Droplet encapsulation of particles in different regimes and sorting of particle-encapsulating-droplets from empty droplets.

Encapsulation of microparticles in droplets has profound applications in biochemical assays. We investigate encapsulation of rigid particles (polystyrene beads) and deformable particles (biological cells) inside aqueous droplets in various droplet generation regimes, namely, squeezing, dripping, and jetting. Our study reveals that the size of the positive (particle-encapsulating) droplets is larger or smaller compared to that of the negative (empty) droplets in the dripping and jetting regimes but no size contrast is observed in the squeezing regime. The size contrast of the positive and negative droplets in the different regimes is characterized in terms of capillary number C a and stream width ratio ω (i.e., ratio of stream width at the throat to particle diameter ω = w / d p ). While for deformable particles, the positive droplets are always larger compared to the negative droplets, for rigid particles, the positive droplets are larger in the dripping and jetting regimes for 0.50 ≤ ω ≤ 0.80 but smaller in the jetting regime for ω < 0.50 . We exploit the size contrast of positive and negative droplets for sorting across the fluid-fluid interface based on noninertial lift force (at R e ≪ 1 ), which is a strong function of droplet size. We demonstrate sorting of the positive droplets encapsulating polystyrene beads and biological cells from the negative droplets with an efficiency of ∼95% and purity of ∼65%. The proposed study will find relevance in single-cell studies, where positive droplets need to be isolated from the empty droplets prior to downstream processing.

Non-inertial lift induced migration for label-free sorting of cells in a co-flowing aqueous two-phase system.

Label free sorting of circulating tumor cells (CTCs) often remains a challenge due to their rarity in peripheral blood and identical morphology to white blood cells. We present a novel label-free passive microfluidic technique for isolation of cancer cells (EpCAM+ and CD45-) from peripheral blood mononuclear cells (PBMCs) (CD45+ and EpCAM-) in aqueous two-phase system (ATPS). Our technique involves non-inertial lift induced lateral cell migration across liquid-liquid interface that is employed for sorting cells of different size and stiffness. The interplay between lift force and interfacial tension (IFT) force governs cell migration phenomena. We estimate the order of magnitude of the lift force and find it to be higher than the IFT for cancer cells above a critical strain rate parameter ([small gamma, Greek, dot above]/h). The effect of spreading parameter and viscoelastic force was found to have negligible effect on lateral migration of cells. We demonstrated isolation of two different types of cancer cells, namely, MCF-7 and MDA-MB-231 from PBMCs and quantify our sorting results by tagging the cells with EpCAM and CD45 and using fluorescence imaging. With 102-104 cancer cells in 105-107 PBMCs, we achieved a processing rate of >25 000 cells per min at a sorting efficiency of ∼99%. Moreover, we demonstrated that cancer cells isolated from PBMCs using the proposed technique remain viable and can be cultured for downstream analysis.

Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method.

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0-100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 µM - 125 µM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.

Self-Transport and Manipulation of Aqueous Droplets on Oil-Submerged Diverging Groove.

We report experimental study of self-transport of aqueous droplets along an oil-submerged diverging groove structure. The migration phenomenon is illustrated, and the effect of various parameters such as droplet size d, oil layer thickness h, groove angle 2θ, and groove thickness δ on the droplet transport behavior (i.e., migration velocity and length) is investigated. Our study reveals that complete engulfment of aqueous droplets in the oil layer, that is attributed to a positive spreading parameter ( S > 0), is a prerequisite for the droplet transport. The results show that only droplets of diameter larger than the oil layer thickness (i.e., d ≥ h) get transported owing to a differential Laplace pressure between the leading and trailing faces of a droplet because of the diverging groove. Using experimental data, the variation of droplet migration velocity with distance along the diverging groove is correlated as U( x) = ψ x-0.9, where ψ = d0.32θ-2.2 h-1.5δ0.7. The submerged groove structure was used to demonstrate simultaneous and sequential coalescence and transport of multiple droplets. Finally, the submerged groove structure was employed for extraction of aqueous droplets from oil. The proposed technique opens up a new avenue for evaporation and contamination free transport and coalescence of droplets for chemical and biological applications.

Continuous splitting of aqueous droplets at the interface of co-flowing immiscible oil streams in a microchannel.

We report the continuous splitting of aqueous droplets at the interface between two co-flowing immiscible oil streams in a microchannel. The aqueous droplets initially present in a primary continuous stream (CP1) migrate into a secondary continuous stream (CP2) when the ratio of the non-inertial lift force to the interfacial tension force exceeds a critical value (K. S. Jayaprakash, U. Banerjee and A. K. Sen, Langmuir, 2016, 32, 2136-2143). Here, experiments were performed to understand the droplet splitting phenomenon and demonstrate the splitting of droplets encapsulating microbeads and cells. The results showed that the droplet splitting phenomenon is governed by the capillary number Ca, which is a function of the average shear stress across the channel, interfacial tension σ between the CP1 and the droplet phase and the droplet length-scale L. Irrespective of the individual values of these parameters, droplet splitting was observed when the capillary number Ca exceeds a critical value Cacr, which was found to be a function of droplet to CP2 viscosity ratio λ. The Cacr was found to be minimum for λ ≈ 1 but higher for droplets of λ ≫ 1 and λ ≪ 1. The sizes of the primary and secondary daughter and migrated droplets (i.e. Lp|sD and Lp|sM) were found to increase linearly with the increase in the size of the primary or secondary parent droplets (Lp|sP). Splitting of parent droplets encapsulating a single microbead or PBMC showed that after splitting, the presence of the microbead or PBMC in the daughter or migrated droplets depends on the ratio of the size of the migrated droplets to that of the parent droplet (i.e. VM/VP). Finally, splitting of parent droplets containing two or more microbeads or cells into droplets containing a single particle or cell was demonstrated. A new paradigm of droplet splitting is reported that could find applications in soft matter and single-cell studies.

Droplet Demulsification Using Ultralow Voltage-Based Electrocoalescence.

Demulsification of droplets stabilized with surfactant is very challenging due to their low surface energy. We report ultralow voltage-based electrocoalescence phenomenon for the demulsification of aqueous droplets with an aqueous stream. In the absence of electric field, due to the disjoining pressure resulting from the tail-tail interaction between the surfactant molecules present on the aqueous droplets and interface, coalescence of aqueous droplets with the aqueous stream is prevented. However, above a critical electric field, the electrical stress overcomes the disjoining pressure, thus leading to the droplet coalescence. The influence of surfactant concentration, droplet diameter, and velocity on the electrocoalescence phenomena is studied. The macroscopic contact between the aqueous droplet with the aqueous stream enables droplet coalescence at much lower voltage (10 to 90 V), which is at least two orders of magnitude smaller than voltages used in prior works (1.0 to 3.0 kV). The electrocoalescence phenomena is used for the extraction of microparticles encapsulated in aqueous droplets into the aqueous stream and size-based selective demulsification. A new paradigm of droplet electrocoalescence and content extraction is presented that would find significant applications in chemistry and biology.

Dynamics of rigid microparticles at the interface of co-flowing immiscible liquids in a microchannel.

We report the dynamical migration behavior of rigid polystyrene microparticles at an interface of co-flowing streams of primary CP1 (aqueous) and secondary CP2 (oils) immiscible phases at low Reynolds numbers (Re) in a microchannel. The microparticles initially suspended in the CP1 either continue to flow in the bulk CP1 or migrate across the interface into CP2, when the stream width of the CP1 approaches the diameter of the microparticles. Experiments were performed with different secondary phases and it is found that the migration criterion depends on the sign of the spreading parameter S and the presence of surfactant at the interface. To substantiate the migration criterion, experiments were also carried out by suspending the microparticles in CP2 (oil phase). Our study reveals that in case of aqueous-silicone oil combination, the microparticles get attached to the interface since S<0 and the three phase contact angle, θ>90°. For complete detachment of microparticles from the interface into the secondary phase, additional energy ΔG is needed. We discuss the role of interfacial perturbation, which causes detachment of microparticles from the interface. In case of mineral and olive oils, the surfactants present at the interface prevents attachment of the microparticles to the interface due to the repulsive disjoining pressure. Finally, using a aqueous-silicone oil system, we demonstrate size based sorting of microparticles of size 25µm and 15µm respectively from that of 15µm and 10µm and study the variation of separation efficiency η with the ratio of the width of the aqueous stream to the diameter of the microparticles ρ.

Dynamics of Aqueous Droplets at the Interface of Coflowing Immiscible Oils in a Microchannel.

We report the dynamics of aqueous droplets of different size and viscosity at the interface of a coflowing stream of immiscible oils (i.e., primary and secondary continuous phases) in a microchannel, at low Re. The lateral migration of droplets introduced into the primary continuous phase toward the interface and subsequent selective migration of droplets across the interface into the secondary continuous phase is investigated. The interplay between the competing noninertial lift and interfacial tension forces, which govern the interfacial migration of the droplets, is presented and discussed. The velocity and strain rate profiles, and interface location, which are critical for calculating the lift force and migration behavior of droplets, are presented. The trajectories of droplets of different size and viscosity in the primary continuous phase are obtained for different interface locations. During interfacial migration, the deformation behavior of droplets of different viscosities is studied. Finally, sorting of droplets based on size contrast is demonstrated and sorting efficiency is found. A new paradigm of migration and sorting of droplets is reported, which could find importance in chemical and biological applications.