Parallel Laminar Flow Research Articles

Diffusion based kinetic selectivity modulation of enzymatic proteolysis in a microfluidic reactor: experimental analysis and stochastic modeling

Microreactors enable new experimental ways for enzyme engineering. In this context, we show that the liquid–liquid parallel laminar flows in microchannels cause a kinetic selectivity modification of proteolytic enzymatic reaction involving hemoglobin and pepsin, reaction that generates numerous bioactive peptides at different advancement state. Here we show that this diffusion based kinetic modulation induces an altered peptides appearance kinetics for a part of the initial substrate population. Indeed, microfluidic and conventional batch experiments performed in the same reaction conditions lead to strong differences in the resulting peptidic profiles obtained by reversed-phase high-performance liquid chromatography. Such differences are explained by the laminar flow diffusive conditions inside microchannels and are supported by a stochastic algorithm based on the Michaelis–Menten equation. Several bioactive peptides are identified by mass spectrometry and show the potential of such methodology in peptides screening from a complex proteolysis but also for selective peptides preparation by microfluidics.

電気化学マイクロリアクターを活用する新規有機電解合成プロセスの開発

Electron transfer is one of the most typical driving factors for organic reaction, and organic electrosynthesis serves as a straightforward and powerful method for organic electron transfer processes. On the other hand, microreactor technology for organic synthesis has been significantly investigated for the past two decades, however, the integrated use of microreactor technology with organic electrosynthesis has been quite limited so far. We report here that the integrated use of microreactor technology with organic electrosynthesis which offers one of the most sophisticated processes in organic chemistry. In particular, (a) Organic electrosynthetic processes without using intentionally added supporting electrolyte, (b) Organic electrosynthetic processes utilizing liquid-liquid parallel laminar flow, (c) Novel paired electrolytic processes, and (d) Multi-step reaction systems via electrogenerated species, have been described.

On the stationary Navier-Stokes flow with isotropic streamlines in all latitudes on a sphere or a 2D hyperbolic space

In this paper, we show the existence of real-analytic stationary Navier-Stokes flows with isotropic streamlines in all latitudes in some simply-connected flow region on a rotating round sphere. We also exclude the possibility of having a Poiseuille's flow profile to be one of these stationary Navier-Stokes flows with isotropic streamlines. When the sphere is replaced by a 2-dimensional hyperbolic space, we also give the analog existence result for stationary parallel laminar Navier-Stokes flows along a circular-arc boundary portion of some compact obstacle in the 2-D hyperbolic space. The existence of stationary parallel laminar Navier-Stokes flows along a straight boundary of some obstacle in the 2-D hyperbolic space is also studied. In any one of these cases, we show that a parallel laminar flow with a Poiseuille's flow profile ceases to be a stationary Navier-Stokes flow, due to the curvature of the background manifold.

High-purity and label-free isolation of circulating tumor cells (CTCs) in a microfluidic platform by using optically-induced-dielectrophoretic (ODEP) force

Negative selection-based circulating tumor cell (CTC) isolation is believed valuable to harvest more native, and in particular all possible CTCs without biases relevant to the properties of surface antigens on the CTCs. Under such a cell isolation strategy, however, the CTC purity is normally compromised. To address this issue, this study reports the integration of optically-induced-dielectrophoretic (ODEP) force-based cell manipulation, and a laminar flow regime in a microfluidic platform for the isolation of untreated, and highly pure CTCs after conventional negative selection-based CTC isolation. In the design, six sections of moving light-bar screens were continuously and simultaneously exerted in two parallel laminar flows to concurrently separate the cancer cells from the leukocytes based on their size difference and electric properties. The separated cell populations were further partitioned, delivered, and collected through the two flows. With this approach, the cancer cells can be isolated in a continuous, effective, and efficient manner. In this study, the operating conditions of ODEP for the manipulation of prostate cancer (PC-3) and human oral cancer (OEC-M1) cells, and leukocytes with minor cell aggregation phenomenon were first characterized. Moreover, performances of the proposed method for the isolation of cancer cells were experimentally investigated. The results showed that the presented CTC isolation scheme was able to isolate PC-3 cells or OEC-M1 cells from a leukocyte background with high recovery rate (PC-3 cells: 76-83%, OEC-M1 cells: 61-68%), and high purity (PC-3 cells: 74-82%, OEC-M1 cells: 64-66%) (set flow rate: 0.1 μl min(-1) and sample volume: 1 μl). The latter is beyond what is currently possible in the conventional CTC isolations. Moreover, the viability of isolated cancer cells was evaluated to be as high as 94 ± 2%, and 95 ± 3% for the PC-3, and OEC-M1 cells, respectively. Furthermore, the isolated cancer cells were also shown to preserve their proliferative capability. As a whole, this study has presented an ODEP-based microfluidic platform that is capable of isolating CTCs in a continuous, label-free, cell-friendly, and particularly highly pure manner. All these traits are found particularly meaningful for exploiting the harvested CTCs for the subsequent cell-based, or biochemical assays.

Phase transfer catalyzed esterification: modeling and experimental studies in a microreactor under parallel flow conditions

A liquid–liquid phase transfer catalyzed (PTC) esterification reaction of 4-t-butylphenol in aqueous phase (1 M sodium hydroxide solution) and 4-methoxybenzoyl chloride in organic phase (dichloromethane) in a microchannel under parallel laminar flow conditions was studied in this work. Tetrabutylammonium bromide was used as the PTC. Stable liquid–liquid hydrodynamic flow and a defined specific interfacial area in a microreactor offer considerable benefits over conventional batch reactors and are crucial to study interactions between kinetics and mass transfer effects. Mentioned features were used to develop a 3D mathematical model considering convection in the flow direction, diffusion in all spatial directions, and reactions in organic and aqueous phases. Results have shown a much higher mass transfer rate of the PTC between both phases as the one predicted by the 3D mathematical model. It may be assumed that the instability of parallel flow, along with the mass transfer of catalyst between both phases, causes rippling and erratic pulsation at the interface which then leads to interfacial convection and increased mass transfer rates. With a proposed correlation for mass transfer enhancement due to interfacial convection, all the experimental data were successfully predicted by the model.

On-chip cell migration assay for quantifying the effect of ethanol on MCF-7 human breast cancer cells

Ethanol consumption is associated with the risk of breast cancer progression; however, the mechanism of relationship has not yet been fully explained. Research on breast cancer cell migration after ethanol stimulation may give hope for a better understanding of the disease and oncotherapy. Conventional cell migration assays such as Boyden chamber and wound-healing assays are easy to conduct for this purpose; however, these assays have inherent limitations. In this study, we quantified the effect of ethanol on MCF-7 hunam breast cancer cells using a microfluidics-based wound-healing assay. Wounds were prepared by partially digesting a confluent cell sheet using parallel laminar flows in the presence of protease trypsin. The cells at the leading edge of the wound remained intact. Cell image analysis indicates that all the cells cultured in the microdevice took on a good morphology and monolayer growth status. Cell viability assay demonstrates that cell viability decreased with the increase in ethanol concentration and treatment time. For 0, 22, 43, and 65 mmol/l of ethanol, cell viability after being cultured for 24 h was 100%, 99.6%, 99.4%, and 98.4%, respectively. Studying MCF-7 human breast cancer cell migration when treated with different ethanol concentrations revealed that the cell migration distance is directly proportional with ethanol concentration. After being cultured for 24 h at 37°C and 5% CO2, the maximal cell migration distance was 231, 283, and 332 μm for 22, 43, and 65 mmol/l ethanol, respectively; all results were higher than the blank test (i.e., ethanol-free test, 218 μm). These findings will be beneficial in developing microfluidic device applications for future research on breast tumor therapy in a biomimetic microenvironment and for developing new methods for breast cancer therapy.

Product selectivity control induced by using liquid–liquid parallel laminar flow in a microreactor

Product selectivity control based on a liquid-liquid parallel laminar flow has been successfully demonstrated by using a microreactor. Our electrochemical microreactor system enables regioselective cross-coupling reaction of aldehyde with allylic chloride via chemoselective cathodic reduction of substrate by the combined use of suitable flow mode and corresponding cathode material. The formation of liquid-liquid parallel laminar flow in the microreactor was supported by the estimation of benzaldehyde diffusion coefficient and computational fluid dynamics simulation. The diffusion coefficient for benzaldehyde in Bu(4)NClO(4)-HMPA medium was determined to be 1.32 × 10(-7) cm(2) s(-1) by electrochemical measurements, and the flow simulation using this value revealed the formation of clear concentration gradient of benzaldehyde in the microreactor channel over a specific channel length. In addition, the necessity of the liquid-liquid parallel laminar flow was confirmed by flow mode experiments.

Phase separation of parallel laminar flow for aqueous two phase systems in branched microchannel

Aqueous two phase systems (ATPSs) have good biocompatibility and special selectivity. Their phase equilibrium and applications in biological analysis have received much attention. Herein, parallel laminar flow (PLF) in the microchannel can provide an effective platform to enhance mass transfer and preserve separate phases simultaneously. As fundamentals in feasible and convenient sampling of PLF for ATPS, the phase separation methods and rules in branched microchannel were studied in this work, selecting PEG 4000 + Na2SO4 + H2O as a model system. The exploration of flow pattern showed that a stable PFL was easily to form in the shallow microchannel of 200 μm (depth) × 600 μm (width), as long as the velocity of lower phase was higher than 0.51 mm/s. The phase interface of PLF could be easily controlled by the flow ratio of two phases. Single-phase separation could be reliably achieved in T-junction outlets when the flow rate of outlet ascertains to be smaller or larger than that of inlet on the same side. The trifurcate outlets with an extra middle channel could help realize a simultaneous two-phase separation. The flow rate of the extra channel is the key for the phase separation performance, the range of which available for simultaneous two-phase separation is determined by the resistance balance and the flow rates deviation offsetting as well. It is favor for increasing phase separation efficiency to make the products of flow rate, viscosity, and the length of corresponding outlet channel close with each other for the upper phase and the lower phase. The adjustable lengths of three channels can provide flexible choices to enhance simultaneous two-phase separation of diversified ATPSs at various operating flow ratios. A multiport microchip with T-junction inlets and trifurcate outlets designed for adjusting the lengths of branched channels on-chip is a convenient tool for PLF contact and in situ phase separation of ATPSs in varieties of application.

Focus on nanoscience and nanotechnology

Focus on nanoscience and nanotechnology

Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of β-catenin signaling

In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of β-catenin signaling was chosen as a case study. The highly conserved Wnt-activated β-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of β-catenin pathway, leading to translocation of β-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/β-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the β-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the β-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of β-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells.

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.

A Novel Electrosynthetic System for Anodic Substitution Reactions by Using Parallel Laminar Flow in a Microflow Reactor

We have developed a novel electrosynthetic system for anodic substitution reactions by using parallel laminar flow in a microflow reactor. This system enables nucleophilic reactions to overcome the restraint, such as the oxidation potential of nucleophiles and the stability of cationic intermediates, by the combined use of ionic liquids as reaction media and the parallel laminar flow in the microflow reactor. By using this novel electrosynthetic system, the anodic substitution reaction of carbamates, especially of cyclic carbamates, with allyltrimethylsilane were carried out to provide the corresponding products in moderate to good conversion yields in a single flow-through operation at ambient temperature (without the need for low-temperature conditions).

Self-Supported Paired Electrosynthesis Using a Microflow Reactor Without Intentionally Added Electrolyte

We have developed a paired electrosynthetic system using a microflow reactor. This system enables paired electrochemical reactions composed of chloride reduction/alcohol oxidation combination to progress without intentionally added electrolyte. In addition, the use of parallel laminar flow in the microflow reactor resulted in a further improvement of the desired product yields in the paired electrosynthesis.

A New Approach to Anodic Substitution Reaction of Amines with Allyltrimethylsilane using Parallel Laminar Flow

not Available.

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells. Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).

Parallel and non-parallel laminar mixed convection flow in an inclined tube: The effect of the boundary conditions

The necessary condition for the onset of parallel flow in the fully developed region of an inclined duct is applied to the case of a circular tube. Parallel flow in inclined ducts is an uncommon regime, since in most cases buoyancy tends to produce the onset of secondary flow. The present study shows how proper thermal boundary conditions may preserve parallel flow regime. Mixed convection flow is studied for a special non-axisymmetric thermal boundary condition that, with a proper choice of a switch parameter, may be compatible with parallel flow. More precisely, a circumferentially variable heat flux distribution is prescribed on the tube wall, expressed as a sinusoidal function of the azimuthal coordinate ϑ with period 2π. A π/2 rotation in the position of the maximum heat flux, achieved by setting the switch parameter, may allow or not the existence of parallel flow. Two cases are considered corresponding to parallel and non-parallel flow. In the first case, the governing balance equations allow a simple analytical solution. On the contrary, in the second case, the local balance equations are solved numerically by employing a finite element method.

A New Approach to Anodic Substitution Reaction Using Parallel Laminar Flow in a Micro-Flow Reactor

We have developed a novel electrosynthetic system for anodic substitution reaction using a micro-flow reactor. This system enables nucleophilic reactions to overcome the restraints such as the oxidation potential of nucleophiles and the stability of carbocations by the combined use of ionic liquids as reaction media and the parallel laminar flow in the reactor.

Laser-Induced Mixing in Microfluidic Channels

We demonstrate a novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems. This method utilizes highly focused nanosecond laser pulses from a Q-switched Nd:YAG laser at lambda = 532 nm to generate cavitation bubbles within 100- and 200-microm-wide microfluidic channels containing the parallel laminar flow of two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. We use time-resolved imaging and fluorescence detection methods to visualize the mixing process and to estimate both the volume of mixed fluid and the time scale for the re-establishment of laminar flow. The results show that mixing is initiated by liquid jets that form upon cavitation bubble collapse and occurs approximately 20 micros following the delivery of the laser pulse. The images also reveal that mixing occurs on the millisecond time scale and that laminar flow is re-established on a 50-ms time scale. This process results in a locally mixed fluid volume in the range of 0.5-1.5 nL that is convected downstream with the main flow in the microchannel. We demonstrate the use of this mixing technique by initiating the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to yield fluorescent resorufin. This approach to generate the mixing of adjacent fluids may prove advantageous in many microfluidic applications as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation.

In Situ Fabrication of Cross‐Linked Protein Membranes by Using Microfluidics

We report a novel technique for preparing cross-linked protein membranes within microchannels by using an interfacial cross-linking reaction. Glass microchannels with a Y input were assembled by using a simple adhesive bonding technique to achieve dual, parallel laminar flows. Membrane formation utilised an interfacial reaction at the liquid-liquid interface, which involved bovine serum albumin (aqueous solution with a flow rate of 300 microL min(-1)) and terephthaloyl chloride (xylene solution with a flow rate of 700 microL min(-1)), to form thin ( approximately 25 microm) cross-linked films along the length of the channel under the continuous pressure-driven laminar flow. Such microfabricated membranes could extend the separation potential of any microfluidic structure to provide a stable barrier layer. Furthermore, degradation of the membrane was possible by using an alkali sodium dodecyl sulfate solution, which led to the complete disappearance of the membrane. These membranes could facilitate additional modification to allow for different permeability properties by controlled degradation. The one-step in situ membrane-fabrication methodology reported here generated precisely localised membranes and avoided the complexities of subcomponent assembly, which require complicated alignment of small, preformed membranes. This methodology could become the basis for sophisticated microseparation systems, biosensors and several "lab-on-a-chip" devices.

Characterization of a laminar flow cell for the prevention of biosensor fouling

A flow cell using dual parallel laminar flows was designed and fabricated with the aim to prevent biosensor fouling. Y-shaped entrances allowed the introduction of sample and electrolyte streams, with the electrolyte serving as a mobile protective layer near the biosensor. Potassium permanganate was used to quantify the diffusion in the flow cell. Optical intensity analysis of potassium permanganate along a series of transverse lines across the flow cell was carried out under different flow conditions. It was found that the error function, erf( y/√( Dt)), where y was the position along the transverse line, D the diffusion coefficient of the solute and t was the time, gave reasonable approximation to the diffusion of potassium permanganate in the cell. The diffusion coefficient of potassium permanganate was determined in stop–flow measurements and the value, D = 4.0 × 10 −5 cm 2/s, agreed to previously reported values. Velocity distribution in the flow cell was simulated numerically to reveal the development of two inflows into one single laminar flow. Results from the study provided preliminary data on solute diffusion characteristics in the flow cell and supported the working principle of laminar flow cells in preventing biosensor fouling.