基礎講座 電気泳動(Electrophoresis)

Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.

Environmental sensing through focal adhesions

Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

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Reactions in Droplets in Microfluidic Channels

This critical review summarizes developments in microfluidic platforms that enable the miniaturization, integration, automation and parallelization of (bio-)chemical assays (see S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094–1110, for an earlier review). In contrast to isolated application-specific solutions, a microfluidic platform provides a set of fluidic unit operations, which are designed for easy combination within a well-defined fabrication technology. This allows the easy, fast, and cost-efficient implementation of different application-specific (bio-)chemical processes. In our review we focus on recent developments from the last decade (2000s). We start with a brief introduction into technical advances, major market segments and promising applications. We continue with a detailed characterization of different microfluidic platforms, comprising a short definition, the functional principle, microfluidic unit operations, application examples as well as strengths and limitations of every platform. The microfluidic platforms in focus are lateral flow tests, linear actuated devices, pressure driven laminar flow, microfluidic large scale integration, segmented flow microfluidics, centrifugal microfluidics, electrokinetics, electrowetting, surface acoustic waves, and dedicated systems for massively parallel analysis. This review concludes with the attempt to provide a selection scheme for microfluidic platforms which is based on their characteristics according to key requirements of different applications and market segments. Applied selection criteria comprise portability, costs of instrument and disposability, sample throughput, number of parameters per sample, reagent consumption, precision, diversity of microfluidic unit operations and the flexibility in programming different liquid handling protocols (295 references).

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Microfluidic lab-on-a-chip platforms: requirements, characteristics and applications

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

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Single-cell analysis and sorting using droplet-based microfluidics

We demonstrate a high-throughput drop sorter for microfluidic devices that uses dielectrophoretic forces. Microelectrodes underneath a polydimethylsiloxane channel produce forces of more than 10nN on a water drop in an inert oil, resulting in sorting rates greater than 1.6kHz. We investigate the dependence of such forces on drop size and flow. Alternate designs with electrodes on either side of a symmetric channel Y junction provide refined control over droplet selection.

Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices

This paper describes a miniaturized, integrated, microfluidic device that can pull molecules and living cells bound to magnetic particles from one laminar flow path to another by applying a local magnetic field gradient, and thus selectively remove them from flowing biological fluids without any wash steps. To accomplish this, a microfabricated high-gradient magnetic field concentrator (HGMC) was integrated at one side of a microfluidic channel with two inlets and outlets. When magnetic micro- or nano-particles were introduced into one flow path, they remained limited to that flow stream. In contrast, when the HGMC was magnetized, the magnetic beads were efficiently pulled from the initial flow path into the collection stream, thereby cleansing the original fluid. Using this microdevice, living E. coli bacteria bound to magnetic nanoparticles were efficiently removed from flowing solutions containing densities of red blood cells similar to that found in blood. Because this microdevice allows large numbers of beads and cells to be sorted simultaneously, has no capacity limit, and does not lose separation efficiency as particles are removed, it may be especially useful for separations from blood or other clinical samples. This on-chip HGMC-microfluidic separator technology may potentially allow cell separations to be carried out in the field outside of hospitals and clinical laboratories.

Combined microfluidic-micromagnetic separation of living cells in continuous flow.

We present an integrated circuit/microfluidic chip that traps and moves individual living biological cells and chemical droplets along programmable paths using dielectrophoresis (DEP). Our chip combines the biocompatibility of microfluidics with the programmability and complexity of integrated circuits (ICs). The chip is capable of simultaneously and independently controlling the location of thousands of dielectric objects, such as cells and chemical droplets. The chip consists of an array of 128 × 256 pixels, 11 × 11 µm2 in size, controlled by built-in SRAM memory; each pixel can be energized by a radio frequency (RF) voltage of up to 5 Vpp. The IC was built in a commercial foundry and the microfluidic chamber was fabricated on its top surface at Harvard. Using this hybrid chip, we have moved yeast and mammalian cells through a microfluidic chamber at speeds up to 30 µm sec–1. Thousands of cells can be individually trapped and simultaneously positioned in controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one. Our IC/microfluidic chip provides a versatile platform to trap and move large numbers of cells and fluid droplets individually for lab-on-a-chip applications.

Integrated circuit/microfluidic chip to programmably trap and move cells and droplets with dielectrophoresis

Local physical interactions between cells and extracellular matrix (ECM) influence directional cell motility that is critical for tissue development, wound repair, and cancer metastasis. Here we test the possibility that the precise spatial positioning of focal adhesions governs the direction in which cells spread and move. NIH 3T3 cells were cultured on circular or linear ECM islands, which were created using a microcontact printing technique and were 1 μm wide and of various lengths (1 to 8 μm) and separated by 1 to 4.5 μm wide nonadhesive barrier regions. Cells could be driven proactively to spread and move in particular directions by altering either the interisland spacing or the shape of similar-sized ECM islands. Immunofluorescence microscopy confirmed that focal adhesions assembled preferentially above the ECM islands, with the greatest staining intensity being observed at adhesion sites along the cell periphery. Rac-FRET analysis of living cells revealed that Rac became activated within 2 min afte...

Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation

Positioning single cells is of utmost importance in areas of biomedical research as diverse as in vitro fertilization, cell-cell interaction, cell adhesion, embryology, microbiology, stem cell research, and single cell transfection. Here we describe dielectrophoretic tweezers, a sharp glass tip with electrodes on either side, capable of trapping single cells with electric fields. Mounted on a micromanipulator, dielectrophoresis tweezers can position a single cell in three dimensions, holding the cell against fluid flow of hundreds of microns per second with more than 10 pN of force. We model the electric field produced by the tweezers and the field produced by coaxial microelectrodes. We show that cells are trapped without harm while they divide in the trap. In addition, dielectrophoretic tweezers offer the possibility for trapping, electroporating, and microinjecting a single cell with one probe.

Thomas Hunt

Papers

Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices

Combined microfluidic-micromagnetic separation of living cells in continuous flow.

Integrated circuit/microfluidic chip to programmably trap and move cells and droplets with dielectrophoresis

Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation

Dielectrophoresis tweezers for single cell manipulation.