Processing of nanolitre liquid plugs for microfluidic cell-based assays

Abstract

Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.     

Microprocessing of liquid plugs for bio/chemical analyses

A microfluidic device and operation to handle liquid plugs for biochemical analyses were developed for efficient handling of plugs of many solutions. A major part of the device was a T-junction consisting of a main flow channel and a handling flow channel. Unit operations including attachment of plugs, division of a plug, sorting of plugs, and formation of plugs of various lengths enabled controlled sequential reactions in a microflow channel. Rapid mixing could easily be achieved by moving a plug formed by merging two plugs back and forth. The device could be used for efficient characterization of performance in bio/chemical sensing. In experiments using L-glutamate oxidase, plugs containing an enzyme or a substrate were formed, mixed sequentially, and the intensity of fluorescence from plugs of different concentrations of L-glutamate or pHs could be measured simultaneously. Cross-contamination of plugs by neighboring plugs poses a problem in using the same flow channel repeatedly. However, the influence could be minimized by using a cleansing plug placed between them in a sufficiently hydrophobic flow channel and by processing the plugs at a low velocity. The device can be a critical component for microprocessing in various bio/chemical analyses.     

Sampling and electrophoretic analysis of segmented flow streams using virtual walls in a microfluidic device

A method for sampling and electrophoretic analysis of aqueous plugs segmented in a stream of immiscible oil is described. In the method, an aqueous buffer and oil stream flow parallel to each other to form a stable virtual wall in a microfabricated K-shaped fluidic element. As aqueous sample plugs in the oil stream make contact with the virtual wall, coalescence occurs and sample is electrokinetically transferred to the aqueous stream. Using this virtual wall, two methods of injection for channel electrophoresis were developed. In the first, discrete sample zones flow past the inlet of an electrophoresis channel and a portion is injected by electroosmotic flow, termed the "discrete injector". With this approach at least 800 plugs could be injected without interruption from a continuous segmented stream with 5.1% RSD in peak area. This method generated up to 1,050 theoretical plates, although analysis of the injector suggested that improvements may be possible. In a second method, aqueous plugs are sampled in a way that allows them to form a continuous stream that is directed to a microfluidic cross-style injector, termed the "desegmenting injector". This method does not analyze each individual plug but instead allows periodic sampling of a high-frequency stream of plugs. Using this system at least 1000 injections could be performed sequentially with 5.8% RSD in peak area and 53,500 theoretical plates. This method was demonstrated to be useful for monitoring concentration changes from a sampling device with 10 s temporal resolution. Aqueous plugs in segmented flows have been applied to many different chemical manipulations including synthesis, assays, sampling processing and sampling. Nearly all such studies have used optical methods to analyze plug contents. This method offers a new way to analyze such samples and should enable new applications of segmented flow systems.     

Characterisation of porous ceramic plugs for use in electrochemical sensors

In electrochemical sensors like pH-, reference- or ion-selective electrodes a porous ceramic plug (or diaphragm) maintains the conducting junction with the test solution. These liquid junctions should have a resistance as low as possible meanwhile avoiding leakage through the junction. Five porous magnesium stabilised zirconium oxide plugs with different porosity's and pore size (distribution) were investigated as liquid junctions. The physical properties of these porous plugs were investigated with SEM and Mercury Intrusion Porosimetry. Important working conditions of these porous plugs are the resistance of the porous plug filled with an electrolyte and the contamination speed through these porous plugs, both for the test solution as the reference solution. The first property was measured by a 4-wire resistance measurement. The second property was measured by measuring the flow through rate of the reference electrolyte through the plug. It was shown that an optimal plug i.e., low leakage and high conductivity through the membrane, had a high porosity and relative small pores (0.25 m). A simple mathematical model based on the Hagen-Poiseuille equation was developed to describe the porous plug characteristics. It was shown that mathematical calculation of the porous plug resistance was in good agreement with experimental results.     

High-Frequency Method of Removal of Paraffin Plugs in the Equipment of Oil Wells and Oil Pipelines

The processes of heating and melting of paraffin plugs in the shafts of oil wells and in oil pipelines by high-frequency electromagnetic radiation have been investigated. The evolution of the temperature fields in the volume of the plugs has been investigated for various physical conditions and various geometries of the plugs. The times of melting of a through channel in a plug and the times of its complete liquidation have been determined. Unlike the works carried out earlier, elimination of paraffin plugs was investigated with regard for the nonuniformity of the distribution of high-frequency power over the cross section of a plug and the absorption of a high-frequency wave in the metallic walls of a well or a pipeline.     

Integrated microdevice for long-term automated perfusion culture without shear stress and real-time electrochemical monitoring of cells

Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers' release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "circular-centre" and "bottom-up" perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.     

Inline injection microdevice for attomole-scale sanger DNA sequencing

A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA sequencing separations. The microdevice comprises three axial domains for nanoliter-scale sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel. Extension fragments selectively hybridize and concentrate at the polymer interface while residual primer, nucleotides, and salts electrophorese out of the system. The plug is thermally released and injected into the CE channel by direct application of the separation voltage. To evaluate this system, 30 nL of sequencing sample prepared from 100 amol (60 million molecules) of human mitochondrial hypervariable region II amplicon was introduced into the microchip, purified, concentrated, and injected, generating a read length of 365 bases with 99% accuracy. This efficient inline injection system obviates the need for the excess sample that is required by cross-injection techniques, thereby enabling Sanger sequencing and other high-performance genetic analysis using DNA quantities approaching theoretical detection limits.     

Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system

Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor kappaB (NFkappaB) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NFkappaB activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrode-integrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method.     

Solid-support sample loading for DNA sequencing

We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of <100 pg total suspended weight match the signal strength of our previous conventional injections at >10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of approximately 1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.     

Superfluid plug as a control device for helium coolant

New results have been obtained on the characteristics of the superfluid plug as a nonmechanical control device for supplying cold helium vapour on demand from a container of superfluid helium. The superfluid plug is a device which has been proposed for space applications to serve as a phase separator for liquid helium in the superfluid state. Typical plugs are made of a porous material having pores of one to ten μm in diameter. The experimental arrangement is such that one side of the plug is in contact with the superfluid liquid helium while vapour at a low pressure (of order 1 to 10 torr) is maintained on the other side.The data reported here are for a plug with approximately 5 μm diameter pores. Temperatures, pressures, and flow rate were monitored during the experiment. A theoretical background and steady state data are presented on mass flow rates and pressures as a function of liquid temperature.The typical response of the flow rate to a change in heat input from a heater is an exponential rise or fall to an equilibrium flow rate which is proportional to the amount of heat input. The time constants of the exponential changes were measured for two heater control modes under study. The study has included an investigation of the important parameters effecting the dynamic response of the plug including the superfluid properties, plug material properties, plug pore size and plug permeability. Operating temperatures from 1.5 K to the lambda point were investigated and heating rates up to two watts were applied. These tests serve to demonstrate that the superfluid plug can be employed as a flow control device in a control system designed to provide coolant on demand.     

Uniform threshold intensity distribution-based quantitative multivariate imaging cytometry

Implementation of quantitative analytical approaches to image-based cellular assays remains a major challenge. We disclose a tool to achieve automatic rapid quantitative cellular imaging analysis based on uniform threshold intensity distribution. An acousto-optic tunable filter-based, quantitative multivariate imaging cytometer was set up to elucidate drug-induced cell death dynamics via cell viability and apoptosis/necrosis measurements in the human myeloid leukemia cell line, HL-60. Cells were treated with various drugs (camptothecin, naringenin, sodium salicylate) at different concentrations and time intervals. The developed protocol can directly depict and quantitate targeted cellular moieties, subsequently enabling a method that is applicable to various cellular assays with special reference to next generation drug discovery screening. This may also complement certain flow-cytometric measurements in studying quantitative physiology of cellular systems.     

Simulation of Gas-Solid Flow in Vertical Pipe by Hard-Sphere Model

This article presents a two-dimensional study of the gas-solid flow in a vertical pneumatic conveying pipe by means of a hard-sphere model where the motion of individual particles can be traced. Simulations were performed for a pipe of height 0.9 m and width 0.06 m, with air as gas phase and particles of density 900 kg/m³ and diameter 0.003 m as solid phase. Periodic boundary conditions were applied to the solid phase in the axial direction. Different cases were simulated to examine the effects of the number of particles used, superficial gas velocity, and restitution coefficient. The results show that the main features of plug flow can be reasonably captured by the proposed simulation technique. That is, increasing the number of particles in a simulation will increase the length of plugs but does not change the velocity of plugs; the solid fraction of a plug is relatively low if the number of particles is small. In particular, it is shown that increasing superficial gas velocity will increase the velocity of plugs and the frequency of plugs, and the pressure drop through a rising plug increases linearly with the plug length, suggesting that the total pressure of a conveying system with a given length can be quantified from the information of plug length and plug frequency. Increasing the restitution coefficient can promote the momentum transfer between particles and hence the raining down of particles from the back of a plug in vertical pneumatic conveying. The simulation offers a useful technique to understand the fundamentals governing the gas-solid flow under pneumatic conveying conditions.     

Electrokinetic focusing injection methods on microfluidic devices

This paper presents an experimental and numerical investigation into electrokinetic focusing injection on microfluidic chips. The valving characteristics on microfluidic devices are controlled through appropriate manipulations of the electric potential strengths during the sample loading and dispensing steps. The present study also addresses the design and testing of various injection systems used to deliver a sample plug. A novel double-cross injection microfluidic chip is fabricated, which employs electrokinetic focusing to deliver sample plugs of variable volume. The proposed design combines several functions of traditional sample plug injection systems on a single microfluidic chip. The injection technique uses an unique sequence of loading steps with different electric potential distributions and magnitudes within the various channels to effectuate a virtual valve.     

Simulation of Gas-Solid Flow in Vertical Pipe by Hard-Sphere Model

ABSTRACT

This article presents a two-dimensional study of the gas-solid flow in a vertical pneumatic conveying pipe by means of a hard-sphere model where the motion of individual particles can be traced. Simulations were performed for a pipe of height 0.9 m and width 0.06 m, with air as gas phase and particles of density 900 kg/m³ and diameter 0.003 m as solid phase. Periodic boundary conditions were applied to the solid phase in the axial direction. Different cases were simulated to examine the effects of the number of particles used, superficial gas velocity, and restitution coefficient. The results show that the main features of plug flow can be reasonably captured by the proposed simulation technique. That is, increasing the number of particles in a simulation will increase the length of plugs but does not change the velocity of plugs; the solid fraction of a plug is relatively low if the number of particles is small. In particular, it is shown that increasing superficial gas velocity will increase the velocity of plugs and the frequency of plugs, and the pressure drop through a rising plug increases linearly with the plug length, suggesting that the total pressure of a conveying system with a given length can be quantified from the information of plug length and plug frequency. Increasing the restitution coefficient can promote the momentum transfer between particles and hence the raining down of particles from the back of a plug in vertical pneumatic conveying. The simulation offers a useful technique to understand the fundamentals governing the gas-solid flow under pneumatic conveying conditions.     

Reproducibility and robustness of a real-time microfluidic cell toxicity assay

Numerous opportunities exist to apply microfluidic technology to high-throughput and high-content cell-based assays. However, maximizing the value of microfluidic assays for applications such as drug discovery, screening, or toxicity evaluation will require assurance of within-device repeatability, day-to-day reproducibility, and robustness to variations in conditions that might occur from laboratory to laboratory. This report describes a study of the performance and variability of a cell-based toxicity assay in microfluidic devices made of poly(dimethylsiloxane) (PDMS). The assay involves expression of destabilized green fluorescent protein (GFP) as a reporter of intracellular protein synthesis and degradation. Reduction in cellular GFP due to inhibition of ribosome activity by cycloheximide (CHX) was quantified with real-time quantitative fluorescence imaging. Assay repeatability was measured within a 64-chamber microfluidic device. Assay performance across a range of cell loading densities within a single device was assessed, as was replication of measurements in microfluidic devices prepared on different days. Assay robustness was tested using different fluorescence illumination sources and reservoir-to-device tubing choices. Both microfluidic and larger scale assay conditions showed comparable GFP decay rates upon CHX exposure, but the microfluidic data provided the higher level of confidence.     

游动芯头拉拔模具失效分析

为了改善游动芯头拉拔过程中芯头模具的磨损,揭示芯头在拉拔过程中不同形式的失效机理.利用扫描电镜,金相显微镜对不同失效形式下模具进行观察及成分分析,同时对拉拔润滑油和铜管进行密度和黏度测量.结果表明:芯头模具表面局部Co元素的缺失或其黏合作用相对较小,导致WC颗粒在模具表面形成竖道划痕;芯头模具由于受到管材变形时压应力,在温升和润滑不良的情况下形成环形沟槽缺陷;管坯中的气孔在变形过程中贯穿连接,以及模具表面粗糙度的提升,导致管材上的铜粘附在模具表面.     

Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays

Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies.     

Optical forces for noninvasive cellular analysis

A novel, noninvasive measurement technique for quantitative cellular analysis is presented that utilizes the forces generated by an optical beam to evaluate the physical properties of live cells in suspension. In this analysis, a focused, near-infrared laser line with a high cross-sectional intensity gradient is rapidly scanned across a field of cells, and the interaction of those cells with the beam is monitored. The response of each cell to the laser depends on its size, structure, morphology, composition, and surface membrane properties; therefore, with this technique, cell populations of different type, treatment, or biological state can be compared. To demonstrate the utility of this cell analysis platform, we evaluated the early stages of apoptosis induced in the U937 cancer cell line by the drug camptothecin and compared the results with established reference assays. Measurements on our platform show detection of cellular changes earlier than either of the fluorescence-based Annexin V or caspase assays. Because no labeling or additional cell processing is required and because accurate assays can be performed with a small number of cells, this measurement technique may find suitable applications in cell research, medical diagnostics, and drug discovery.     

Axial and Radial Pressure Drops of Dense Phase Plugs

An experimental technique to measure various characteristics of plug flow in dense phase pneumatic conveying systems based on the unique characteristics of plug flow, i.e., the fluctuation of axial pressure drop along a pipeline and pressure difference in the radial direction at the back of a plug, was developed by Li et al. (2002). Based on this work, a further experimental study combined with numerical modeling was carried out to describe the structure of plugs through the analysis of the measurements of pressure difference in both axial and radial directions. A theoretical explanation of these pressure differences was proposed and agrees very well with the recorded signals of pressure difference from differential transducers. This explanation will prove useful in understanding plug structures in industrial applications.     

贵州塘寨电厂取水口岩塞爆破

贵州塘寨电厂采用竖井加平洞方案从索风营水库取水,平洞施工采用岩塞挡水,洞内施工完成后,爆除岩塞实现取水.根据水库水位及平洞布置情况,采用上倾角圆形岩塞(30°倾角)冲碴方案.进行了岩塞爆破设计,采用排孔爆破的布孔方式、周边采用预裂爆破、选用防水乳化炸药和国产高精度电子雷管的毫秒延时起爆网路进行了岩塞爆破,取得了理想的爆...