Nanoporous micro-element arrays for particle interception in microfluidic cell separation

The ability to control cell-surface interactions in order to achieve binding of specific cell types is a major challenge for microfluidic immunoaffinity cell capture systems. In the majority of existing systems, the functionalized capture surface is constructed of solid materials, where flow stagnation at the solid-liquid interface is detrimental to the convection of cells to the surface. We study the use of ultra-high porosity (99%) nanoporous micro-posts in microfluidic channels for enhancing interception efficiency of particles in flow. We show using both modelling and experiment that nanoporous posts improve particle interception compared to solid posts through two distinct mechanisms: the increase of direct interception, and the reduction of near-surface hydrodynamic resistance. We provide initial validation that the improvement of interception efficiency also results in an increase in capture efficiency when comparing nanoporous vertically aligned carbon nanotube (VACNT) post arrays with solid PDMS post arrays of the same geometry. Using both bacteria (～1 μm) and cancer cell lines (～15 μm) as model systems, we found capture efficiency increases by 6-fold and 4-fold respectively. The combined model and experimental platform presents a new generation of nanoporous microfluidic devices for cell isolation.     

Automated cell culture in high density tubeless microfluidic device arrays

Microfluidics is poised to have an impact on life sciences research. However, current microfluidic methods are not compatible with existing laboratory liquid dispensing and detection infrastructure. This incompatibility is a barrier to adoption of microfluidic systems and calls for improved approaches that will enhance performance and promote acceptance of microfluidic systems in the life sciences. Ease of use, standardized interfaces and automation remain critical challenges. We present a platform based on surface tension effects, where the difference in pressure inside drops of unequal volume drives flow in passive structures. We show integration with existing laboratory infrastructure, microfluidic operations such as pumping, routing and compartmentalization without discrete micro-components as well as cell patterning in both monolayer and three-dimensional cell culture.     

Novel multi-depth microfluidic chip for single cell analysis

A novel multi-depth microfluidic chip was fabricated on glass substrate by use of conventional lithography and three-step etching technology. The sampling channel on the microchip was 37 microm deep, while the separation channel was 12 microm deep. A 1mm long weir was constructed in the separation channel, 300 microm down the channel crossing. The channel at the weir section was 6 microm deep. By using the multi-depth microfluidic chip, human carcinoma cells, which easily aggregate, settle and adhere to the surface of the channel, can be driven from the sample reservoir to the sample waste reservoir by hydrostatic pressure generated by the difference of liquid level between sample and sample waste reservoirs. Single cell loading into the separation channel was achieved by applying a set of pinching potentials at the four reservoirs. The loaded cell was stopped by the weir and precisely positioned within the separation channel. The trapped cell was lysed by sodium dodecyl sulfate (SDS) containing buffer solution in 20s. This approach reduced the lysing time and improved the reproducibility of chip-based electrophoresis separations. Reduced glutathione (GSH) and reactive oxygen species (ROS) were used as model intracellular components in single human carcinoma cells, and the constituents were separated by chip-based electrophoresis and detected by laser-induced fluorescence (LIF). A throughput of 15 samples/h, a migration time precision of 3.1% RSD for ROS and 4.9% RSD for GSH were obtained for 10 consecutively injected cells.     

Multi-channel microfluidic chip-mass spectrometry platform for cell analysis

In this review, we highlight the latest development of multi-channel microfluidic chip-mass spectrometry (chip-MS) in cell analysis and metabolite detection. Following a brief introduction about history and development of multi-channel microchip and MS combination, we will elaborate the key issues of constructing chip-MS platform interface. Then exciting progresses made in this field should be reviewed with well exemplified works, including chip-MS technology for cell introduction, pretreatment of cell secretions and cell metabolite analysis. We will also describe the development of integrated total analysis systems proposed by our group. We hope this brief review will inspire interested readers and provide knowledge about chip-MS platform in the bioanalysis field, particularly in cell analysis and metabolite identifying applications.     

Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.     

A linear dilution microfluidic device for cytotoxicity assays

A two-layer polymer microfluidic device is presented which creates nine linear dilutions from two input fluid streams mixed in varying volumetric proportions. The linearity of the nine dilutions is conserved when the flow rate is held constant at 1.0 microl min(-1) (R(2) = 0.9995) and when it is varied from 0.5-16 microl min(-1) (R(2) = 0.9998). An analytical expression is presented for designing microfluidic devices with arbitrary numbers of linear dilutions. To demonstrate the efficacy of this device, primary human epidermal keratinocytes (HEK) were stained with nine dilutions of calcein, resulting in a linear spread of fluorescent intensities (R(2) = 0.94). The operating principles of the device can be scaled up to incorporate any number of linear dilutions. This scalability, coupled with an intrinsic ability to create linear dilutions under a variety of operating conditions, makes the device applicable to high throughput screening applications such as combinatorial chemistry or cytotoxicity assays.     

Combining microfluidic networks and peptide arrays for multi-enzyme assays

This paper reports the use of microfluidic networks (muFNs) to both prepare peptide microarrays and carry out label-free enzyme assays on self-assembled monolayers (SAMs) of alkanethiolates on gold. A poly(dimethylsiloxane) (PDMS) stamp fabricated with microchannels is used to immobilize a linear array of cysteine-terminated peptides onto SAMs presenting maleimide groups. The stamp is then reapplied to the SAM in a perpendicular direction to introduce enzyme solutions so that each solution can interact with an identical linear array of immobilized peptides. The muFNs enable multiple enzyme-substrate interactions to be simultaneously evaluated at a submicroliter scale, while the use of SAMs enables the use of MALDI mass spectrometry (MS) to analyze the enzyme activities. This paper demonstrates applications of this system for assaying multiple kinases and for profiling the activities of kinases and phosphatases in human K562 cell extracts. The combination of muFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples.     

A digital microfluidic platform for primary cell culture and analysis

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.     

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.     

CMOS arrays as chemiluminescence detectors on microfluidic devices

A simple, low-cost process to integrate complementary metal oxide semiconductor array detectors (CMOSAD) for chemiluminescence is presented, evaluated, and applied to the determination of nitrite in ground water samples. CMOS arrays of different brands (obtained from commercial image sensors) were adapted as chemiluminescence detectors on microfluidic devices. The performance of the CMOSADs was evaluated in the visible zone of the spectrum using a tungsten halogen lamp as light source. Intrinsic parameters assessed included signal stability, spectral response, dark current, and signal-to-noise ratio. Thereafter, the CMOSADs were integrated on microfluidic devices and their performances in quantitative analysis were assessed with the chemiluminometric reaction of hydrogen peroxide with luminol, catalyzed with hexacyanoferrate (III). The parameters assessed were sensitivity, linear range, detection limit, reproducibility, correlation coefficient of the calibration curves, and baseline drift during measurements. The CMOSAD with the best performance was selected to assess the applicability of the developed microfluidic devices with the integrated detector. The microfluidic system permitted the determination of nitrite with both good precision and good recovery values in the analysis of ground water samples. Integration was easily achieved and enabled the development of a simple, low-cost, and feasible alternative to conventional detectors.     

Cell culture arrays using magnetic force-based cell patterning for dynamic single cell analysis

In order to understand the behavior of individual cells, single cell analyses have attracted attention since most cell-based assays provide data with values averaged across a large number of cells. Techniques for the manipulation and analysis of single cells are crucial for understanding the behavior of individual cells. In the present study, we have developed single cell culture arrays using magnetic force and a pin holder, which enables the allocation of the magnetically labeled cells on arrays, and have analyzed their dynamics. The pin holder was made from magnetic soft iron and contained more than 6000 pillars on its surface. The pin holder was placed on a magnet to concentrate the magnetic flux density above the pillars. NIH/3T3 fibroblasts that were labeled with magnetite cationic liposomes (MCLs) were seeded into a culture dish, and the dish was placed over the pin holder with the magnet. The magnetically labeled cells were guided on the surface where the pillars were positioned and allocated on the arrays with a high resolution. Single-cell patterning was achieved by adjusting the number of cells seeded, and the target cell was collected by a micromanipulator after removing the pin holder with the magnet. Furthermore, change in the morphology of magnetically patterned cells was analyzed by microscopic observation, and cell spreading on the array was observed with time duration. Magnetic force-based cell patterning on cell culture arrays would be a suitable technique for the analysis of cell behavior in studies of cell-cell variation and cell-cell interactions.     

Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems

A one-step immunomagnetic separation technique was performed on a microfluidic platform for the isolation of specific cells from blood samples. The cell isolation and purification studies targeted T cells, as a model for low abundance cells (about 1:10,000 cells), with more dilute cells as the ultimate goal. T cells were successfully separated on-chip from human blood and from reconstituted blood samples. Quantitative polymerase chain reaction analysis of the captured cells was used to characterize the efficiency of T cell capture in a variety of flow path designs. Employing many (4-8), 50 microm deep narrow channels, with the same overall cross section as a single, 3 mm wide channel, was much more effective in structuring dense enough magnetic bead beds to trap cells in a flowing stream. The use of 8-multiple bifurcated flow paths increased capture efficiencies from approximately 20 up to 37%, when compared to a straight 8-way split design, indicating the value of ensuring uniform flow distribution into each channel in a flow manifold for effective cell capture. Sample flow rates of up to 3 microL min(-1) were evaluated in these capture beds.     

基于微流控芯片-质谱联用的细胞分析研究进展

微流控芯片与质谱联用为细胞研究提供了一个很好的研究平台.质谱的高灵敏度和对化合物独特的鉴别能力可以从复杂的化学信息背景中筛选识别出微量目标物,是细胞分析理想的检测手段.本文重点综述了近年来基于微流控芯片-质谱联用技术的细胞研究进展,从芯片-电喷雾质谱(ESI-MS)接口技术、集成化的样品前处理技术、细胞的药物代谢和细胞相互作用研究及基质辅助激光解吸电离质谱(MALDI-MS)的细胞分析应用等方面总结了最新的方法和技术发展.并展望了芯片-质谱联用新技术应用于细胞分析的可能性.     

Dual-electrode microfluidic cell for characterizing electrocatalysts

In this paper we introduce a microelectrochemical cell configured for generation-collection experiments and designed primarily for examining the kinetics of electrocatalysts. The heart of the device consists of two, closely spaced, pyrolyzed photoresist microband electrodes enclosed within a microchannel. The cell is suitable for evaluating the efficiency of electrocatalysts under an unprecedented range of conditions. Specifically, compared to the gold-standard rotating ring-disk electrode (RRDE), this device offers four major advantages. First, collection efficiencies of 97% are easily achieved, compared to values of 20-37% that are characteristic of RRDEs. Second, mass transfer coefficients of 0.5 cm s(-1) are accessible for typical redox species, which is significantly higher than RRDEs (up to 0.01 cm s(-1)). Third, we show that the device can operate effectively at temperatures up to 70 °C, which is important for measuring electrochemical kinetics that are relevant to fuel cell catalysts. Finally, much less catalyst and much smaller volumes of electrolyte solution are required to make kinetic measurements using the microelectrochemical device compared to the RRDE. Here, we present the simple procedure used to fabricate the device, fundamental electroanalytical characterization, and electrocatalytic measurements relevant to the oxygen reduction reaction.     

Rapid prototyping for injection moulded integrated microfluidic devices and diffractive element arrays

This paper describes two fabrication procedures that makes it possible to design, fabricate and injection mold a microfluidic system with an on board coupling element or an optical array platform in less than four hours. Epoxy masters for the array and a single diffractive element were produced using conventional soft lithography techniques and a commercially available UV curable epoxy. The fabrication of the master for the integrated microfluidic device utilized the surface chemistry of polyester and its interaction with the anionic surfactant sodium dodecyl sulfate (SDS), to selectively inhibit the adhesion between the epoxy and the polyester film during the curing reaction. The transfer of a microfluidic design and the required coupling element (632 nm holographic grating) along the base of the channel was completed in a single step. The turnaround time from design to injection molded device whether a microchannel or array was 3.5 h.     

Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening

Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development.     

Integration of nanocapillary arrays into microfluidic devices for use as analyte concentrators

A nanocapillary array was integrated into a microfluidic device and its ability to concentrate analytes was characterized. Through the application of an electric field across the channel, large molecules were concentrated in front of the nanocapillary array, and a concentrated analyte band was ejected from the channel by reversing the polarity of the electric field. The effects of nanocapillary diameter, analyte charge, analyte concentration, analyte plug length, and analyte relative mobility were investigated. Concentration factors up to 300-fold were measured for fluorescein. By concentrating anionic FITC-labeled peptides, it was demonstrated that the magnitude of the electrophoretic mobility did not have a measurable effect on the concentration factor. Therefore, multiple analytes can be concentrated in front of the same nanocapillary array without adjusting the conditions, provided the analytes have the same net charge. In the presence of an electric field, a charge trapping effect was observed; small anionic molecules can be concentrated in front of nanocapillary array with channel diameters which are orders of magnitude above the molecular weight cut-offs for hydrodynamically driven systems. The concentrating process was found to be very efficient for fluorescein, as no leakage through the nanocapillary array or sorption of fluorescein to the nanocapillary array was observed. Due to their flexibility and efficiency, it is anticipated that nanocapillary arrays will find increased utility in electrokinetically driven microfluidic systems.     

Polymeric microfluidic system for DNA analysis

The application of micro total analysis system (μTAS) has grown exponentially in the past decade. DNA analysis is one of the primary applications of μTAS technology. This review mainly focuses on the recent development of the polymeric microfluidic devices for DNA analysis. After a brief introduction of material characteristics of polymers, the various microfabrication methods are presented. The most recent developments and trends in the area of DNA analysis are then explored. We focus on the rapidly developing fields of cell sorting, cell lysis, DNA extraction and purification, polymerase chain reaction (PCR), DNA separation and detection. Lastly, commercially available polymer-based microdevices are included.     

A microfluidic electroporation device for cell lysis

We demonstrate a micro-electroporation device for cell lysis prior to subcellular analysis. Simple circuit models show that electrical lysis method is advantageous because it is selective towards plasma membrane while leaving organelle membrane undamaged. In addition, miniaturization of this concept leads to negligible heat generation and bubble formation. The designed microdevices were fabricated using a combination of photolithography, metal-film deposition, and electroplating. We demonstrate the electro-lysis of human carcinoma cells in these devices to release the subcellular materials.     

High-density fabrication of normally closed microfluidic valves by patterned deactivation of oxidized polydimethylsiloxane

The use of polydimethylsiloxane (PDMS) in microfluidic devices is extensive in academic research. One of the most fundamental treatments is to expose PDMS to plasma oxidation in order to render its surface temporarily hydrophilic and capable of permanent bonding. Here, we show that changes in the surface chemistry induced by plasma oxidation can spatially be counteracted very cleanly and reliably in a scalable manner by subsequent microcontact printing of residual oligomers from a PDMS stamp. We characterize the surface modifications through contact angle, atomic force microscopy, X-ray photoelectron spectroscopy, and bond-strength measurements. We utilize this approach for negating the bonding of a flexible membrane layer within an elastomeric valve and demonstrate its effectiveness by integration of over one thousand normally closed elastomeric valves within a single substrate. In addition, we demonstrate that surface energy patterning can be used for "open microfluidic" applications that utilize spatial control of surface wetting.