since the drag force is negligible.
At Qin > 30 μL min-1, another regime onset. Indeed, for increasing bulk velocity Qin, the pressure drop along the capsules (propelling force) would be expected to be higher than the pressure drop felt by the counterpart flow free of capsules [49]. Hence, the capsule velocity should overtake the fluid velocity. All of this is consistent with the experimental results obtained under capillary-driven motion of B1, where speed up of the fluid motion was observed for reactive capsules in absence of H2O2 [37]. The least square approach leads to a polynomial law with a Qin5/12 dependence (Figure 3d). The not linear dependence of ∆P on the capillary number Ca (and consequently on the flow speed Qin) has been already reported in the case of bubbles flowing along a rectangular channel [50] and attributed to the deformability of the bubbles [51][52][53].
In the case of a fluid transporting bubbles along a rectangular channel, the total pressure drop ∆Ptot can be decomposed as the sum of the pressure drops around each bubble, the body of the bubble and the region of the channel free of bubbles [50,52,53]:  Figure   4b). The graph also highlights the superior performance of B1 PMCs, where the slight higher amount of Ru4POM sandwiched within the multilayer shell. Because of the difficulties to fit the original data at low flow rates, where the reaction effects predominate and O2 bubbles create a strong turbulence with occurrence of often negative counter-flows, it was not possible to estimate Qout for each set Qin and to perform least square fits (data not shown). Under these conditions no theories can be elaborated due to the chaotic flow generated.

Mixing experiments
Based on the results of the above model, we performed mixing experiments under hydrodynamic pumping regime at Qin = 50 L min-1 to favor conditions with dominance of the transport mechanism of the capsules by the background fluid. Because of low Reynolds number in microfluidic channels, involving laminar and uniaxial fluid flow, the mixing between streams in the flow is purely diffusive.
Hence, short resident time (Rt) and low mixing efficiency can result when the interface between fluid streams lies at the center of a T-shape microchannel [56]. As shown in Figure 5a, in the absence of any turbulence source, two aqueous solutions (H2O and eosin containing 5M H2O2), both injected at 50 L min-1 flow rate, clearly show no mixing effect in the optical images acquired in correspondence of the inlet region (Rt1 = 0 s), where the solutions are clearly separated. Unlike the T-junction region, a good mixing was observed in the middle of the T-shaped serpentine microreactor (corresponding to Rt3 = 3.24 s, see Figure 5e). This is also confirmed by the intensity profile plotted in Figure 5d, black curve. Indeed, a sharp transition of the color profile from 0 % to 100 % (in the half-section channel indicated by the arrow in Figure 5a) is straightforward at Rt1, while the transition is more gradual at Rt3 (Figure 5e and black plot of Figure 5h). To evaluate the mixing efficiency, the coefficient of variation (CoV) was measured at Rt1 and at Rt3 . It was found a larger value (i.e., CoV= PMCs were introduced into the microchannels. The intensity profiles were measured as for the reference sample at Rt1 (Figures 5b and c) where the two flows meet for the first time, and at Rt3 where the mixing effect appears good from the optical images (Figures 5f and g).
The red solution, containing 5M H2O2 and B3 (Figure 5b) or the most reactive batch B1 (Figure 5c), was injected at Qin = 50 L min-1 and mixed along the serpentine microreactor with H2O injected from the other inlet at the same speed. By comparison with the same experiment in absence of PMCs ( Figure 5a), it seems that the addition of PMCs immediately shifts the fluids interface far from the middle of the channel at the T-junction level. In both cases (for B3 and B1), we also observed that the transition of color concentration (red and blue curves, Figure 5d) was less sharp with respect to the pure aqueous solution (black curve, Figure 5d) at Rt1. In these cases, the CoV values were 0.74 ± 0.12 and 0.52 ± 0.13 for B3 and B1, respectively. The mixing efficiency was further improved at Rt3.
Indeed, the fluids interfaces were dissipated in the optical images (Figure 5f and g) and the color transitions lost the sharp step typical of the reference sample (see blue and red plots in Figure 5h).
This was also confirmed by the decrease of CoV to 0.11 ± 0.02 for B3 and to 0.11 ± 0.01 for B1.
Then, the viscoelastic effects due to the PMCs caused a perturbation of the laminar regime so that a good enough mixing occurred at Rt3. In the case of reactive PMCs, large error bars can be observed

Conclusions
The work aims at demonstrating the effect of viscoelastic soft micromotors on diffusion processes in microfluidic environments.
As model system, pristine PMCs and catalytic PMCs (PMCs embedding a polyoxometalate into a shell structure) were studied.
The micromotors presented in this study use a Ru-substituted polyoxometalate (Ru4POM) that act as catalytic trigger to dismutate hydrogen peroxide into water and oxygen. Notably, hydrogen peroxide is considered toxic to the in vivo environment and unsuitable for use in biological applications, such as LOCs and OOCs [57,58]. Furthermore, oxygen at high concentration could interfere with cell metabolism in organ-on-a-chip (OOC) applications. However, thanks to the high versatility of the Layer-by-Layer (LbL) assembly, when required, the Ru4POM catalyst could be easily replaced by other catalyst-fed systems (i.e. metal and photocatalytic systems for water splitting) even generating other products (i.e. acid-activated micromotors containing carbonate particles, already used for in vivo drug delivery in the gastric tract of a mouse model, or enzyme-based micromotors [32]) or micromotors responding to physical stimuli (light, acoustic or magnetic [59]). In this way, the combination of a biologically compatible catalyst with the LbL assembly method still allows for efficient mixing while avoiding altering the cells survival and metabolism. On the basis of these results, we believe that viscoelastic micromotors can be considered an interesting tool to improve mass transport in experimental conditions purely dominated by diffusion.
In particular, they could be useful to enhance mixing between fluids containing reagents or nutrients in LOC and OOC platforms to achieve a rapid mixing, a fast diagnosis or to guarantee efficient transport of nutrients for cell survival.    (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)