Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

Tri-fluid mixing in a microchannel for nanoparticle synthesis.

It is becoming more difficult to use bulk mixing and bi-fluid micromixing in multi-step continuous-flow reactions, multicomponent reactions, and nanoparticle synthesis because they typically involve multiple reactants. To date, most micromixing studies, both passive and active, have focused on how to efficiently mix two fluids, while micromixing of three or more fluids together (multi-fluid mixing) has been rarely explored. This study is the first on tri-fluid mixing in microchannels. We investigated tri-fluid mixing in three microchannel models: a straight channel, a classical staggered herringbone mixing (SHM) channel, and a three-dimensional (3D) X-crossing microchannel. Numerical simulations and experiments were jointly conducted. A two-step experimental process was performed to determine the tri-fluid mixing efficiencies of these microchannels. We found that the SHM cannot significantly enhance mixing of three streams especially for a Reynolds number (Re) higher than 10. However, the 3D X-crossing channel based on splitting-and-recombination (SAR) showed effective tri-mixing performance over a wide Re range up to 275 (with a corresponding flow rate of 1972.5 µL min-1), thereby enabling high microchannel throughput. Furthermore, this tri-fluid micromixing process was used to synthesize a kind of Si-based nanoparticle. This achieved a narrower particle size distribution than traditional bulk mixing. Therefore, SAR-based tri-fluid mixing is an alternative for chemical and biochemical reactions where three reactants need to be mixed.

Microparticle separation using asymmetrical induced-charge electro-osmotic vortices on an arc-edge-based floating electrode.

Five arc-shaped gaps were designed on the bipolar electrode to actuate alternately opposite-direction asymmetrical induced-charge electro-osmosis (AICEO) vortices, and we developed a microfluidic device using such asymmetrical vortices to realize particle separation. When the buoyancy force dominates in the vertical direction, particles stay at the channel bottom, experiencing a left deflection under the vortices in the convex arc areas. In contrast, when the levitation force induced by AICEO vortices overcomes the buoyancy force, particles are elevated to a high level and captured by right vortices, undergoing a right deflection under the vortices in the concave arc areas. Moreover, when particles pass through the concave or convex arc areas every time, their right or left deflections are enlarged gradually and the separation becomes more complete. Remarkably, as the light/small particles at low voltage, heavy/large particles can be elevated to a new high level and undergo right deflection by increasing the voltage. We first explicitly proved the separation principle and analyzed numerically its capability in density- and size-based separation. Depending on the study of the voltage-dependent AICEO characterization of 4 µm silica and 4 µm PMMA particles, we experimentally verified the feasibility of our device in density-based separation. According to the investigation of sensitivity to particle size, we separated multi-sized yeast cells to confirm the capability of our device in size-based separation. Finally, we extracted yeast cells from impeding particles, obtaining 96% purity. Additionally, we designed a 500 µm distance between the focusing and separation region to circumvent the problems caused by electric-field interaction. Our AICEO-based separation method holds potential to serve as a useful tool in transesterification of microalgal lipids to biodiesel and solar cell processing because of its outstanding advantages, such as gentle conditions, contact-free separation, high-sensitivity and high-efficiency separation capability.

Induced charge electro-osmotic particle separation.

Vortex-based separation is a promising method in particle-particle separation and has only been demonstrated theoretically some years ago. To date, a continuous-flow separation device based on vortices has not been conceived because many known vortices were either unstable or controlling them lacked precision. Electro-convection from induced charge electro-osmosis (ICEO) has advantages, such as adjustable flow profiles, long-range actuation, and long-lived vortices, and offers an alternative means of particle separation. We found though a different ICEO focusing behaviour of particles whereby particles were trapped and concentrated in two vortex cores. Encouraged by these features of ICEO vortices, we proposed a direct method for particle separation in continuous flow. In various experiments, we first characterized the ICEO-induced focusing performances of various kinds of particle samples in a straight channel embedded with an individual central bipolar electrode, presenting a justifiable explanation. Second, the combined dependences of ICEO particle separation on the sample size and mass density were investigated. Third, an application to cell purification was performed in which we obtained a purity surpassing 98%. Finally, we investigated the ICEO characteristics of nanoparticles, exploiting our method in isolating nanoscale objects by separating 500 nm and 5 µm polystyrene beads, gaining clear separation. Certain features of this method, such as having ease of operation, simple structure, and continuous flow, and being prefocusing free and physical property-based, indicate its good potential in tackling environmental monitoring, cell sorting, chemical analysis, isolation of uniform-sized graphene and transesterification of micro-algal lipids to biodiesel.

Flexible Continuous Particle Beam Switching via External-Field-Reconfigurable Asymmetric Induced-Charge Electroosmosis.

Continuous sample switching is an essential process for developing an integrated platform incorporating multiple functionality with applications typically ranging from chemical to biological assays. Herein we propose a unique method of external-field-reconfigurable symmetry breaking in induced-charge electroosmosis above a simple planar bipolar electrode for continuous particle beam switching. In the proposed system, the spatial symmetry of a nonlinear electroosmotic vortex flow can be artificially reordered to achieve an asymmetric electrically floating-electrode polarization by regulating the configurations of the external ac signals, thus contributing to flexible particle beam switching. This switching system comprises an upstream flow-focusing region where particles are prefocused into a beam on the bipolar electrode by transversal electroconvective mass transfer, and a deflecting region in which the resulting particle beam is deflected to generate a steerable lateral displacement to enter the desired region via the action of an asymmetric polarization-induced reshapable electroosmotic flow stagnation line in a controllable background field gradient. A lateral particle displacement on the order of hundreds of micrometers can be achieved in a deterministic manner by varying the voltage, frequency, and inlet flow rate, thereby enabling multichannel particle switching. Furthermore, the versatility of the switching mechanism is extended by successfully accomplishing fluorescent nanoparticle beam switching, yeast cell switching, five-outlet particle switching, and simultaneous switching of two particle types. The proposed switching approach provides a promising technique for flexible electrokinetic sample preconcentration prior to any subsequent analysis and can be conveniently integrated with other micro/nanofluidic components into a complete functional on-chip platform owing to its simple electrode structure.

A Simplified Microfluidic Device for Particle Separation with Two Consecutive Steps: Induced Charge Electro-osmotic Prefocusing and Dielectrophoretic Separation.

Continuous dielectrophoretic separation is recognized as a powerful technique for a large number of applications including early stage cancer diagnosis, water quality analysis, and stem-cell-based therapy. Generally, the prefocusing of a particle mixture into a stream is an essential process to ensure all particles are subjected to the same electric field geometry in the separation region. However, accomplishing this focusing process either requires hydrodynamic squeezing, which requires an encumbering peripheral system and a complicated operation to drive and control the fluid motion, or depends on dielectrophoretic forces, which are highly sensitive to the dielectric characterization of particles. An alternative focusing technique, induced charge electro-osmosis (ICEO), has been demonstrated to be effective in focusing an incoming mixture into a particle stream as well as nonselective regarding the particles of interest. Encouraged by these aspects, we propose a hybrid method for microparticle separation based on a delicate combination of ICEO focusing and dielectrophoretic deflection. This method involves two steps: focusing the mixture into a thin particle stream via ICEO vortex flow and separating the particles of differing dielectic properties through dielectrophoresis. To demonstrate the feasibility of the method proposed, we designed and fabricated a microfluidic chip and separated a mixture consisting of yeast cells and silica particles with an efficiency exceeding 96%. This method has good potential for flexible integration into other microfluidic chips in the future.

Continuously Electrotriggered Core Coalescence of Double-Emulsion Drops for Microreactions.

Microfluidically generated double emulsions are promising templates for microreactions, which protect the reaction from external disturbance and enable in vitro analyses with large-scale samples. Controlled combination of their inner droplets in a continuous manner is an essential requirement toward truly applications. Here, we first generate dual-cored double-emulsion drops with different inner encapsulants using a capillary microfluidic device; next, we transfer the emulsion drops into another electrode-integrated polydimethylsiloxane microfluidic device and utilize external AC electric field to continuously trigger the coalescence of inner cores inside these emulsion drops in continuous flow. Hundreds of thousands of monodisperse microreactions with nanoliter-scale reagents can be conducted using this approach. The performance of core coalescence is investigated as a function of flow rate, applied electrical signal, and core conductivity. The coalescence efficiency can reach up to 95%. We demonstrate the utility of this technology for accommodating microreactions by analyzing an enzyme catalyzed reaction and by fabricating cell-laden hydrogel particles. The presented method can be readily used for the controlled triggering of microreactions with high flexibility for a wide range of applications, especially for continuous chemical or cell assays.

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM.

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms.

Effect of the crossing-structure sequence on mixing performance within three-dimensional micromixers.

The geometry of crossing structure formed by two-layer microchannels determines the axial and transverse movements of contact interface between two liquid streams, which gives us a new method for promoting the micromixers. Hence, we designed four different three-dimensional micromixers by selecting two different crossing structures as basic units (one unit is a crossing structure called "X" and the other is a reversed crossing structure called "rX"). In order to find out how the crossing-structure sequence affects the mixing performance within three-dimensional micromixers, we organized these four mixers in different ways, i.e., the first combination is X-rX-X-rX-…, the second is X-rX-rX-X-…, the third is X-X-rX-X-…, and the last one is X-X-X-X…. Consequently, quite distinct mixing phenomena are engendered. Furthermore, experiments were also conducted using the first and the last models to verify the simulation results. We infer that the last mixer is more likely to trigger chaos and convection by rotating the contact surface than the first one that merely swings the surface even when the flow rates and viscosities of the two liquid streams are increased.

An effective splitting-and-recombination micromixer with self-rotated contact surface for wide Reynolds number range applications.

It is difficult to mix two liquids on a microfluidic chip because the small dimensions and velocities effectively prevent the turbulence. This paper describes two 2-layer PDMS passive micromixers based on the concept of splitting and recombining the flow that exploits a self-rotated contact surface to increase the concentration gradients to obtain fast and efficient mixing. The designed micromixers were simulated and the mixing performance was assessed. The mixers have shown excellent mixing efficiency over a wide range of Reynolds number. The mixers were reasonably fabricated by multilayer soft lithography, and the experimental measurements were performed to qualify the mixing performance of the realized mixer. The results show that the mixing efficiency for one realized mixer is from 91.8% to 87.7% when the Reynolds number increases from 0.3 to 60, while the corresponding value for another mixer is from 89.4% to 72.9%. It is rather interesting that the main mechanism for the rapid mixing is from diffusion to chaotic advection when the flow rate increases, but the mixing efficiency has not obvious decline. The smart geometry of the mixers with total length of 10.25 mm makes it possible to be integrated with many microfluidic devices for various applications in µ-TAS and Lab-on-a-chip systems.