Investigation of hydrodynamic focusing in a microfluidic coulter counter device

The Coulter technique enables rapid analysis of particles or cells suspended in a fluid stream. In this technique, the cells are suspended in an electrically conductive solution, which is hydrodynamically focused by nonconducting sheath flows. The cells produce a characteristic voltage signal when they interrupt an electrical path. The population and size of the cells can be obtained through analyzing the voltage signal. In a microfluidic Coulter counter device, the hydrodynamic focusing technique is used to position the conducting sample stream and the cells and also to separate close cells to generate distinct signals for each cell and avoid signal jam. The performance of hydrodynamic focusing depends on the relative flow ratio between the sample stream and sheath stream. We use a numerical approach to study the hydrodynamic focusing in a microfluidic Coulter counter device. In this approach, the flow field is described by solving the incompressible Navier-Stokes equations. The sample stream concentration is modeled by an advection-diffusion equation. The motion of the cells is governed by the Newton-Euler equations of motion. Particle motion through the flow field is handled using an overlapping grid technique. A numerical model for studying a microfluidic Coulter counter has been validated. Using the model, the impact of relative flow rate on the performance of hydrodynamic focusing was studied. Our numerical results show that the position of the sample stream can be controlled by adjusting the relative flow rate. Our simulations also show that particles can be focused into the stream and initially close particles can be separated by the hydrodynamic focusing. From our study, we conclude that hydrodynamic focusing provides an effective way to control the position of the sample stream and cells and it also can be used to separate cells to avoid signal jam.     

Continuous scalable blood filtration device using inertial microfluidics

Cell separation is broadly useful for applications in clinical diagnostics, biological research, and potentially regenerative medicine. Recent attention has been paid to label‐free size‐based techniques that may avoid the costs or clogging issues associated with centrifugation and mechanical filtration. We present for the first time a massively parallel microfluidic device that passively separates pathogenic bacteria cells from diluted blood with macroscale performance. The device was designed to process large sample volumes in a high‐throughput, continuous manner using 40 single microchannels placed in a radial array with one inlet and two rings of outlets. Each single channel consists of a short focusing, gradual expansion and collection region and uses unique differential transit times due to size‐dependent inertial lift forces as a method of cell separation. The gradual channel expansion region is shown to manipulate cell equilibrium positions close to the microchannel walls, critical for higher efficiency collection. We demonstrate >80% removal of pathogenic bacteria from blood after two passes of the single channel system. The massively parallel device can process 240 mL/h with a throughput of 400 million cells/min. We expect that this parallelizable, robust, and label‐free approach would be useful for filtration of blood as well as for other cell separation and concentration applications from large volume samples. Biotechnol. Bioeng. 2010;107: 302–311. © 2010 Wiley Periodicals, Inc.     

基于微流控技术的微生物细胞梯度稀释分离方法

随着微流控分析技术的快速发展,集成化的微流控芯片在满足实验高通量的同时,还在微生物细胞分离领域呈现出独特的优势。本研究基于微流控技术,制备了以聚二甲基硅氧烷(PDMS)、玻片为材料的细菌细胞梯度稀释分离芯片。该芯片的核心是通过一系列复杂的梯度网络来实现对细菌悬液的连续稀释,最终被分离的细菌细胞进入通道末端的存储孔内。结果显示,该方法能分离出的最少细菌细胞数低于10个。此芯片平台操作简单、耗时短、成本低,为微生物单细胞研究提供了新的途径。     

Sample flow switching techniques on microfluidic chips

This paper presents an experimental investigation into electrokinetically focused flow injection for bio-analytical applications. A novel microfluidic device for microfluidic sample handling is presented. The microfluidic chip is fabricated on glass substrates using conventional photolithographic and chemical etching processes and is bonded using a high-temperature fusion method. The proposed valve-less device is capable not only of directing a single sample flow to a specified output port, but also of driving multiple samples to separate outlet channels or even to a single outlet to facilitate sample mixing. The experimental results confirm that the sample flow can be electrokinetically pre-focused into a narrow stream and guided to the desired outlet port by means of a simple control voltage model. The microchip presented within this paper has considerable potential for use in a variety of applications, including high-throughput chemical analysis, cell fusion, fraction collection, sample mixing, and many other applications within the micro-total-analysis systems field.     

Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel

Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 μm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations.     

Electrophoresis today and tomorrow: Helping biologists' dreams come true

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two‐dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High‐throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser‐induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH‐driven mobilization, provides efficient separations and on‐line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single‐cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single‐molecule detection.     

Influences of Woody Debris on Flow Patterns and Channel Morphology in a Low Energy,Sand‐Bed Stream Reach

In lowland areas, such as the glacial landscapes of eastern Germany, sand‐bed streams are the most common stream type. They have low gradients and their hydrological regime is often subdued due to the frequent interruption by lakes. Very few is known about the influence of woody debris in these streams, since nearly all previous studies are from high‐gradient conditions, where streams have coarse bed sediments and harsh hydrological regimes. The research objectives of this study were first to assess the quasi‐natural quantity and quality of wood in a lowland sand‐bed stream and second to understand the influence of wood on the channel morphology and the flow patterns at base‐flow. The three‐dimensional stream bed relief was surveyed by electronic distance measurement. The position and the size of large woody debris was assessed by close‐up photography. An acoustic Doppler velocimeter was used to record the patterns of flow velocity and turbulence. Overlay and analysis of the spatial data was done using a Geographic Information System. The standing stock of wood was 1.9 m³ and 39 woody elements per 100 m² of stream bed. The flow pattern was clearly controlled by the wood. Woody elements elevated above the stream bed deflected flow and locally caused strong secondary current, high turbulence, and scour of the stream bed at baseflow. Wood resting directly on the stream bed, which contributed the majority of the wood inside the bank‐full channel, determined the roughness of the stream bed. Near‐bed flow patterns observed were isolated roughness flow and wake interference flow, which was registered inside the accumulations of wood. 68% of the stream bed had shear stress above critical. Hence, the secondary morphological structures of the sand‐bed were controlled at base‐flow by the flow which was determined by the woody debris distribution.     

Sheath fluid control to permit stable flow in rapid mix flow cytometry.

BACKGROUND: Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. METHODS: We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. RESULTS: With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. CONCLUSIONS: This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.     

Genetic structure of a native cyprinid in a reservoir‐altered stream network

1. Reservoirs modify riverine ecosystems worldwide, and often with deleterious impacts on native biota. The immediate effects of reservoirs on native fish species below dams and in impounded reaches have received considerable attention, but it is unclear how reservoirs may affect fish species at larger spatial and temporal scales. Documented declines of stream fish populations in direct tributaries of reservoirs suggest reservoir pools may reduce gene flow among historically connected populations. 2. Because of increased predator densities in reservoirs and the extent of habitat alteration in impounded reaches, I predicted reservoir habitats would reduce gene flow among small‐bodied fish populations separated by reservoir habitat. I used microsatellite markers to assess the spatial genetic structure of populations of the red shiner (Cyprinella lutrensis), in a reservoir‐fragmented stream network (Lake Texoma, U.S.A.). I also tested the prediction that populations in two direct tributaries that have experienced population declines would have low genetic diversity. Individuals were collected from six sites upstream of the reservoir, three sites in the reservoir and three sites in direct tributaries of the reservoir during 2008 and 2009. 3. Results indicate that most populations were isolated by distance with little divergence among populations. In one direct tributary population, however, there was substantial genetic divergence, and genetic diversity was significantly lower than in other populations. Gene flow also seemed to be lower in reservoir habitats than in intact stream habitats, suggesting reservoir habitats may be reducing gene flow among the reservoir‐separated populations. These results indicate that reservoirs may reduce gene flow among reservoir‐fragmented stream fish populations, altering the evolutionary trajectories of fragmented populations.     

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A “sheath” fluid passing through a microfluidic channel at low Reynolds number can be directed around another “core” stream and used to dictate the shape as well as the diameter of a core stream. Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid. By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed. Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid. Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers. Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light. Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.     

A temporary immersion plant propagation bioreactor with decoupled gas and liquid flows for enhanced control of gas phase

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic‐driven TIB (Hy‐TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO₂ supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ～0.0002 atm) caused by flexing of non‐rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337–345, 2016     

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.     

Selective bioparticle retention and characterization in a chip‐integrated confocal ultrasonic cavity

We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the chamber center, where the cells are trapped. We investigate the resonant modes in the expansion chamber and its connecting inlet channel by theoretical modeling and experimental verification during no‐flow conditions. Furthermore, by triple‐frequency ultrasonic actuation during continuous microfluidic sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass—injection—aggregation and retention—positioning. Finally, we demonstrate transillumination microscopy imaging of ultrasonically trapped COS‐7 cell aggregates. Biotechnol. Bioeng. 2009;103: 323–328. © 2009 Wiley Periodicals, Inc.     

Separation of apoptotic cells using a microfluidic device

A highly sensitive microfluidic device has been developed to separate apoptotic cells. Apoptotic Jurkat cells were selectively labeled with magnetic beads (0.8 μm diam) using the C2A protein which recognizes phosphatidylserine. The cell mixture was flowed through a microfluidic channel and apoptotic cells were separated by a 0.3 T permanent magnet. Separations using our device showed 96% agreement with those of a commercial flow cytometer, indicating our device can be used to sort apoptotic cells in a miniaturized system.     

A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage

Interest in electrical lysis of biological cells on a microfludic platform has increased because it allows for the rapid recovery of intracellular contents without introducing lytic agents. In this study we demonstrated a simple microfluidic flow-through device which lysed Escherichia coli cells under a continuous dc voltage. The E. coli cells had previously been modified to express green fluorescent protein (GFP). In our design, the cell lysis only happened in a defined section of a microfluidic channel due to the local field amplification by geometric modification. The geometric modification also effectively decreased the required voltage for lysis by several folds. We found that local field strength of 1000-1500 V/cm was required for nearly 100% cell death. This threshold field strength was considerably lower than the value reported in the literature, possibly due to the longer duration of the field [Lee, S.W., Tai, Y.C., 1999. Sens. Actuators A: Phys. 73, 74-79]. Cell lysis was detected by both plate count and fluorescence spectroscopy. The cell membrane was completely disintegrated in the lysis section of the microfluidic device, when the field strength was higher than 2000 V/cm. The devices were fabricated using low-cost soft lithography with channel widths considerably larger than the cell size to avoid clogging and ensure stable performance. Our tool will be ideal for high throughput processing of bacterial cells for chemical analysis of intracellular contents such as DNA and proteins. The application of continuous dc voltage greatly simplified the instrumentation compared to devices using electrical pulses for similar purposes. In principle, the same approach can also be applied for lysis of mammalian cells and electroporative transfection.     

Simple approaches to close the open structure of microfluidic chips and connecting them to the macro-world

Microchip electrophoresis has a great potential to improve the speed and throughput of chemical and biochemical analyses. Conventional electrophoresis microchip fabrication methods comprise the main steps of channel formation, cover plate binding and access hole construction. While the fabrication of appropriate cover plates and their bonding process are quite essential to the creation of closed microfluidic networks, connection means of microchips to the macro-world is one of the most important parts of microchip fabrication. In this paper the most commonly used approaches are discussed for cover plate connector fabrication in conjunction with high and low temperature glue-less binding processes. The microchannels in the glass substrate were fabricated by sawing and powder blasting under regular laboratory settings, i.e., not necessitating the use of a clean-room environment, making in this way broader availability for electrophoresis microchip technology.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

Microfluidic synthesis of composite cross‐gradient materials for investigating cell–biomaterial interactions

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high‐throughput screening of cell–material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed “hit”) materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross‐gradients of gelatin and chitosan for investigating cell–biomaterial interactions. The microfluidic synthesis of the cross‐gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross‐gradients were characterized by Fourier transform infrared spectrometry and X‐ray photoelectron spectrometry. The three‐dimensional (3D) porous gelatin/chitosan cross‐gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient‐dependent manner. We envision that our microfluidic cross‐gradient platform may accelerate the material development processes involved in a wide range of biomedical applications. Biotechnol. Bioeng. 2011; 108:175–185. © 2010 Wiley Periodicals, Inc.     

Optical measurement of transverse molecular diffusion in a microchannel

Quantitative analysis of molecular diffusion is a necessity for the efficient design of most microfluidic devices as well as an important biophysical method in its own right. This study demonstrates the rapid measurement of diffusion coefficients of large and small molecules in a microfluidic device, the T-sensor, by means of conventional epifluorescence microscopy. Data were collected by monitoring the transverse flux of analyte from a sample stream into a second stream flowing alongside it. As indicated by the low Reynolds numbers of the system (< 1), flow is laminar, and molecular transport between streams occurs only by diffusion. Quantitative determinations were made by fitting data with predictions of a one-dimensional model. Analysis was made of the flow development and its effect on the distribution of diffusing analyte using a three-dimensional modeling software package. Diffusion coefficients were measured for four fluorescently labeled molecules: fluorescein-biotin, insulin, ovalbumin, and streptavidin. The resulting values differed from accepted results by an average of 2.4%. Microfluidic system parameters can be selected to achieve accurate diffusion coefficient measurements and to optimize other microfluidic devices that rely on precise transverse transport of molecules.     

Detergent screening of the human voltage‐gated proton channel using fluorescence‐detection size‐exclusion chromatography

The human voltage‐gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino‐ and carboxy‐termini. The protein is activated by membrane depolarization, similar to other voltage‐sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three‐dimensional structures for homologues of this voltage‐gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino‐terminus of the human Hv1 proton channel (termed EGFP‐Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence‐detection size‐exclusion chromatography. The EGFP‐Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3–12 and the nonionic n‐dodecyl‐β‐d ‐maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size‐exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full‐length human Hv1 proton channel.