Time-resolved cryo-EM (TRCEM) sample preparation using a PDMS-based microfluidic chip assembly.

Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO-coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid.

Passage through micro-sprayer increases functional activity - implications for activity assays in time-resolved cryo-EM.

This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a micro-sprayer. We find a reproducible , rather than the expected in GFP production as monitored by fluorescence, which suggests heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.