Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.     

Chemotaxis assays of mouse sperm on microfluidic devices

Sperm chemotaxis is an area of significant interest to scientists involved in reproductive science. Understanding how and when sperm cells are attracted to the egg could have profound effects on reproduction and contraception. In an effort to systematically study this problem, we have fabricated and evaluated a microfluidic device to measure sperm chemotaxis. The device was designed with a flow-through configuration using a spatially and temporally stable chemical gradient. Mouse sperm cells were introduced into the chemotaxis chamber between confluent flows of mouse ovary extract and buffer. The sperm experiencing chemotaxis swam toward the extract and were counted relative to those that swam toward the buffer. The ovary extracts were diluted from 10(2) to 10(7) times, and each extract dilution was screened for chemotaxis. Four out of six ovaries showed a strong chemotactic response at extract dilutions of 10(-3) to 10(-5). This device provided a convenient, disposable platform on which to conduct chemotaxis assays, and the flow-through design overcomes difficulties associated with distinguishing chemotaxis from trapping.     

Static and dynamic acute cytotoxicity assays on microfluidic devices

Static and dynamic acute toxicity assays of cells were performed on microfluidic devices where materials were hydraulically transported. Static assays were performed by incubating cells with an agent in a microchip reservoir and optically interrogating the cells after hydrodynamic focusing at a cross intersection. Dynamic assays were performed on a microchip with a 25-cm-long spiral channel where the cells were mixed with an agent and optically monitored 0.1, 12, and 22 cm from the point of mixing. The incubation time was determined by the time needed for cells to transit from the mixing location to the point of detection. Cell viability was determined using the ratio of fluorescence signals from membrane permeant (calcein) and membrane impermeant (propidium iodide) stains. The model system used in this study was the viability of Jurkat cells in the presence of the agent Triton X-100). An average LC50 value of 138 microM for Triton X-100 was obtained for an incubation period of 7-12 min using the static assay. LC50 values obtained with the dynamic assay for 25- and 47-s incubation times were 290 and 250 microM Triton X-100, respectively. Higher LC50 values for the dynamic assay were expected due to the shorter incubation times.     

Voltammetric monitoring of transient hydrodynamic flow profiles in microfluidic flow cells

We consider the transition to steady-state flow in the inlet region of a hydrodynamic channel cell and show that a microelectrode positioned within this inlet region allows chronoamperometric results to be recorded, from which information about the extent of the development of the flow profile may be deduced as well as information about the precise dimensions of the microfluidic channel.     

Influence of maintenance policies on multi-stage manufacturing systems in dynamic conditions

Manufacturing systems are subject to a degradation process that leads to machine failure if no action is taken. Machine failures reduce the performance of the manufacturing system with loss of profits. The research proposed here concerns the evaluation of the manufacturing system performance in dynamic conditions when different maintenance policies are implemented in a multi-machine manufacturing system controlled by multi-agent-architecture. There are two extreme maintenance policies that can be applied: no preventive maintenance, where action is taken on the failure state, and intensive preventive maintenance, which can eliminate unforeseen failures, but at a high cost. Dynamic policy maintenance is proposed to reduce the number of maintenance operations of the preventive policy. A discrete simulation environment has been developed in order to investigate the performance measures and the indexes of the costs of maintenance policies. The simulations have been conducted for several levels of mix, product demand and working time uncertainty. The simulation results show that the proposed approach leads to better performance for the manufacturing system and reduces the number of maintenance operations (cost index of the maintenance policy), except in the case of the mean time between failure, which is characterised by a very low standard deviation.     

Processing of nanolitre liquid plugs for microfluidic cell-based assays

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.     

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.     

Highly-efficient single-cell capture in microfluidic array chips using differential hydrodynamic guiding structures

This paper presents a highly efficient single cell capture scheme using hydrodynamic guiding structures in a microwell array. The implemented structure has a capturing efficiency of >80%, and has a capacity to place individual cells into separated microwells, allowing for the time-lapse monitoring on single cell behavior. Feasibility was tested by injecting microbeads (15 μm in diameter) and prostate cancer PC3 cells in an 8×8 microwell array chip and >80% of the microwells were occupied by single ones. Using the chips, the number of cells required for cell assays can be dramatically reduced and this will facilitate overcoming a huddle of assays with scarce supply of cells.     

Integrated membrane filters for minimizing hydrodynamic flow and filtering in microfluidic devices

Microfluidic devices have gained significant scientific interest due to the potential to develop portable, inexpensive analytical tools capable of quick analyses with low sample consumption. These qualities make microfluidic devices attractive for point-of-use measurements where traditional techniques have limited functionality. Many samples of interest in biological and environmental analysis, however, contain insoluble particles that can block microchannels, and manual filtration prior to analysis is not desirable for point-of-use applications. Similarly, some situations involve limited control of the sample volume, potentially causing unwanted hydrodynamic flow due to differential fluid heads. Here, we present the successful inclusion of track-etched polycarbonate membrane filters into the reservoirs of poly(dimethylsiloxane) capillary electrophoresis microchips. The membranes were shown to filter insoluble particles with selectivity based on the membrane pore diameter. Electrophoretic separations with membrane-containing microchips were performed on cations, anions, and amino acids and monitored using conductivity and fluorescence detection. The dependence of peak areas on head pressure in gated injection was shown to be reduced by up to 92%. Results indicate that separation performance is not hindered by the addition of membranes. Incorporating membranes into the reservoirs of microfluidic devices will allow for improved analysis of complex solutions and samples with poorly controlled volume.     

Development of quantitative cell-based enzyme assays in microdroplets

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.     

Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device

We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards). We investigated scaling properties and sensitivity for readout of 12plex PCR reactions. The sensitive detection, stemming from confocal optics, allowed reduction of the PCR cycle number by approximately five cycles compared to commercial fluorometric readout. This increased sensitivity appears to allow quantitative PCR over a dynamic range of >9 log2 abundance ratio in multiplex reactions exceeding 20plexes. We argue that the combination of mapping, multiplexing, and an internal standard, improves the per-well efficiency of quantitative expression analysis by a factor of 50-100 relative to fluorometric qPCR readout. Therefore, the approach is attractive for analysis of large gene networks at reduced cost.     

Influence of the operating conditions on highly oxidative radicals generation in Fenton's systems

In this work, an indirect method for estimating the total amount and concentration of oxidative radicals in aqueous and slurry-phase Fenton's systems was developed. This method, based on the use of benzoic acid as probe compound, was applied for evaluating the effect of the operating conditions on the radicals amount produced, their production efficiency (i.e. moles of radicals generated per mole H2O2 and their concentration. A Rotatable Central Composite design (RCC) was used to select the operating conditions in order to get a statistically meaningful data set. Hydrogen peroxide and ferrous ion concentrations ranged between 0.2-1mM and 0.2-0.5mM, respectively; humic acid concentration between 0 and 15mg/L, whereas the soil/water weight ratio in slurry-phase systems between 1:10 and 9:10. The probe compound concentration was 9 or 0.1mM in experiments aimed to evaluate the total amount or concentration of oxidative radicals, respectively. The obtained results indicated that the amount of radicals generated in both aqueous and soil slurry Fenton's system increased with higher H2O2 concentration and, more specifically, that their production efficiency increased with increasing Fe(II):H2O2 molar ratio. Addition of dissolved organic compounds as humic acid did not notably affect the oxidative radicals amount and concentration. On the contrary, a one order of magnitude reduction in both radicals amount generated and concentration was observed when soil was added to the reaction environment.     

Achieving uniform mixing in a microfluidic device: hydrodynamic focusing prior to mixing

We describe a microfluidic mixer that is well-suited for kinetic studies of macromolecular conformational change under a broad range of experimental conditions. The mixer exploits hydrodynamic focusing to create a thin jet containing the macromolecules of interest. Kinetic reactions are triggered by molecular diffusion into the jet from adjacent flow layers. The ultimate time resolution of these devices can be restricted by premature contact between co-flowing solutions during the focusing process. Here, we describe the design and characterization of a mixer in which hydrodynamic focusing is decoupled from the diffusion of reactants, so that the focusing region is free from undesirable contact between the reactants. Uniform mixing on the microsecond time scale is demonstrated using a device fabricated by imprinting optical-grade plastic. Device characterization is carried out using fluorescence correlation spectroscopy (FCS) and two-photon microscopy to measure flow speeds and to quantify diffusive mixing by monitoring the collisional fluorescence quenching, respectively. Criteria for achieving microsecond time resolution are described and modeled.     

Numerical investigations of strong hydrodynamic interaction between neighboring particles inertially driven in microfluidic flows

Dispersed particles traveling at a high throughput in microchannels laterally migrate and focus into a streamline at each channel face. The focusing attractors within the cross-section are determined by the balance between the lift forces. However, particles in close proximity (e.g. due to high concentration and abrupt particle contact) suffer a breakdown of distinct focusing due to excessive hydrodynamic interaction. Here, I present numerical investigations into the effects of the strong hydrodynamic interaction on the inertial focusing. The direct numerical simulation is used to calculate the focusing/defocusing of particles, specifically since the particle-induced disturbance flows vary at the particle scale and hence affect the individual particle motion. The simulated defocusing of many-body systems prefer finite inter-particle separation, in contrast with sedimentation of two mobile particles, whereby the trailing particle catches up with the leading particle due to reduced drag in its wake. I numerically demonstrate that the finite separation between nearest neighbors is a consequence of hydrodynamic repulsive motion unique to wall-bound shear flows. The author further presents direct demonstrations of the effects of the strong hydrodynamic interaction on the inertial focusing in an experimentally unachievable manner. The excessive hydrodynamic interaction drastically dissipates the near-wall focusing attractors and thus causes irreversible defocusing by breaking the balance between the lift forces. Unexpectedly, I also find that moderate hydrodynamic interaction can alter focusing speed on specific conditions, suggesting that an optimum concentration may significantly boost the inertial focusing in microfluidic-based applications.     

Controlled mixing in microfluidic systems using bacterial chemotaxis

We demonstrate the use of Escherichia coli and their chemotactic characteristics to enhance mixing in a microchannel in a controlled and bi-directional manner. The presence of a chemoattractant in one arm of a three-junction microchannel results in an asymmetric increase in the effective diffusion coefficient of extremely high molecular weight TMR-Dextran (MW 2 000 000), which rises linearly with the concentration of attractant from a baseline value of 8-42 microm(2)/s at a concentration of 0.1 M. The response to a repellent is similar, with the opposite bias.     

Influence of temperature on the hydrodynamic resistance reduction effect

The effect of temperature on hydrodynamic resistance of aqueous solutions of polyethylene oxide is established experimentally.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 37, No. 6, pp. 1012–1014, December, 1979.     

Metabolomics-on-a-chip and predictive systems toxicology in microfluidic bioartificial organs

The world faces complex challenges for chemical hazard assessment. Microfluidic bioartificial organs enable the spatial and temporal control of cell growth and biochemistry, critical for organ-specific metabolic functions and particularly relevant to testing the metabolic dose-response signatures associated with both pharmaceutical and environmental toxicity. Here we present an approach combining a microfluidic system with (1)H NMR-based metabolomic footprinting, as a high-throughput small-molecule screening approach. We characterized the toxicity of several molecules: ammonia (NH(3)), an environmental pollutant leading to metabolic acidosis and liver and kidney toxicity; dimethylsulfoxide (DMSO), a free radical-scavenging solvent; and N-acetyl-para-aminophenol (APAP, or paracetamol), a hepatotoxic analgesic drug. We report organ-specific NH(3) dose-dependent metabolic responses in several microfluidic bioartificial organs (liver, kidney, and cocultures), as well as predictive (99% accuracy for NH(3) and 94% for APAP) compound-specific signatures. Our integration of microtechnology, cell culture in microfluidic biochips, and metabolic profiling opens the development of so-called "metabolomics-on-a-chip" assays in pharmaceutical and environmental toxicology.     

Recent developments and patents on biological sensing using nanoparticles in microfluidic systems

Microfluidic systems provide a total solution of biological and chemical analysis from the sample application to the display of the analysis results. A lot of developments on the point-of-care diagnostic applications have been reported and the commercial possibility is shown. To achieve sensitive and specific biological sensing, nanoparticles may provide a promising tool because they have similar length scale with the biomolecules. The nano-sensing technology suggests a molecular level detection of the biomolecules to pursue higher performance. In this review, recent developments and patents on the biological sensing using nanoparticles in microfluidic systems are discussed. An updated, systematic and rapid reference in the field of nano-biological sensing is provided.     

Design and characterization of immobilized enzymes in microfluidic systems.

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.     

Investigation of the hydrodynamic conditions of jet heat transfer

An impact jet model that can be used to explain the regularities of heat transfer is proposed.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 33, No. 2, pp. 210–212, August, 1977.