Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

Controllable Synthesis of Multicompartmental Particles Using 3D Microfluidics

A microfluidic assembly method based on a microfluidic chip and capillary device was developed to create multicompartmental particles. The microfluidic chip design endows the particles with regulable internal structure. By adjusting the microstructure of the chip, the diameter of the capillary, the gap length between the two microfluidic components, and the flow rates, the size of the particles and the number or the ratio of different regions within the particle could be widely varied. As a proof of concept, we have produced some complicated particles that even contain 20 compartments. Furthermore, the potential applications of the anisotropic particles are explored by encapsulating magnetic beads, fluorescent nanoparticles, and the cells into different compartments of the microparticles. We believe that this method will open new avenues for the design and application of multicompartmental particles.     

A microfluidic diffusion chamber for reversible environmental changes around flaccid lipid vesicles

The reversible environmental changes around flaccid lipid vesicles represent a considerable experimental challenge, particularly because of remarkable softness of flaccid membranes, which can warp irreversibly under the slightest hydrodynamic flow. As a result, we have developed a microfluidic device for the controlled analysis of individual flaccid, giant lipid vesicles in a changing chemical environment. The setup combines the advantages of a flow-free microfluidic diffusion chamber and optical tweezers, which are used to load the sample vesicles into the chamber. After a vesicle is loaded into the diffusion chamber, its chemical environment is controllably and reversibly changed solely by means of diffusion. The chamber is designed as a 250 micrometres-long and 100 micrometres-wide dead-end microchannel, which extends from a T-junction of the main microchannels. Measurements of the flow-velocity profile in the chamber show that the flow rate decreases exponentially and scales linearly with the flow rate in the main channel. The characteristic length of the exponential decrease is 15 (1 ± 0.13) micrometres, meaning that a large part of the diffusion chamber is effectively flow-free. The diffusion properties are assessed by monitoring the diffusion of a dye into the chamber. It was found that a simple 1D diffusion model fits well to the experimental data. The time needed for the exchange of solutes in the chamber is of the order of minutes, depending on the solute's molecular weight. Here, we demonstrate how the diffusion chamber can be used for reversible environmental changes around flaccid, giant lipid vesicles and membrane tethers (nanotubes).     

An inertia enhanced passive pumping mechanism for fluid flow in microfluidic devices

We describe and characterize a pumping mechanism that leverages the momentum present in small droplets ejected from a micro-nozzle to drive flow in an open microfluidic device. This approach allows driving flow in a microfluidic device in a regime that offers unique features different to those achievable with typical passive pumping or syringe-pump driven flow. Two flow regimes with specific flow characteristics are described: inertia enhanced passive pumping, in which fluid exchange times in the channel are significantly reduced, and inertia actuated flow, in which it is possible to initiate flow in an empty channel or against natural pressure gradients. Momentum is leveraged to create rapid fluid exchanges, instantaneous flow reversal, filling and mixing inside the microfluidic device.     

基于微流控芯片的细胞外囊泡分离技术研究进展

细胞外囊泡（extracellular vesicles，EVs）是脂质双分子层包绕形成的半球状囊泡。研究表明EVs存在重要的生物学功能，同时EVs排放的数量、种类以及内含蛋白质、脂质或RNA等构成变化与疾病密切相关。EVs的研究将有助于理解其生物学功能和作用机制，同时也有望用于疾病的诊断和治疗，因此拥有巨大的临床应用前景。从复杂的体液样品中分离捕获EVs是实现基于EVs开展医学研究以及临床诊断的前提，但是目前绝大多数的EVs分离捕获仍然是采用传统分离手段，纯度低、效率差，迫切需要高效和高选择性的EVs分离手段。先进的微流控芯片技术具有微型化、集成化和自动化的优势，利用微流控芯片的EVs分离技术研究已成热点，本文围绕相关研究的最新进展进行了综述。     

Specific Transport of Target Molecules by Motor Proteins in Microfluidic Channels

Direct transport powered by motor proteins can alleviate the challenges presented by miniaturization of microfluidic systems. There have been several recent attempts to build motor‐protein‐driven transport systems based on simple capturing or transport mechanisms. However, to achieve a multifunctional device for practical applications, a more complex sorting/transport system should be realized. Herein, the proof of concept of a sorting device employing selective capture of distinct target molecules and transport of the sorted molecules to different predefined directions is presented. By combining the bottom‐up functionality of biological systems with the top‐down handling capabilities of micro‐electromechanical systems technology, highly selective molecular recognition and motor‐protein‐based transport is integrated in a microfluidic channel network.     

Electro-optical BLM chips enabling dynamic imaging of ordered lipid domains

Studies of lipid rafts, ordered microdomains of sphingolipids and cholesterol within cell membranes, are essential in probing the relationships between membrane organization and cellular function. While in vitro studies of lipid phase separation are commonly performed using spherical vesicles as model membranes, the utility of these models is limited by a number of factors. Here we present a microfluidic device that supports simultaneous electrical measurements and confocal imaging of on-chip bilayer lipid membranes (BLMs), enabling real-time multi-domain imaging of membrane organization. The chips further support closed microfluidic access to both sides of the membrane, allowing the membrane boundary conditions to be rapidly changed and providing a mechanism for dynamically adjusting membrane curvature through application of a transmembrane pressure gradient. Here we demonstrate the platform through the study of dynamic generation and dissolution of ordered lipid domains as membrane components are transported to and from the supporting annulus containing solvated lipids and cholesterol.     

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.     

Soft lithographic patterning of supported lipid bilayers onto a surface and inside microfluidic channels

We present simple soft lithographic methods for patterning supported lipid bilayer (SLB) membranes onto a surface and inside microfluidic channels. Micropatterns of polyethylene glycol (PEG)-based polymers were fabricated on glass substrates by microcontact printing or capillary moulding. The patterned PEG surfaces have shown 97 +/- 0.5% reduction in lipid adsorption onto two dimensional surfaces and 95 +/- 1.2% reduction inside microfluidic channels in comparison to glass control. Atomic force microscopy measurements indicated that the deposition of lipid vesicles led to the formation of SLB membranes by vesicle fusion due to hydrophilic interactions with the exposed substrate. Furthermore, the functionality of the patterned SLBs was tested by measuring the binding interactions between biotin (ligand)-labeled lipid bilayer and streptavidin (receptor). SLB arrays were fabricated with spatial resolution down to approximately 500 nm on flat substrate and approximately 1 microm inside microfluidic channels, respectively.     

Culturing and investigation of stress-induced lipid accumulation in microalgae using a microfluidic device

There is increasing interest in using microalgae as a lipid feedstock for the production of biofuels. Lipids used for these purposes are triacylglycerols that can be converted to fatty acid methyl esters (biodiesel) or decarboxylated to “green diesel.” Lipid accumulation in most microalgal species is dependent on environmental stress and culturing conditions, and these conditions are currently optimized using slow, labor-intensive screening processes. Increasing the screening throughput would help reduce the development cost and time to commercial production. Here, we demonstrated an initial step towards this goal in the development of a glass/poly(dimethylsiloxane) (PDMS) microfluidic device capable of screening microalgal culturing and stress conditions. The device contained power-free valves to isolate microalgae in a microfluidic growth chamber for culturing and stress experiments. Initial experiments involved determining the biocompatibility and culturing capability of the device using the microalga Tetraselmis chuii. With this device, T. chuii could be successfully cultured for up to 3 weeks on-chip. Following these experiments, the device was used to investigate lipid accumulation in the microalga Neochloris oleabundans. It was shown that this microalga could be stressed to accumulate cytosolic lipids in a microfluidic environment, as evidenced with fluorescence lipid staining. This work represents the first example of microalgal culturing in a microfluidic device and signifies an important expansion of microfluidics into the biofuels research arena.     

Microfluidic electroporation of robust 10-microm vesicles for manipulation of picoliter volumes

We present a new way to transport and handle picoliter volumes of analytes in a microfluidic context through electrically monitored electroporation of 10-25 microm vesicles. In this method, giant vesicles are used to isolate analytes in a microfluidic environment. Once encapsulated inside a vesicle, contents will not diffuse and become diluted when exposed to pressure-driven flow. Two vesicle compositions have been developed that are robust enough to withstand electrical and mechanical manipulation in a microfluidic context. These vesicles can be guided and trapped, with controllable transfer of material into or out of their confined environment. Through electroporation, vesicles can serve as containers that can be opened when mixing and diffusion are desired, and closed during transport and analysis. Both vesicle compositions contain lecithin, an ethoxylated phospholipid, and a polyelectrolyte. Their performance is compared using a prototype microfluidic device and a simple circuit model. It was observed that the energy density threshold required to induce breakdown was statistically equivalent between compositions, 10.2+/-5.0 mJ/m2 for the first composition and 10.5+/-1.8 mJ/m2 for the second. This work demonstrates the feasibility of using giant, robust vesicles with microfluidic electroporation technology to manipulate picoliter volumes on-chip.     

Wirelessly powered microfluidic dielectrophoresis devices using printable RF circuits

We report the first microfluidic device integrated with a printed RF circuit so the device can be wirelessly powered by a commercially available RFID reader. For conventional dielectrophoresis devices, electrical wires are needed to connect the electric components on the microchip to external equipment such as power supplies, amplifiers, function generators, etc. Such a procedure is unfamiliar to most clinicians and pathologists who are used to working with a microscope for examination of samples on microscope slides. The wirelessly powered device reported here eliminates the entire need for wire attachments and external instruments so the operators can use the device in essentially the same manner as they do with microscope slides. The integrated circuit can be fabricated on a flexible plastic substrate at very low cost using a roll-to-roll printing method. Electrical power at 13.56 MHz transmitted by a radio-frequency identification (RFID) reader is inductively coupled to the printed RFIC and converted into 10 V DC (direct current) output, which provides sufficient power to drive a microfluidic device to manipulate biological particles such as beads and proteins via the DC dielectrophoresis (DC-DEP) effect. To our best knowledge, this is the first wirelessly powered microfluidic dielectrophoresis device. Although the work is preliminary, the device concept, the architecture, and the core technology are expected to stimulate many efforts in the future and transform the technology to a wide range of clinical and point-of-care applications.     

Miniaturized planar lipid bilayer: increased stability, low electric noise and fast fluid perfusion

A microfluidic device was designed allowing the formation of a planar lipid bilayer across a micron-sized aperture in a glass slide sandwiched between two polydimethylsiloxane channel systems. By flushing giant unilamellar vesicles through a 500-μm-wide channel above the hole, we were able to form a planar lipid bilayer across the hole, resulting in a giga-seal. We demonstrate incorporation of biological nanopores into the bilayer. This miniaturized system offers noise recordings comparable to open headstage noise (under 1 pA RMS at 10 kHz), fast precision perfusion on each side of the membrane and the use of nanoliter analyte volumes. This technique shows a promising potential for automation and parallelization of electrophysiological setups.     

Fluidic shear-assisted formation of actuating multilamellar lipid tubes using microfabricated nozzle array device

Molecular self-assemblies exhibiting automatic motions have received much attention as potential artificial models of living organisms. We have developed a microfluidic picolitre nozzle-array device to form multilamellar lipid tubes (MLTs) under fluidic shear stress, which transformed into different two patterns (yarn-balls and double-helixes) and also exhibited unique self-actuation behaviors.     

Massively parallel production of lipid microstructures

In this paper we describe a simple and inexpensive microfluidic system for the production of lipid tubules and vesicles. The system incorporates a central microporous membrane for interfacing lipid films with aqueous flows. Hydrodynamic drag was used for the parallel elongation of high axial ratio lipid tubules with uniform 1.5 +/- 0.5 microm diameters. Alternatively, electrokinetic operation was used for the rapid and continuous production of vast numbers of lipid vesicles with diameters ranging from 1 to 3 microm.     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

An integrated microfluidic device for the high-throughput screening of microalgal cell culture conditions that induce high growth rate and lipid content

This study describes the development of a microfluidic device for the high-throughput screening of culture conditions, such as the optimum sodium acetate concentration for promoting rapid growth and high lipid accumulation of Chlamydomonas reinhardtii. An analysis of the microalgal growth on the microfluidic device revealed an optimum sodium acetate concentration of 5.72 g L^?1. The lipid content, determined by the 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY® 505/515) staining method, increased with the sodium acetate concentration. The results were found to be statistically reproducible with respect to cell growth and lipid production. Other nutrient conditions, including the nitrogen and phosphorus concentrations, can also be optimized on the same microfluidic platform. The microfluidic device performance results agreed well with the results obtained from the flask-scale experiments, validating that the culture conditions were scalable. Finally, we, for the first time, established a method for the absolute quantification of the microalgal lipid content in the picoliter culture volumes by comparing the on-chip and off-chip data. In conclusion, we successfully demonstrated the high-throughput screening of sodium acetate concentrations that induced high growth rates and high lipid contents in C. reinhardtii cells on the microfluidic device.

Characterization of a membrane-based gradient generator for use in cell-signaling studies

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.     

AC electrokinetic isolation and detection of extracellular vesicles from dental pulp stem cells: Theoretical simulation incorporating fluid mechanics

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles involved in intracellular communication and the transportation of biomarkers. EVs released by mesenchymal stem cells have been recently reported to play a role in cell-free therapy of many diseases. However, the demand for better research tools to replace the tedious conventional methods used to study EVs is getting stronger. EVs' manipulation using alternating current (AC) electrokinetic forces in a microfluidic device has appeared to be a reliable and sensitive diagnosis and trapping technique. Given that different AC electrokinetic forces may contribute to the overall motion of particles and fluids in a microfluidic device, EVs' electrokinetic trapping must be examined considering all dominant forces involved depending on the experimental conditions. In this paper, AC electrokinetic trapping of EVs using an interdigitated electrode arrays is investigated. A 2D numerical simulation incorporating the two significant AC electrokinetic phenomena (Dielectrophoresis and AC electroosmosis) has been performed. Theoretical predictions are then compared with experimental results and allow for a plausible explanation of observations inconsistent with DEP theory. It is demonstrated that the inconsistencies can be attributed to a significant extent to the contribution of the AC electroosmotic effect.     

Air-exposure technique for the formation of artificial lipid bilayers in microsystems

To develop a reliable method for on-chip bilayer lipid membrane (BLM) formation, which could be employed for use in a biosensor array platform, a polymer microfluidic device has been constructed, and the formation of suspended BLMs within it has been investigated. A simple, yet reproducible BLM formation protocol has been developed, in which a brief air-exposure period is employed to induce the rapid thinning of an initially thick lipid-solvent layer. The technique is rapid, reproducible, and amenable to the simple injection of proteins or analytes, as well as to buffer exchange on both sides of the membrane. Scaling up the technique for use in an array platform is also straightforward, the simultaneous formation of three individually addressable BLMs being demonstrated.