A hybrid poly(dimethylsiloxane) microsystem for on-chip whole blood filtration optimized for steroid screening

Miniaturized biochemical devices in glass, silicon and polymer materials are starting to find their way from the academic laboratories to real-life applications. However, most attention has been given to miniaturize the downstream functions of various microfluidic systems, leaving the sample introduction and preparation steps to more conventional, bulkier solutions. For point-of-care diagnostics in particular, it becomes crucial to be able to handle complex human samples in a miniaturized format. In this work, we report on a microsystem for on-chip sample preparation that is able to remove blood cells from whole blood. The hybrid system consists of a commercially available membrane filter incorporated into a poly(dimethylsiloxane) (PDMS) casted device. Membrane materials were evaluated on the bases of low nonspecific adsorption of free and protein-bound testosterone as analyte substance. The hybrid system including a hydrophilic polypropylene filter successfully removed blood cells from diluted human whole blood. Surface oxidation was sufficient to make the plasma filtrate flow through the membrane filter and the channel system by capillary force alone and thus no external pumping source was needed.     

Fast and continuous plasma extraction from whole human blood based on expanding cell-free layer devices

This paper presents promising microfluidic devices designed for continuous and passive extraction of plasma from whole human blood. These designs are based on red cells lateral migration and the resulting cell-free layer locally expanded by geometric singularities such as an enlargement of the channel or a cavity adjacent to the channel. After an explanation of flow patterns, different tests are described that confirm the advantages of both proposed singularities, providing a 1.5 and 2X increase in extraction yield compared to a reference device, for 1:20 diluted blood at 100 μL/min. Devices have also been successively optimized, with extraction yields up to 17.8%, and biologically validated for plasma extraction, with no protein loss or denaturation, no hemolysis and with excellent cell purity. Finally, the dilution effect has been experimentally investigated.     

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.     

Separation of Simulants of Biological Warfare Agents from Blood by a Miniaturized Dielectrophoresis Device

Separation of simulants of biological warfare agents from blood using dielectrophoresis (DEP) was demonstrated in a miniaturized DEP device. The device was fabricated by laminating five different layers (all 40 mm×40 mm) including a polycarbonate substrate, a pressure sensitive acrylic adhesive (PSA) layer, a patterned polyimide layer with a flip-chip bonded dielectrophoresis chip (DEP chip), a PSA layer with microfluidic channel, and a glass cover plate. The DEP chip consisted of repetitive interdigitated electrodes with characteristic dimension of 50 m. This device was employed to separate different simulants of biological warfare agents (BWA), namely Bacillus cereus (B. cereus), Escherichia coli (E. coli) and Listeria monocytogenes (L. monocytogenes), from blood, individually or simultaneously. PCR amplification, which was inhibited by blood components in pre-separation samples, successfully revealed bands in post-separation samples containing single or multiple BWA. Up to 97% efficiency of separation was achieved as demonstrated by culturing post-separation E. coli cells. The DEP device described here can potentially be used to reduce sample complexity for detection of infectious disease pathogens and biological warfare agents.     

Embryonic body culturing in an all-glass microfluidic device with laser-processed 4 μm thick ultra-thin glass sheet filter

In this paper, we report the development and demonstration of a method to fabricate an all-glass microfluidic cell culturing device without circulation flow. On-chip microfluidic cell culturing is an indispensable technique for cellular replacement therapies and experimental cell biology. Polydimethylsiloxane (PDMS) have become a popular material for fabricating microfluidic cell culture devices because it is a transparent, biocompatible, deformable, easy-to-mold, and gas-permeable. However, PDMS is also a chemically and physically unstable material. For example, PDMS undergoes aging easily even in room temperature conditions. Therefore, it is difficult to control long term experimental culturing conditions. On the other hand, glass is expected to be stable not only in physically but also chemically even in the presence of organic solvents. However, cell culturing still requires substance exchanges such as gases and nutrients, and so on, which cannot be done in a closed space of a glass device without circulation flow that may influence cell behavior. Thus, we introduce a filter structure with micropores onto a glass device to improve permeability to the cell culture space. Normally, it is extremely difficult to fabricate a filter structure on a normal glass plate by using a conventional fabrication method. Here, we demonstrated a method for fabricating an all-glass microfluidic cell culturing device having filters structure. The function of this all-glass culturing device was confirmed by culturing HeLa, fibroblast and ES cells. Compared with the closed glass devices without a filter structure, the numbers of cells in our device increased and embryonic bodies (EBs) were formed. This method offers a new tool in microfluidic cell culture technology for biological analysis and it expands the field of microfluidic cell culture.     

Integrated siphon-based metering and sedimentation of whole blood on a hydrophilic lab-on-a-disk

In this paper, we present a novel and fully integrated centrifugal microfluidic “lab-on-a-disk” for rapid colorimetric assays in human whole blood. All essential steps comprising blood sampling, metering, plasma extraction and the final optical detection are conducted within t = 150 s in passive, globally hydrophilized structures which obviate the need for intricate local hydrophobic surface patterning. Our technology features a plasma extraction structure (V = 500 nL, CV < 5%) where the purified plasma (c _RBC < 0.11%) is centrifugally separated, metered by an overflow and subsequently extracted by a siphon-based principle through a hydrophilic extraction channel into the detection chamber.     

A Microfluidic Device for Electrofusion of Biological Vesicles

This paper reports a microfabricated device with high aspect-ratio electrodes and low power consumption for the electrofusion of liposomes and cells. The applications may range from gene transfection or cell tracking to biophysical studies of membrane proteins. The device consists of 250 microm thick silicon electrodes bonded to a glass substrate and covered by a PDMS-coated glass slide. Liposomes were first aligned by AC voltage at 300 kHz and then fused with short DC pulses. The fusion yield can reach 75% and is globally better for liposome diameters larger than 10 microm. The encapsulation of microbeads inside liposomes has also been demonstrated and opens up the route towards fusion-based delivery of artificial microstructures into cells.     

A pneumatically-driven microfluidic system for size-tunable generation of uniform cell-encapsulating collagen microbeads with the ultrastructure similar to native collagen

This study reports a microfluidic system for high throughput, uniform, and size-tunable generation of cell-containing collagen microbeads. The principle is based on two pneumatically-driven mechanisms to achieve multi-channel mixture suspension transportation, and to actuate the spotting actions of micro-vibrators that continuously generate tiny collagen micro-droplets into a thin oil layer and then a sterile Pluronic® F127 surfactant solution located below. The temporarily formed collagen microdroplets are then thermally gelatinized. By regulating the feeding rate of cells/collagen suspension, and the spotting frequency of micro-vibrator, the size of the collagen microbeads can be manipulated. One of the key technical features is its capability to generate uniform collagen microbeads (coefficient of variation: 5.4–8.6 %) with sizes ranging from 73.9 to 349.3 μm in diameter. This is currently difficult to achieve using the existing methods particularly the generation of cell-encapsulating collagen microbeads with diameter less than 100 μm. Another advantageous trait is that the ultrastructure of the generated collagen microbeads is similar to that found in native collagen. In this study, moreover, the use of the proposed device for the microencapsulation of 3T3 cells in collagen microbeads has been successfully demonstrated showing that the encapsulated cells maintained high cell viability (96?±?2 %). Furthermore, a reasonable proliferative capability of the encapsulated cells was observed during 7 days culture. As a whole, the proposed device has opened up a new route to generate cell-containing collagen microbeads, which is found particularly meaningful for biomedical applications.     

Force spectroscopy study of the adhesion of plasma proteins to the surface of a dialysis membrane: role of the nanoscale surface hydrophobicity and topography

A mechanochemical study of the process of adhesion of plasma proteins to the surface of dialysis membranes was carried out with a scanning force microscope (SFM) in the force spectroscopy mode. Three representative blood plasma proteins (fibronectin, fibrinogen, and albumin) covalently were grafted to a SFM probe, and the adhesion forces of these proteins to cellulosic and synthetic dialysis membranes were measured. The experiment was tailored to apply a controlled load on the protein molecules adsorbed onto the surface in order to simulate the squeezing forces exerted on them during blood filtration. The de-adhesion forces, measured using this new approach for studying the interaction between a protein and dialysis membranes, suggest that the membrane's topography, at a nanometer scale, plays a critical role in the adhesion process. This result was strongly supported by parallel experiments performed on a flattened glass surface with the same dominant hydrophilic character as dialysis membranes. In contrast, a hydrophobic polystyrene surface led to de-adhesion forces at least one order of magnitude greater, overwhelming any possible shape recognition process between the protein molecules and the surface.     

A microfluidic platform to isolate avian erythrocytes infected with Plasmodium gallinaceum malaria parasites based on surface morphological changes

This paper reports on a microfluidic platform to isolate and study avian red blood cells (RBCs) infected to various degrees by the malaria parasite Plasmodium gallinaceum. The experimental findings point to the feasibility of using the morphological changes on the surface of the malaria infected avian RBC (miaRBCs) as biomarkers for diagnosis. A glass substrate with a controlled surface roughness was used as part of a polydimethylsiloxane (PDMS) microfluidic channels. When whole-blood samples were introduced into the channels, the miaRBCs would be preferentially slowed and eventually become immobilized on the roughened surface. The surface lesions and furrow-like structures on the miaRBC surfaces offered a markedly higher probability to interact with the roughened substrate and allowed the cells to become imobilized on the surface. The captured miaRBCs were from blood samples at various degrees of infection at 3.2%, 3.9%, 9.1%, 13.4%, 20.1%, 28%, and 37%. It was observed that the miaRBCs could be selectively captured under a wall shear rate between 2.1 to 3.2 s^?1, which was directly proportional to the flow rate through the channels. This capture rate could be improved by increasing the channel length and finer flow control. It was also found that a roughened glass substrate with ten-point-height larger than the depth of surface lesions and furrow-like structures of miaRBCs showed a substantial enhancement on the number of immobilized infected RBCs. These findings indicated that surface morphologies, including surface lesions and furrow-like structures, can serve as an alternative biomarker for malaria diagnosis.     

Micropatterned surfaces prepared using a liquid crystal projector-modified photopolymerization device and microfluidics

A commercial liquid crystal device projector was modified for photopolymerization using its on-board intense light source and a precision optical control circuit. This device projects reduced images generated by a typical personal computer onto the stage where photopolymerization on a surface occurs. This all-in-one device does not require expensive photomasks and external light sources. However, light scattering and diffraction through glass substrates resulted in undesired reactions in areas corresponding to masked (black) domains in mask patterns, limiting pattern resolution. To overcome this shortcoming, two-step surface patterning was developed. First, three-dimensional microstructures of crosslinked silicone elastomer were fabricated with this device and adhered onto silanized glass substrate surfaces, forming microchannels in patterns on the glass support. Then, acrylamide monomer solution containing photoreactive initiator was flowed into these micromold channels and reacted in situ. The resultant polyacrylamide layer was highly hydrophilic and repelled protein adsorption. Cell seeding on these patterns in serum-supplemented culture medium produced cells selectively adhered to different patterns: cells attached and spread only on unpolymerized silanized glass surfaces, not on the photopolymerized acrylamide surfaces. This technique should prove useful for inexpensive, rapid prototyping of surface micropatterns from polymer materials.     

Computational and functional evaluation of a microfluidic blood flow device

The development of microfluidic devices supporting physiological blood flow has the potential to yield biomedical technologies emulating human organ function. However, advances in this area have been constrained by the fact that artificial microchannels constructed for such devices need to achieve maximum chemical diffusion as well as hemocompatibility. To address this issue, we designed an elastomeric microfluidic flow device composed of poly (dimethylsiloxane) to emulate the geometry and flow properties of the pulmonary microcirculation. Our chip design is characterized by high aspect ratio (width > height) channels in an orthogonally interconnected configuration. Finite element simulations of blood flow through the network design chip demonstrated that the apparent pressure drop varied in a linear manner with flow rate. For simulated flow rates <250 mul min, the simulated pressure drop was <2000 Pa, the flow was laminar, and hemolysis was minimal. Hemolysis rate, assayed in terms of [total plasma hemoglobin (TPH) (sample - control)/(TPH control)] during 6 and 12 hour perfusions at 250 mul/min, was <5.0% through the entire period of device perfusion. There was no evidence of microscopic thrombus at any channel segment or junction under these perfusion conditions. We conclude that a microfluidic blood flow device possessing asymmetric and interconnected microchannels exhibits uniform flow properties and preliminary hemocompatibility. Such technology should foster the development of miniature oxygenators and similar biomedical devices requiring both a microscale reaction volume and physiological blood flow.     

Continuous-Flow Fractionation of Animal Cells in Microfluidic Device Using Aqueous Two-Phase Extraction

Monitoring of live cells is important in the field of medical science, diagnostics, biology, and the pharmaceutical industry. In this study, live and dead CHO-K1 (Chinese Hamster Ovary) cells were fractionated by continuous-flow extraction in a microfluidic device using immiscible aqueous two-phase extraction technique. The polymer solutions offered stable two-phase flows in microchannel without diffusive mixing. The fundamentals of aqueous two-phase extraction can support stable and reproducible recovery and separation of biomolecules in microfluidic devices. Polyethylene glycol 8000 (PEG 8000, 4%) and dextran T500 (5%) were selected as model polymer solutions. The appropriate flow rates of polymer and cell solutions were suggested. The fractionation efficiency of live and dead CHO K-1 cells from the culture broth was compared in normal macroscale system and microfluidic device. The optimum pH for the fractionation was 6.6 in both the normal and micro-scale systems. The loss of target live cells by sedimentation was circumvented in microfluidic device because of the negligible effect of gravity on the sedimentation. Most live cells were distributed to PEG-rich phase, while dead cells were found at the interface of two polymer solutions in microchannel. In this case, the recovery and fractionation efficiency of live cells in the PDMS-based microfluidic device was 100% and 97.0%, respectively.     

Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices: cell culture and flow studies with glial cells

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.     

Blood plasma separation in microfluidic channels using flow rate control

Several studies have clearly shown that cardiac surgery induces systemic inflammatory responses, particularly when cardiopulmonary bypass (CPB) is used. CPB induces complex inflammatory responses. Considerable evidence suggests that systemic inflammation causes many postoperative complications. Currently, there is no effective method to prevent this systemic inflammatory response syndrome in patients undergoing CPB. The ability to clinically intervene in inflammation, or even study the inflammatory response to CPB, is limited by the lack of timely measurements of inflammatory responses. In this study, a microfluidic device for continuous, real-time blood plasma separation, which may be integrated with downstream plasma analysis device, is introduced. This device is designed to have a whole blood inlet, a purified plasma outlet, and a concentrated blood cell outlet. The device is designed to separate plasma with up to 45% hematocrit of the inlet blood and is analyzed using computational fluid dynamics simulation. The simulation results show that 27% and 25% of plasma can be collected from the total inlet blood volume for 45% and 39% hematocrit, respectively. The device's functionality was demonstrated using defibrinated sheep blood (hematocrit=39%). During the experiment, all the blood cells traveled through the device toward the concentrated blood outlet while only the plasma flowed towards the plasma outlet without any clogging or lysis of cells. Because of its simple structure and control mechanism, this microdevice is expected to be used for highly efficient, realtime, continuous cell-free plasma separation.     

Microfluidic Cell Separation by 2-dimensional Dielectrophoresis

We describe a microfluidic device for separating cells according to their dielectric properties by combining 2-dimensional dielectrophoretic forces with field-flow-fractionation. The device comprises a thin chamber in which a travelling-wave electrical field is generated by a planar, multilayer microelectrode array at the bottom. Under the balance of gravitational and dielectrophoretic levitation forces, cells introduced into the device are positioned at different equilibrium heights in a velocity profile established inside the chamber, and thereby transported at different velocities by the fluid. Simultaneously, cells are subjected to a horizontal travelling-wave dielectrophoretic force that deflects them across the flow stream. The 2-dimensional dielectrophoretic forces acting on cells and the associated velocities in the fluid-flow and travelling-field directions depend sensitively on cell dielectric properties. The responses of cultured MDA-435 human breast cancer, HL-60 human leukemia and DS19 murine erythroleukemia cells, and of peripheral blood mononuclear (PBMN) cells were studied as functions of the frequency and voltage of the applied electric signals, and of the fluid flow rate. Significant differences were observed between the responses of different cell types. Cell separation was demonstrated by the differential redistribution of MDA-435 and PBMN cells as they flowed through the device. The device can be readily integrated with other microfluidic components for microscale sample preparation and analysis.     

Generation of monodisperse alginate microbeads and <Emphasis Type="Italic">in situ</Emphasis> encapsulation of cell in microfluidic device

A microfluidic method for the in situ production of monodispersed alginate hydrogels using chaotic mixing is described. Aqueous droplets comprising of alginate and calcium as a cross-linking agent were formed as an immiscible continuous phase, and then the alginate and calcium in the droplet came into contact and were rapidly mixed. Gelation of the hydrogel was achieved in situ by the chaotic mixing of the droplets in the microfluidic device. Important operating parameters included: the capillary number (Ca) and the flow rate of the continuous phase, which mainly influenced the formation of three distinctive flow regimes, such as fluctuation, stable droplets, and laminar flow. Under the stable formation of droplets regime, monodispersed alginate microbeads having a narrow size distribution (below 3% of CV) were produced in the microfluidic device and the size of the microbeads, ranging from 60 to 95 μm, could be easily modulated by varying the flow rate, viscosity, and interfacial tension. In addition, this approach can be applied to the encapsulation of yeast cells in alginate hydrogels with a high monodispersity. This simple microfluidic technique for the production of monodispersed hydrogels and encapsulation of biomolecules shows strong potential for use in biosensors, cell sensors, drug delivery systems, and cell transplantation applications.     

Design Considerations for a Microfluidic Device to Quantify the Platelet Adhesion to Collagen at Physiological Shear Rates

Accurate assessment of blood platelet function is essential in understanding thrombus formation which plays a central role in cardiovascular disease. Parallel plate flow chambers have been widely used as they allow for platelet adhesion on a collagen surface at physiologically relevant fluid mechanical forces. Standard parallel plate flow chambers typically need several milliliters of blood, which is substantially more than can be obtained from small animals. We designed, fabricated, and assessed the functionality of a microfluidic channel with a width of 500 μm and a height of 50 μm in which a wall shear rate of 1000 s⁻¹ can be achieved with a flow rate of 15 μL/min. The velocity distribution in the microchannel predicted from the equations of motion was compared to experimentally measured velocities of fluorescent beads. This analysis showed that the motion of beads was quite similar to the predicted motion. Adhesion of platelets from whole blood at a shear rate of 1000 s⁻¹ onto a collagen surface using the microfluidic flow channel was qualitatively similar to platelet adhesion observed with a standard sized parallel plate flow chamber. After 5 min flow the surface coverage of platelets in the microfluidic device was about 55% while in a traditional size flow chamber the surface coverage was about 75%. This suggests that the microfluidic flow chamber can be used to quantify platelet adhesion for system where only very small amounts of blood are available.     

Microfluidic particle sorting utilizing inertial lift force

A simple passive microfluidic device that continuously separates microparticles is presented. Its development is motivated by the need for specific size micro perfluorocarbon (PFC) droplets to be used for a novel gas embolotherapy method. The device consists of a rectangular channel in which inertial lift forces are utilized to separate particles in lateral distance. At the entrance of the channel, particles are introduced at the center by focusing the flow from a center channel with flow from two side channels. Downstream, large particles will occupy a lateral equilibrium position in shorter axial distance than small particles. At the exit of the channel, flow containing large particles is separated from flow containing small particles. It is shown that 10.2-μm diameter microspheres can be separated from 3.0-μm diameter microspheres with a separation efficiency of 69–78% and a throughput in the order of 2 ·10⁴ particles per minute. Computational Fluid Dynamics (CFD) calculations were done to calculate flow fields and verify theoretical particle trajectories. Theory underlying this research shows that higher separation efficiencies for very specific diameter cut-off are possible. This microfluidic channel design has a simple structure and can operate without external forces which makes it feasible for lab-on-a-chip (LOC) applications.     

有核红细胞全血分选微管芯片实验研究

目的 设计微流控芯片分离全血中胎儿有核红细胞（fetal nucleated red blood cell, fNRBCs）,实现有核红细胞的快速便捷获取。方法 利用有核细胞在血流作用下的边集效应以及细胞抗原抗体特异性黏附特点,设计微管流控芯片,分离全血中的fNRBCs。以脐血全血为例,通过免疫荧光计数,分析不同剪切率对fNRBCs富集效果的影响。结果 相较于简单静置黏附,增加剪切率可以增加直微管黏附有核红细胞的数量,细胞的富集效果随血流剪切率的增大先增大后减小。结论 利用直微管能够实现fNRBCs的全血快速有效捕获。研究结果 为无创产前诊断的发展以及胎儿细胞转移机制的探究提供实验参考。