Rapid Microfluidic Mixing Research Articles

Combining Rapid Microfluidic Mixing and Three-Color Single-Molecule FRET for Probing the Kinetics of Protein Conformational Changes.

Single-molecule Förster resonance energy transfer (FRET) is well suited for studying the kinetics of protein conformational changes, owing to its high sensitivity and ability to resolve individual subpopulations in heterogeneous systems. However, the most common approach employing two fluorophores can only monitor one distance at a time, and the use of three fluorophores for simultaneously monitoring multiple distances has largely been limited to equilibrium fluctuations. Here we show that three-color single-molecule FRET can be combined with rapid microfluidic mixing to investigate conformational changes in a protein from milliseconds to minutes. In combination with manual mixing, we extended the kinetics to 1 h, corresponding to a total range of 5 orders of magnitude in time. We studied the monomer-to-protomer conversion of the pore-forming toxin cytolysin A (ClyA), one of the largest protein conformational transitions known. Site-specific labeling of ClyA with three fluorophores enabled us to follow the kinetics of three intramolecular distances at the same time and revealed a previously undetected intermediate. The combination of three-color single-molecule FRET with rapid microfluidic mixing thus provides an approach for probing the mechanisms of complex biomolecular processes with high time resolution.

2D acoustofluidic patterns in an ultrasonic chamber modulated by phononic crystal structures

Controllable manipulation of micro/nano-particles and biological organisms are essential for the engineering development of miniaturized lab-on-a-chip systems in the application of physical, chemical, and biological researches. In this paper, a series of phononic crystal structure based acoustofluidic devices, which are actuated by incident plane wave at different frequencies, have been proposed and numerically investigated for micro-particle manipulation. The interaction between different phononic crystal structures and ultrasonic waves, providing reflection, scattering and diffraction, can generate diverse spatial variations of sound field distribution along the wave propagation path. The combination of phononic crystal structures and lab-on-a-chip devices is beneficial to overcome the monotonousness of the acoustofluidic field distribution for various physical and biochemical applications. The movement trajectories of micro-particles under the influence of acoustic radiation forces and acoustic streaming induced drag forces are also simulated to demonstrate the particle manipulation capability of the designed acoustofluidic device. Our simulation results suggest the possibility of considering phononic crystal structures as an effective ingredient to customize acoustofluidic field for constituting diverse lab-on-a-chip devices in the investigation of rapid microfluidic mixing and non-invasive manipulation of bio-organisms.

Diversity of 2D Acoustofluidic Fields in an Ultrasonic Cavity Generated by Multiple Vibration Sources.

Two-dimensional acoustofluidic fields in an ultrasonic chamber actuated by segmented ring-shaped vibration sources with different excitation phases are simulated by COMSOL Multiphysics. Diverse acoustic streaming patterns, including aggregation and rotational modes, can be feasibly generated by the excitation of several sessile ultrasonic sources which only vibrate along radial direction. Numerical simulation of particle trajectory driven by acoustic radiation force and streaming-induced drag force also demonstrates that micro-scale particles suspended in the acoustofluidic chamber can be trapped in the velocity potential well of fluid flow or can rotate around the cavity center with the circumferential acoustic streaming field. Preliminary investigation of simple Russian doll- or Matryoshka-type configurations (double-layer vibration sources) provide a novel method of multifarious structure design in future researches on the combination of phononic crystals and acoustic streaming fields. The implementation of multiple segmented ring-shaped vibration sources offers flexibility for the control of acoustic streaming fields in microfluidic devices for various applications. We believe that this kind of acoustofluidic design is expected to be a promising tool for the investigation of rapid microfluidic mixing on a chip and contactless rotational manipulation of biosamples, such as cells or nematodes.

Quantifying kinetics from time series of single-molecule Förster resonance energy transfer efficiency histograms

Single-molecule fluorescence spectroscopy is a powerful approach for probing biomolecular structure and dynamics, including protein folding. For the investigation of nonequilibrium kinetics, Förster resonance energy transfer combined with confocal multiparameter detection has proven particularly versatile, owing to the large number of observables and the broad range of accessible timescales, especially in combination with rapid microfluidic mixing. However, a comprehensive kinetic analysis of the resulting time series of transfer efficiency histograms and complementary observables can be challenging owing to the complexity of the data. Here we present and compare three different methods for the analysis of such kinetic data: singular value decomposition, multivariate curve resolution with alternating least square fitting, and model-based peak fitting, where an explicit model of both the transfer efficiency histogram of each species and the kinetic mechanism of the process is employed. While each of these methods has its merits for specific applications, we conclude that model-based peak fitting is most suitable for a quantitative analysis and comparison of kinetic mechanisms.

Rapid microfluidic mixing via rotating magnetic microbeads

Presented here is a novel method for mixing in a microfluidic channel via an array of rotating magnetic microbeads. The microbeads rotate around circular permalloy (NiFe) features magnetized by a rotating external magnetic field. This system demonstrates rapid mixing in short channel lengths. The effectiveness of this system is quantified by analyzing the degree of mixing of two streams, one with fluorescence and one without. Our experiments and numerical simulations reveal that beads orbiting at a high velocity as compared to flow down the channel cause the fluid to form circular coronae that stretch across the microchannel leading to fluid mixing. Under these conditions, rotating magnetic microbeads were able to fully mix the streams in a 270μm mixing region.

Orbiting magnetic microbeads enable rapid microfluidic mixing

We use three-dimensional numerical simulations and experiments to examine microfluidic mixing induced by orbiting magnetic microbeads in a microfluidic channel. We show that orbiting microbeads can lead to rapid fluid mixing in low Reynolds number flow, and identify two distinct mixing mechanisms. Bulk advection of fluid across the channel occurs due to the flow pattern that is developed when the ratio of flow velocity to bead velocity is low, and leads to rapid mixing. At higher velocity ratios, dispersion of small amounts of fluid across the channel occurs and results in increased mixing. We use simulations to investigate the effect of system parameters on the distance required to achieve a desired mixing level. We develop an experimental continuous-flow device and use it to validate our simulations and to demonstrate rapid microfluidic mixing. This device has the flexibility to also be applied to a mixing chamber or to stop-flow applications for rapid and controllable mixing. In addition to rapid mixing, the use of orbiting magnetic microbeads has the added benefit that functionalized microbeads can be used to capture particles from the fluid solution during mixing, and that they can be extracted from the device for analysis, thus serving multiple functionalities in a single device.

Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents

Previous studies from this group have shown that limit size lipid-based systems – defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents – can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30–40 nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of ∼12 h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50 nm that could lead to development of a novel class of ultra-small drug delivery vehicles.

Experimental observations of flow instabilities and rapid mixing of two dissimilar viscoelastic liquids

Viscoelastically induced flow instabilities, via a simple planar microchannel, were previously used to produce rapid mixing of two dissimilar polymeric liquids (i.e. at least a hundredfold different in shear viscosity) even at a small Reynolds number. The unique advantage of this mixing technology is that viscoelastic liquids are readily found in chemical and biological samples like organic and polymeric liquids, blood and crowded proteins samples; their viscoelastic properties could be exploited. As such, an understanding of the underlying interactions will be important especially in rapid microfluidic mixing involving multiple-stream flow of complex (viscoelastic) fluids in biological assays. Here, we use the same planar device to experimentally show that the elasticity ratio (i.e. the ratio of stored elastic energy to be relaxed) between two liquids indeed plays a crucial role in the entire flow kinematics and the enhanced mixing. We demonstrate here that the polymer stretching dynamics generated in the upstream converging flow and the polymer relaxation events occurring in the downstream channel are not exclusively responsible for the transverse flow mixing, but the elasticity ratio is also equally important. The role of elasticity ratio for transverse flow instability and the associated enhanced mixing were illustrated based on experimental observations. A new parameter Deratio = Deside / Demain (i.e. the ratio of the Deborah number (De) of the sidestream to the mainstream liquids) is introduced to correlate the magnitude of energy discontinuity between the two liquids. A new Deratio-Demain operating space diagram was constructed to present the observation of the effects of both elasticity and energy discontinuity in a compact manner, and for a general classification of the states of flow development.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing

Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.

Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing.

We combined rapid microfluidic mixing with single-molecule Förster Resonance Energy Transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid-mimics and subsequent rapid formation of transient structures within the encounter complex. The method also enabled study of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.

Milliseconds microfluidic chaotic bubble mixer

In this study, we report a rapid microfluidic mixing device based on chaotic advection induced by microbubble–fluid interactions. The device includes inlets for to-be-mixed fluids and nitrogen gas. A side-by-side laminar flow segmented by monodisperse microbubbles is generated when the fluids and the nitrogen are co-injected through a flow focusing micro-orifice. The flow subsequently enters a series of hexagonal expansion chambers, in which the hydrodynamic interaction among the microbubbles results in the stretch and fold of segmented fluid volumes and rapid mixing and homogenization. We characterize the performance of the microfluidic mixer and demonstrate rapid mixing within 20 ms. We further show that bubbles can be conveniently removed from the mixed fluids using a microfluidic comb structure on completion of the mixing.

Rapid microfluidic mixing.

A preformed T-microchannel imprinted in polycarbonate was postmodified with a pulsed UV excimer laser (KrF, 248 nm) to create a series of slanted wells at the junction. The presence of the wells leads to a high degree of lateral transport within the channel and rapid mixing of two confluent streams undergoing electroosmotic flow. Several mixer designs were fabricated and investigated. All designs were relatively successful at low flow rates (0.06 cm/s, > or = 75% mixing), but had varying degrees of success at high flow rates (0.81 cm/s, 45-80% mixing). For example, one design operating at high flow rates was able to split an incoming fluorescent stream into two streams of varying concentrations depending on the number of slanted wells present. The final mixer design was able to overcome stream splitting at high flow rates, and it was shown that the two incoming streams were 80% mixed within 443 microm of the T-junction for a flow rate of 0.81 cm/s. Without the presence of the mixer and at the same high flow rate, a channel length of 2.3 cm would be required to achieve the same extent of mixing when relying upon molecular diffusion entirely, while 6.9 cm would be required for 99% mixing.