Multi-step microfluidic device for studying cancer metastasis

This paper describes a multi-step microfluidic device for studying the deformation and extravasation of primary tumor cells. Prior to extravasation, primary tumor cells undergo sequential steps of deformation through the capillaries, before adhering and transmigrating through the endothelial lining and basement membrane. To study this cascade of events, we fabricated a multi-step microfluidic device whose microgaps were coated with Matrigel to mimic the basement membrane. The microchannel was lined with human microvascular endothelial cells (HMECs) to replicate the endothelial lining. Analysis of deformation, biological and migratory capabilities of various tumor cell lines viz. HepG2, HeLa, and MDA-MB 435S were quantified using the fabricated device. After deformation, the cells' viabilities were significantly reduced and their doubling times were simultaneously increased, indicating changes in their biological capability. However, cell deformation did not significantly reduce their cell motility. Cell motility was co-assessed using the cell's migration rate and the overall population's percentage migration under various conditions (no barrier, Matrigel and Matrigel-HMEC). The device was also used to quantify the effects of Matrigel and the endothelial lining on cell migration. Our results suggest that both played an independent role in inhibiting cell extravasation, with the Matrigel significantly slowing down cell movement and the endothelial lining reducing the total number of transmigrated cells.     

Optical and electrochemical detection techniques for cell-based microfluidic systems

The ability to fabricate microfluidic systems with complex structures and with compatible dimensions between the microfluidics and biological cells have attracted significant attention in the development of microchips for analyzing the biophysical and biochemical functions of cells. Just as cell-based microfluidics have become a versatile tool for biosensing, diagnostics, drug screening and biological research, detector modules for cell-based microfluidics have also undergone major development over the past decade. This review focuses on detection methods commonly used in cell-based microfluidic systems, and provides a general survey and an in-depth look at recent developments in optical and electrochemical detection methods for microfluidic applications for biological systems, particularly cell analysis. Selected examples are used to illustrate applications of these detection systems and their advantages and weaknesses.     

Analysis of the paracrine loop between cancer cells and fibroblasts using a microfluidic chip

We use a microfluidic cell culture chip equipped with pneumatic microvalves to analyze the paracrine loop between lung cancer cells and fibroblasts. In order to assess the cellular responses in the paracrine loop, we measure the migration speeds of cancer cells and the aspect ratios of fibroblasts which reflect the phenotype of myofibroblasts. With well-controlled interaction sequences between these two types of cells, we verify that the cytokines from cancer cells effectively stimulate the fibroblasts into myofibroblasts. The cytokines from myofibroblasts, rather than fibroblasts, increase the migration speeds of cancer cells. We confirm that the transforming growth factor-β1 (TGF-β1) is involved in the interaction between cancer cells and fibroblasts, and we also interrupt this paracrine loop in the cell culture chip by inhibiting the TGF-β1 receptors on fibroblasts.     

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(?) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.     

A microfluidic membrane chip for in situ fouling characterization

A new method for non-invasive in situ monitoring of a microfiltration process is described. In microfiltration systems, local information on the deposition characteristics can be used to determine the cake behavior during a filtration run. Typically, non-invasive methods of fouling study are restricted to specialized membranes, or require highly complex systems. This study employs the use of synthetic embedded channel membranes, with channels separated by a porous structure (active membrane). The characteristics of the active membrane have been analyzed. Deposition on the membrane surface can be observed and monitored optically across the width of the feed channel. This can be used to observe the liquid hydrodynamics in the channel as well as the local cake properties in time. In dead end filtration, it has been observed that with 6 μm particles, the cake initially deposits towards the end of the membrane. However, as filtration continues, the deposition changes with more local deposition towards the channel entrance, leading to a more homogeneous cake layer.     

Simultaneous laser-induced fluorescence and contactless-conductivity detection for microfluidic chip

A combined detection system involving simultaneous LIF and contacfless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conduetivity detector was used in C^4D. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably.     

化学发光检测在微流控芯片中的应用综述

综述了近年来化学发光检测在微流控芯片中的应用.指出微流控芯片(又称为"芯片实验室"或者"微型全分析系统")因具有小型化、集成化和自动化等特点而在近20年来日益受到关注,而化学发光检测具有仪器结构简单、背景噪音低、操作和维护成本低等优点,非常适合用作微流控芯片的检测手段.     

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.     

Bilayer microfluidic chip for diffusion-controlled activation of yeast species

A bilayer microfluidic chip is used, in which multiple laminar streams are generated to define local microenvironments. The bilayer architecture of the microchip separates cell handling and positioning from cell activation by soluble chemicals. Cell activation is diffusion controlled through a porous membrane. By employing time-lapse fluorescence microscopy, gene expression of the enhanced green fluorescent protein (eGFP) in Saccharomyces cerevisiae is studied under various conditions. We demonstrate that the yeast cells remain viable in the microchip for at least 17h, and that gene expression can be initiated by the supply of the inducer galactose at a spatial precision of a few micrometers.     

On-chip connector valve for immunoaffinity chromatography in a microfluidic chip

A miniature valve that operates between a chip port and a tube fitting was developed. The valve functions by means of a rotor, 3 mm in diameter and 1.5 mm in height, made of Teflon, with a 0.2-mm diameter hole at its center that is co-axial with the tube fitting. It also has a radial groove, 0.85 mm long, 0.2 mm wide, and 0.2 mm deep, at the bottom surface, starting at its center. The chip port and the tube fitting have an offset of 0.75 mm, and, thus, the rotation of the rotor can make an on and off connection between the chip port and the groove, which is connected to the tubing. The valve had a pressure resistance of at least 1.0 MPa. The on-chip valve can be placed in position by adding only a single part, a valve rotor, and no changes in the fabrication of the glass microchip are required. Since the valve functions as a part of a connector, we refer to it as an on-chip connector valve. Immunoaffinity chromatography of a fluorescence-labeled recombinant antibody fragment was carried out in a glass microchip using the valves.     

Development of multistage distillation in a microfluidic chip

Although there has been a lot of work on the development of microchemical processing systems such as micro-reactors and micro-sensors, little attention has been paid to micro-separation units, and in particular, microscale distillation. In this paper, various silicon-glass microscale distillation chips with different channel configurations were fabricated and tested. A temperature gradient was setup across the chip by heating and cooling the two ends. The feed was located at the middle of the microchannel. Arrays of micropillars were incorporated in order to guide the liquid flow. It was found that the separation performance was promoted by increasing the length of the microchannel. However, this created an imbalance of the liquid flows at the two sides of the microchannel and caused flooding. This hydrodynamic limitation was addressed by incorporating micropillars on both sides of the channel. The most efficient microdistillation chip consisted of a microchannel with 600 microns width and 40 cm length. Experimental results showed high efficiency for the separation of a 50 mol% acetone-water mixture when the heating and cooling temperature were 95 °C and 42 °C respectively. The concentrations of acetone were 3 mol% in the bottom stream and 95 mol% in the distillate, which was equivalent to at least 4 equilibrium stages at total reflux conditions. Furthermore, a 50 mol% methanol-toluene mixture was separated into nearly pure toluene in the bottom stream and 75 mol% methanol in the distillate. The performance of the microdistillation unit was reproducible in repeated tests.     

Development of a double-layer microfluidic chip with flow medium for chemotherapy resistance analysis of lung cancer

Integration and miniaturization are main advantages of microchip-based systems. Vertical integration of the multiple operations within a multiple-layer chip is expected to satisfy the urgent demand for high-throughput and large-scale applications. This study aimed at establishing a double-layer chip to integrate the operations including the cell culture, the identification of the protein and the detection of the cell viability onto a platform systematically and supplied with flow fresh medium continuously via a syringe pump to mimic the microenvironment in vivo. With this device, human non-small cell lung cancer cell line (SPCA-1) was cultured well; the expression and the activity of multidrug resistance-associated protein (MRP1) were detected by immunofluorescence assay for the cells pretreated with or without MK-571, a known inhibitor of MRP1; apoptosis percentages were assayed for the cells after being treated by the anticancer drug etoposide (VP-16). The results demonstrated that the function of the MRP1 was inhibited by MK-571, and the percentage of apoptotic for the cells pretreated with MK-571 was higher than that of the control (38.2±2.5% versus 12.3±0.85%, p<0.005). All these indicated that the new device could provide a suitable condition for cell culture and functional analysis in biomedical research, and MK-571 is an effective inhibitor of MRP1 associated with the viability of SPCA-1 cell line treated by VP-16.     

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized “in situ” on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.      

A smart surface in a microfluidic chip for controlled protein separation

The smart surface created in a microfluidic chip has shown the capability of adsorbing and releasing proteins under electrical control. The inner surface of the chip channel was first coated by a thin layer of Au through sputtering and was subsequently modified with loosely packed self-assembled monolayers (SAMs) of thiols with terminal carboxylic or amino groups. Upon application of an external electric potential to the gold substrate, reversible conformational transformation between "bent" and "straight" states for the anchored mercapto chains could be modulated, through the electrostatic effect between the ionized terminal groups and the charged gold substrate. Thus, a hydrophobic or hydrophilic channel surface was established and could be reversibly switched electrochemically. Accordingly, the microchips prepared in this way can reversibly and selectively adsorb and release differently charged proteins under electrical control. Two model proteins, avidin and streptavidin, were demonstrated to be readily adsorbed by the smart chips under negative and positive potential, respectively. Also, more than 90 % of the adsorbed proteins could be released upon an electrical command. Furthermore, these chips were applied to the controlled separation of avidin and streptavidin mixtures with 1:1 and 1:1000 molar ratios. Under specific applied potentials, the chips adsorbed a certain protein from the mixture whereas the other protein was allowed to flow out, after which the adsorbed protein could be released by switching the applied potential. Thus, two eluted protein fractions were obtained and the separation of the two proteins was achieved. For the former mixture, each eluted fraction contained up to approximately 80-90 % avidin or streptavidin. For the latter mixture, the resulting separation efficiency indicated that the molar ratio of avidin and streptavidin could be increased from 1:1000 to about 32:1 after five run separations.     

Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.     

Electrophoresis on a microfluidic chip for analysis of fluorescence-labeled human rhinovirus

We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab-on-a-chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (lambda ex 630 nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10 s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size-exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very-low-density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive.     

A microfluidic fluorous solid-phase extraction chip for purification of amino acids

An electrokinetically-driven microfluidic chip was developed to realize beads-based solid-phase extraction (SPE) of amino acids. This chip uses a two-level (deep/shallow) poly(dimethylsiloxane) (PDMS) microchannel network to confine the fluorous reversed-phase silica beads within the SPE chamber. The mixture of fluorous tagged and non-tagged amino acids was carried into the fluorous solid-phase extraction (F-SPE) chamber by electrokinetic pumping and was successfully separated and extracted. By adding a reference material to the sample, the extraction efficiency of the eluted fluorous-tagged amino acid was calculated using the detection results from mass spectrometry (MS). The F-SPE microchips showed good reproducibility and efficiency, yielding an average extraction efficiency of 55% with a RSD of 10.6% under the typical experimental conditions.     

Microalgal motility measurement microfluidic chip for toxicity assessment of heavy metals

A polydimethylsiloxane microfluidic chip has been developed for the estimation of toxic heavy metals based on measurement of mobility of marine microalgae. The chip is mainly composed of an upstream concentration gradient generator and a downstream perfusion-based chemotatic module. The processes of toxic liquid dilution and diffusion, microalgal culturing, cell stimulation, and online screening can be integrated in this chip, which makes it an attractive approach to simplify toxicity testing procedures. The microalgal motility was adopted as a microfluidic bioassay signal and was evaluated as the percentage of motile cells, curvilinear velocity, average path velocity, and straight line velocity. Two mobile marine microalgae, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis, were confined in the chemotatic module and stimulated by the eight concentration gradients of Cu and Cd generated by the concentration gradient generator. In all cases, a toxic response was detected (i.e., a dose-related inhibition of motility was observed). Only 1.5?h was needed to predict EC₅₀ values. Thus, the microfluidic chip developed was proved to be useful as a simple and rapid approach in heavy metal detection and might be expanded as a conventional test method in environmental toxicity assessment.     

A portable and integrated nucleic acid amplification microfluidic chip for identifying bacteria

In this work, we developed a portable integrated microchip of loop-mediated isothermal nucleic acid amplification (LAMP). This chip, with sample-to-answer capability, could perform rapid DNA release, exponential signal amplification and naked-eye result read-out in single or multiplex format. We call it iμLAMP, namely integrated micro-LAMP, which was successfully used for point-of-care identification of bacteria.     

Application of adaptive pressure-driven microfluidic chip in thyroid function measurement

The improvement in accuracy of in vitro diagnosis has always been the focus of early screening of thyroid dysfunction. We constructed a microfluidic chip based on a polystyrene polymer substrate. Total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), and thyrotropin (TSH) in human whole blood samples were analysed by fluorescence immunoassay to evaluate thyroid function. The results indicate that the microfluidic chip shows a good linear relationship in the detection of TT3, TT4, FT3, FT4, and TSH standards, and the correlation coefficient (r) is not less than 0.9900. In addition, the chip also has strong anti-interference (RSD% ≤ 5%) and good repeatability (CV ≤ 8%), and its inter-batch differences are small (CV ≤ 15%). The results of practical application in clinical thyroid function measurement indicated its high accuracy (r ≥ 0.9900). It provides a new method for the determination of thyroid function and lays a foundation for subsequent clinical application.