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.     

Microfluidic device for analyzing preferential chemotaxis and chemoreceptor sensitivity of bacterial cells toward carbon sources

We present a novel microfluidic device that enables high sensitive analyses of the chemotactic response of motile bacterial cells (Escherichia coli) that swim toward a preferred nutrient by sorting and concentrating them. The device consists of the Y-shaped microchannel that has been widely used in chemotaxis studies to attract cells toward a high concentration and a concentrator array integrated with arrowhead-shaped ratchet structures beside the main microchannel to trap and accumulate them. Since the number of accumulated cells in the concentrator array continuously increases with time, the device makes it possible to increase the sensitivity of detecting chemotactic responses of the cells about 10 times greater than Y-shaped channel devices in 60 min. In addition, the device can characterize the relative chemotactic sensitivity of chemoreceptors to chemoeffectors by comparing the number of cells in the concentrator array at different distances from the channel junction. Since the device allows the analysis of both the chemotactic responses and the sensitivity of chemoreceptors with high resolution, we believe that not only can the device be broadly used for various microbial chemotaxis assays but it also can further the advancement of microbiology and even synthetic biology.     

Controlled generation of monodisperse discoid droplets using microchannel arrays

We report a novel technique for generating geometrically confined droplets using a unique microstructure composed of a microchannel (MC) array and a shallow well. Silicon MC array devices were successfully used to generate monodisperse discoid droplets of oil-in-water (O/W) and W/O types by forcing a to-be-dispersed phase through channels into a well filled with a continuous phase. Monodisperse discoid droplets with sizes down to several micrometers were obtained by controlling the channel and well dimensions. The resultant discoid droplets formed a mostly close-packed array in the well. Monodisperse discoid droplets consisting of a silicone oil/water/sodium dodecyl sulfate system did not coalesce during the storage time of seven days. Additionally, MC array plates with many channels can be useful for increasing the droplet productivity of a single microfluidic device.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法

基于微流体脉冲驱动控制技术搭建了电化学微流控芯片的制备系统.首先将纳米银墨水和甘油溶液分别微喷射到玻璃基底表面形成微电极图形和微流道液体阳模图形;然后分别进行烧结和聚二甲基硅氧烷(PDMS)模塑工艺制得微电极和微流道;最后将微电极和微流道键合形成电化学微流控芯片.研究了系统参量对液滴产生的影响以及液滴直径和重叠率对液滴成线的影响,制得的微电极最小线宽为45 μm、厚度为2.2 μm、电阻率为5.2 μΩ·cm,制得的微流道最小线宽为35 μm,流道表面光滑.采用制得的电化学微流控芯片进行了葡萄糖浓度的电化学流动检测.结果表明,葡萄糖溶液的浓度与响应电流具有较高的线性关系,可对一定浓度范围内的葡萄糖溶液进行定量检测.基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法具有微喷射精度高、重复性好,制备系统结构简单、成本低廉等优点,可用于生化分析、生物传感器等领域的芯片制备.     

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.     

Mass transport in nanofluidic devices

Nanofluidics is a recent appearing research field, introduced in 1995 as an analogue of the field of microfluidics, and has been becoming popular in the past few years. The proximity of the channel dimension, the Debye length, and the size of biomolecules such as DNA and proteins gives the unique features of nanofluidic devices. Of various unique properties of the nanofluidics, mass transport in nanochannel plays determining roles in fundamental reaches and practical applications of nanofluidic device. Thus, much work including numerical and experimental researches has been performed to investigate the mass transport behaviors in nanofluidic devices. This review summarizes the fabrication technologies for nanofluidic devices, the mass transport behaviors in nanochannel, and their applications in bioanalysis. The main focus will be laid on the effects of nanochannel size and surface charge on mass transport including electrokinetic transport of charged analytes, diffusion of electric neutral molecules, ionic current rectification, concentration polarization, nonlinear electrokinetic flow at the micro-nanofluidic interfaces.     

Micromachined filter-chamber array with passive valves for biochemical assays on beads

The filter-chamber array presented here enables a real-time parallel analysis of three different samples on beads in a volume of 3 nL, on a 1 cm2 chip. The filter-chamber array is a system containing three filter-chambers, three passive valves at the inlet channels and a common outlet. The design enables parallel sample handling and time-controlled analysis. The device is microfabricated in silicon and sealed with a Pyrex lid to enable real-time analysis. Single nucleotide polymorphism analysis by using pyrosequencing has successfully been performed in single filter-chamber devices. The passive valves consist of plasma-deposited octafluorocyclobutane and show a much higher resistance towards water and surface-active solutions than previous hydrophobic patches. The device is not sensitive to gas bubbles, clogging is rare and reversible, and the filter-chamber array is reusable. More complex (bio)chemical reactions on beads can be performed in the devices with passive valves than in the devices without valves.     

A split microchannel design and analytical model to compensate for electroosmotic instabilities in micro-separations

Organic polymers offer many advantages as materials for the construction of microfluidic devices but suffer frequently from the limitation that the electrodynamic flow they support can exhibit considerable instability. This article describes a split-channel microfluidic device that can be used to compensate for changes in electroosmotic flow. The design of the separation system divides an analyte plug after injection between two separation channels of differing length. The two channels are later recombined for single point detection, eliminating the need for a scanning optical detection system. The utility of this simple design lies in the fact that the migration time of any analyte can be referenced to its twin in the parallel separation channel. This eliminates the need for a separate electroosmotic marker and allows mobilities measured in multiple devices to be compared quantitatively. Using a model adopted from the literature, the data from the split channel system can be used to precisely account for the drift that characterizes electrophoretic separations made in a polymer chip. The relative standard deviations of the analyte mobilities measured for replicate runs on multiple devices were reduced from values as high as 20% to ca. 1% RSD. This internal standardization procedure also appears to address other sources of drift in the electroosmotic flow (EOF) supported by the polymer microchannel, eliminating the need for careful monitoring of either the temperature or reservoir pH between separation runs.     

Gravity-induced convective flow in microfluidic systems: electrochemical characterization and application to enzyme-linked immunosorbent assay tests

A way of using gravity flow to induce a linear convection within a microfluidic system is presented. It is shown and mathematically supported that tilting a 1 cm long covered microchannel is enough to generate flow rates up to 1000 nL.min(-1), which represents a linear velocity of 2.4 mm.s(-1). This paper also presents a method to monitor the microfluidic events occurring in a covered microchannel when a difference of pressure is applied to force a solution to flow in said covered microchannel, thanks to electrodes inserted in the microfluidic device. Gravity-induced flow monitored electrochemically is applied to the performance of a parallel-microchannel enzyme-linked immunosorbent assay (ELISA) of the thyroid-stimulating hormone (TSH) with electrochemical detection. A simple method for generating and monitoring fluid flows is described, which can, for instance, be used for controlling parallel assays in microsystems.     

微流路中利用DNA选择性固定蛋白质

报道了一种可控的通过DNA复合物在微流路中杂交固定蛋白质的方法. 微流路系统中的玻璃基底上固定寡聚核苷酸, 其中的层流提供了不同的DNA-蛋白质复合物. DNA的特异性识别可以将蛋白通过表面寻址固定在基底上. 并且在体系中引入了全内反射荧光技术来追踪整个过程. 此方法的特异性和灵敏度均较高, 且蛋白质的固定和去除可重复. 实验结果显示, 同时检测特异性和非特异性的识别, 可以有效提高生物检测的准确性. 这项技术可以提高具有微流路结构的生物传感器装置的检测质量.     

Selective Protein Immobilization via DNA Conjugation under Microfluidic Laminar Flow

The feasibility of controlled protein immobilization via DNA conjugation by utilizing laminar flow in a microfluidic device was demonstrated. The glass surface in a microchannel was treated by oligonucleotides. The laminar flow brought different protein-DNA conjugates parallel into the microchannel. DNA recognition allows proteins to be delivered to the desired location. The total internal reflection fluorescence was also applied to monitor the process. Both the specificity and sensitivity were high, and the immobilization and removal of the proteins were repeatable. It was shown that with parallel detection of specific and non-specific recognitions, the accuracy of bio-assay would be effectively enhanced. This strategy could improve the performing quality of biosensors in microfluidic devices.     

Micro-/nanofluidic device for tunable generation of a concentration gradient: application to Caenorhabditis elegans chemotaxis

In this study, we propose a novel micro-/nanofluidic device that can generate a chemical concentration gradient using a parallel nanochannel as gradient generator. This device is easy to fabricate, showing high reproducibility. Its main feature is the multiple-nanochannel-based gradient generator, which permits the diffusion of small molecules and tunably generates concentration gradients. The nanopattern for the nanochannels can be rapidly and easily fabricated by wrinkling a diamond-like carbon thin film which is deposited on a polydimethylsiloxane substrate; the generation of the concentration gradient can be adjusted by controlling the dimensions of the nanochannels. The developed gradient generator is embedded into a microfluidic device to study chemotaxis in the nematode Caenorhabditis elegans, which has a highly developed chemosensory system and can detect a wide variety of chemical molecules. This device shows good performance for rapid analysis of C. elegans chemotaxis under sodium chloride stimuli.

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Continuous and precise particle separation by electroosmotic flow control in microfluidic devices

A new scheme has been described for continuous particle separation using EOF in microfluidic devices. We have previously reported a method for particle separation, called "pinched flow fractionation (PFF)", in which size-dependent and continuous particle separation can be achieved by introducing pressure-driven flows with and without particles into a pinched microchannel. In this study, EOF was employed to transport fluid flows inside a microchannel. By controlling the applied voltage to electrodes inserted in each inlet/outlet port, the flow rates from both inlets, and flow rates distributed to each outlet could be accurately tuned, thus enabling more effective separation compared to the pressure-driven scheme. In the experiment, the particle behaviors were compared between EOF and pressure-driven flow schemes. In addition, micrometer- and submicrometer-sized particles were accurately separated and individually collected using a microchannel with multiple outlet branch channels, demonstrating the high efficiency of the presented scheme.     

Concentration landscape generators for shear free dynamic chemical stimulation

In this paper we first introduce a novel fabrication process, which allows for easy integration of thin track-etched nanoporous membranes, within 2D or 3D microchannel networks. In these networks, soluble chemical compounds can diffuse out of the channels through well-defined and spatially organized microfabricated porous openings. Interestingly, multiple micron-scale porous areas can be integrated in the same device and each of these areas can be connected to a different microfluidic channel and reservoir. We then present and characterize several membrane-based microdevices and their use for the generation of stable diffusible concentration gradients and complex dynamic chemical landscapes under shear free conditions. We also demonstrate how a simple flow-focusing geometry can be used to generate "on-demand" concentration profiles. In turn, these devices should provide an ideal experimental framework for high throughput cell-based assays: long term high-resolution video microscopy experiments can be performed, under multiple spatially and temporally controlled chemical conditions, with simple protocols and in a cell-friendly environment.     

Continuous particle separation in a microchannel having asymmetrically arranged multiple branches

A new method for continuous size separation and collection of particles in microfabricated devices, asymmetric pinched flow fractionation (AsPFF), has been proposed and demonstrated. This method improves the separation scheme of pinched flow fractionation (PFF), which utilizes a laminar flow profile inside a microchannel. In this study, multiple branch channels with different channel dimensions were arranged at the end of the pinched segment, so that the flow rate distributions to each branch channel were varied, and a large part of the liquid was forced to go through one branch channel (drain channel). In the proposed channel system, the flow profile inside the microchannel was asymmetrically amplified, enabling the separation of one-order smaller particles compared with PFF. After introducing the method, we examined the effect of the asymmetric amplification by controlling the outlet of the drain channel. Also, a mixture of 1.0 approximately 5.0 microm particles was separated, and erythrocytes were successfully separated from blood. The results indicate that the AsPFF method could be applied to the separation of much smaller-size particles, since more precise separation can be achieved simply by changing the geometries of branch channels.     

Influence of channel position on sample confinement in two-dimensional planar microfluidic devices

We report enhanced sample confinement on microfluidic devices using a combination of electrokinetic flow from adjacent control channels and electric field shaping with an array of channels perpendicular to the sample stream. The basic device design consisted of a single first dimension (1D) channel, intersecting an array of 32 or 96 parallel second dimension (2D) channels. To minimize sample dispersion and leakage into the parallel channels as the sample traversed the sample transfer region, control channels were placed to the left and right of the 1D and waste channels. The electrokinetic flow from the control channels confined the sample stream and acted as a buffer between the sample stream and the 2D channels. To further enhance sample confinement, the electric field was shaped parallel to the sample stream by placing the channel array in close proximity to the sample transfer region. Using COMSOL Multiphysics, initial work focused on simulating the electric fields and fluid flows in various device geometries, and the results guided device design. Following the design phase, we fabricated devices with 40, 80, and 120 microm wide control channels and evaluated the sample stream width as a function of the electric field strength ratio in the control and 1D channels (E(C)/E(1D)). For the 32 channel design, the 40 and 80 microm wide control channels produced the most effective sample confinement with stream widths as narrow as 75 microm, and for the 96 channel design, all three control channel widths generated comparable sample stream widths. Comparison of the 32 and 96 channel designs showed sample confinement scaled easily with the length of the sample transfer region.     

Size-selective concentration and label-free characterization of protein aggregates using a Raman active nanofluidic device

Trace detection and physicochemical characterization of protein aggregates have a large impact in understanding and diagnosing many diseases, such as ageing-related neurodegeneration and systemic amyloidosis, for which the formation of protein aggregates is one of the pathological hallmarks. Here we demonstrate an innovative label-free method for detecting and characterizing small amounts of early stage protein aggregates using a Raman active nanofluidic device. Sub-micrometre channels formed by a novel elastomeric collapse technique enable the separation and concentration of matured protein aggregates from small protein molecules. The Raman enhancement by gold nanoparticle clusters fixed below a micro/nanofluidic junction allows characterization of intrinsic properties of protein aggregates at concentration levels (～fM) much lower than can be done with traditional analytical tools. With our device we show for the first time the concentration dependence of protein aggregation over these low concentration ranges. We expect that our method could facilitate definitive diagnosis and possible therapeutics of diseases at early stages.     

Optimization of sample transfer in two-dimensional microfluidic separation systems

Multidimensional microfluidic separation systems combining a first dimension microchannel with an array of parallel second dimension microchannels can suffer from non-uniform sample transfer between the dimensions, sample leakage, and injection plug tailing within the second dimension array. These factors can significantly reduce overall two-dimensional separation performance. In this paper, numerical and analytical models reveal an optimized chip design which combines multidimensional backbiasing and an angled channel geometry to ensure leakage-free and uniform interdimensional sample transfer, while also minimizing injected sample plug lengths. The optimized design is validated experimentally using a multidimensional chip containing five second dimension channels.