High-density microfluidic arrays for cell cytotoxicity analysis

In this paper, we report on the development of a multilayer elastomeric microfluidic array platform for the high-throughput cell cytotoxicity screening of mammalian cell lines. Microfluidic channels in the platform for cell seeding are orthogonal to channels for toxin exposure, and within each channel intersection is a circular chamber with cell-trapping sieves. Integrated, pneumatically-actuated elastomeric valves within the device isolate the microchannel array within the device into parallel rows and columns for cell seeding and toxin exposure. As a demonstration of the multiplexing capability of the platform, a microfluidic array containing 576 chambers was used to screen three cell types (BALB/3T3, HeLa, and bovine endothelial cells) against a panel of five toxins (digitonin, saponin, CoCl(2), NiCl(2), acrolein). Evaluation of on-chip cell morphology and viability was carried out using fluorescence microscopy, with outcomes comparable to microtiter plate cytotoxicity assays. Using this scalable platform, cell seeding and toxin exposure can be carried out within a single microfluidic device in a multiplexed format, enabling high-density parallel cytotoxicity screening while minimizing reagent consumption.     

On-chip microfluidic buffer swap of biological samples in-line with downstream dielectrophoresis

Microfluidic cell enrichment by dielectrophoresis, based on biophysical and electrophysiology phenotypes, requires that cells be resuspended from their physiological media into a lower conductivity buffer for enhancing force fields and enabling the dielectric contrast needed for separation. To ensure that sensitive cells are not subject to centrifugation for resuspension and spend minimal time outside of their culture media, we present an on-chip microfluidic strategy for swapping cells into media tailored for dielectrophoresis. This strategy transfers cells from physiological media into a 100-fold lower conductivity media by using tangential flows of low media conductivity at 200-fold higher flow rate versus sample flow to promote ion diffusion over the length of a straight channel architecture that maintains laminarity of the flow-focused sample and minimizes cell dispersion across streamlines. Serpentine channels are used downstream from the flow-focusing region to modulate hydrodynamic resistance of the central sample outlet versus flanking outlets that remove excess buffer, so that cell streamlines are collected in the exchanged buffer with minimal dilution in cell numbers and at flow rates that support dielectrophoresis. We envision integration of this on-chip sample preparation platform prior to or post-dielectrophoresis, in-line with on-chip monitoring of the outlet sample for metrics of media conductivity, cell velocity, cell viability, cell position, and collected cell numbers, so that the cell flow rate and streamlines can be tailored for enabling dielectrophoretic separations from heterogeneous samples.     

Microfluidic approaches for cancer cell detection, characterization, and separation

This article reviews the recent developments in microfluidic technologies for in vitro cancer diagnosis. We summarize the working principles and experimental results of key microfluidic platforms for cancer cell detection, characterization, and separation based on cell-affinity micro-chromatography, magnetic activated micro-sorting, and cellular biophysics (e.g., cell size and mechanical and electrical properties). We examine the advantages and limitations of each technique and discuss future research opportunities for improving device throughput and purity, and for enabling on-chip analysis of captured cancer cells.     

Crossing microfluidic streamlines to lyse, label and wash cells

We present a versatile method for continuous-flow, on-chip biological processing of cells, large bio-particles, and functional beads. Using an asymmetric post array in pressure-driven microfluidic flow, we can move particles of interest across multiple, independent chemical streams, enabling sequential chemical operations. With this method, we demonstrate on-chip cell treatments such as labeling and washing, and bacterial lysis and chromosomal extraction. The washing capabilities of this method are particularly valuable because they allow many analytical or treatment procedures to be cascaded on a single device while still effectively isolating their reagents from cross-contamination.     

Fiber-free coupling between bulk laser beams and on-chip polymer-based multimode waveguides

In this paper, we demonstrate the design of a virtually alignment-free optical setup for use with microfluidic applications involving a layered glass/SU-8/PDMS (polydimethylsiloxane) chip. We show how inexpensive external lenses combined with carefully designed on-chip lenses can be used to couple light from a bulk beam to on-chip waveguides and back into a bulk beam again. Using this setup, as much as 20% of the light coming from the source can be retrieved after passing through the on-chip waveguides. The proposed setup is based on a pin-aided alignment system that makes it possible to change chips in the optical train in only a few seconds with a standard deviation of about 2% in the transmitted power. Furthermore, we demonstrate how these optical setups can be combined with microfluidics to create an on-chip flow cytometer enabling detection and counting of polystyrene particles down to 1 μm at a rate of 100 Hz.     

An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models

Conventional cell-based assays in life science and medical applications can be difficult to maintain functionally over long periods. Microfluidics is an emerging technology with potential to provide integrated environments for cell maintenance, continuous perfusion, and monitoring. In this study, we developed an integrated microfluidic device with on-chip pumping and detection functionalities. The microfluidic structure in the device is divided into two independent channels separated by a semipermeable membrane on which cells are inoculated and cultured. Perfusion and fluorescence measurements of culture media for each channel can be conducted by the on-chip pumping system and optical fiber detection system. Performance of the device was examined through long-term culture and monitoring of polarized transport activity of intestinal tissue models (Caco-2 cells). The cells could be cultured for more than two weeks, and monolayer transport of rhodamine 123 was successfully monitored by on-line fluorescent measurement. This device may have applications in toxicity testing and drug screening.     

Microfluidic technologies for MALDI-MS in proteomics

The field of microfluidics continues to offer great promise as an enabling technology for advanced analytical tools. For biomolecular analysis, there is often a critical need to couple on-chip microfluidic sample manipulation with back-end MS. Though interfacing microfluidics to MS has been most often reported through the use of direct ESI-MS, there are compelling reasons for coupling microfluidics to MALDI-MS as an alternative to ESI-MS for both online and offline analysis. The intent of this review is to provide a summary of recent developments in the integration of microfluidic systems with MALDI-MS, with an emphasis on applications in proteomics. Key points are summarized, followed by a review of relevant technologies and a discussion of outlook for the field.     

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.     

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman?, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.     

Ultra wide-field lens-free monitoring of cells on-chip

We experimentally and theoretically demonstrate the proof-of-principle of a new lens-free cell monitoring platform that involves using an opto-electronic sensor array to record the shadow image of cells onto the sensor plane. This technology can monitor/count cells over a field-of-view that is more than two orders of magnitude larger than that of a conventional light microscope. Furthermore, it does not require any mechanical scanning or optical elements, such as microscope objectives or lenses. We also show that this optical approach can conveniently be combined with microfluidic channels, enabling parallel on-chip monitoring of various different cell types, e.g., blood cells, NIH-3T3 fibroblasts, murine embryonic stem cells, AML-12 hepatocytes. An important application of this approach could be a miniaturized point-of-care technology to obtain CD4 T lymphocyte counts of HIV infected patients in resource limited settings.     

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.     

微流控芯片分选富集循环肿瘤细胞的研究进展

近年来,循环肿瘤细胞(CTCs)研究得到了越来越多的关注,许多研究报告已经证实其在肿瘤转移的早期诊断、治疗方案选择、个体化治疗及探索肿瘤转移机制等方面具有潜在的价值,然而CTCs在循环系统中的含量极低,这成为限制其临床相关应用的主要难点。微流控芯片技术具有低成本、快速、高通量及操作简单等优势,利用微流控芯片可实现CTCs的高速、高回收率、高纯度的分选富集,近年来得到广泛的关注。本文综述了近年来在微流控芯片内进行CTCs分选富集的研究并探讨了各种方法的优缺点,并在本研究团队的研究基础上进行了展望。     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

A review on continuous-flow microfluidic PCR in droplets: Advances,challenges and future

Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet technology has been exploited to perform PCR in droplets because of its unique features. For example, it can prevent crossover contamination and PCR inhibition, is suitable for single-cell and single-molecule analyses, and has the potential for system integration and automation. This review will therefore focus on recent developments on droplet-based continuous-flow microfluidic PCR, and the major research challenges. This paper will also discuss a new way of on-chip flow control and a rational design simulation tool, which are required to underpin fully integrated and automated droplet-based microfluidic systems. We will conclude with a scientific speculation of future autonomous scientific discoveries enabled by microfluidic microdroplet technologies.     

Membranes and microfluidics: a review

The integration of mass transport control by means of membrane functionality into microfluidic devices has shown substantial growth over the last 10 years. Many different examples of mass transport control have been reported, demonstrating the versatile use of membranes. This review provides an overview of the developments in this area of research. Furthermore, it aims to bridge the fields of microfabrication and membrane science from a membrane point-of-view. First the basic terminology of membrane science will be discussed. Then the integration of membrane characteristics on-chip will be categorized based on the used fabrication method. Subsequently, applications in various fields will be reviewed. Considerations for the use of membranes will be discussed and a checklist with selection criteria will be provided that can serve as a starting point for those researchers interested in applying membrane-technology on-chip. Finally, opportunities for microfluidics based on proven membrane technology will be outlined. A special focus in this review is made on the membrane properties of polydimethylsiloxane (PDMS), since this material is frequently used nowadays in master replication.     

A switchable digital microfluidic droplet dye-laser

Digital microfluidic devices allow the manipulation of droplets between two parallel electrodes. These electrodes can act as mirrors generating a micro-cavity, which can be exploited for a droplet dye-laser. Three representative laser-dyes with emission wavelengths spanning the whole visible spectrum are chosen to show the applicability of this concept. Sub-microlitre droplets of laser-dye solution are moved in and out of a lasing site on-chip to down-convert the UV-excitation light into blue, green and red laser-pulses.     

Controlled viable release of selectively captured label-free cells in microchannels

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.     

Generation of dynamic chemical signals with microfluidic C-DACs

The utilization of microfluidic "lab-on-a-chip" devices in fundamental medical research, drug discovery and clinical diagnostics has rapidly increased in the past decade. Lab-on-a-chip devices process small volumes of analytes and reagents through on-chip microfluidic signal processing circuits. This paper discusses the implementation of a basic microfluidic circuit block, the concentration digital-to-analog converter (or C-DAC) which produces discretized chemical concentrations in a constant stream of solvent. The chemical concentration is controlled by a time-varying digital word; hence C-DACs are suitable for on-chip generation of arbitrary chemical signals. A 4-bit continuous-flow C-DAC was fabricated in two-level PDMS technology and tested. Several chemical waveforms (sawtooth, cosine, and ramp) were generated at flow rates of 2 microL min(-1) and frequencies of 0.6-4 mHz. The frequency cut off of this C-DAC was approximately 500 mHz.     

Microfluidic DNA amplification—A review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted.