Use of a virtual wall valve in polydimethylsiloxane microfluidic devices for bioanalytical applications

A simple method for micromanipulation of liquids and∕or small groups of cells is presented in this study. Microfabricated sieving structures composed of PDMS (polydimethylsiloxane) were used to segregate aqueous solutions. This microfluidic valving scheme was an application of Cassie-Baxter wetting and was termed "virtual walls" as a nonsolid barrier exists at an air∕water interface. The manipulation of the virtual-air-wall valve was accomplished by controlling the strength of surface-tension and hydrostatic-pressure forces. Virtual walls with a range of feature sizes were designed and characterized by monitoring air and water displacement in response to hydrostatic pressure. Thresholds for the virtual-air-wall valves to be turned on or off were quantified. The walls could also be formed or dissipated by the focused microbeam of a pulsed laser. As an illustration of the virtual wall utility, a series of microfluidic applications were demonstrated. First, the capability of virtual walls to temporarily segregate liquids was integrated into a device utilized to establish a chemical gradient. In a second application, the arraying of nonadherent cells within individual aqueous cavities created by the virtual walls was demonstrated. Individual cells were also released from the cavities on demand using a focused microbeam. The virtual walls were simple and easy-to-fabricate without the requirement for surface treatment or precision alignment, and should find usage in bioanalytical applications.     

Effects of hydraulic pressure on cardiomyoblasts in a microfluidic device

We employed a microfluidic device to study the effects of hydraulic pressure on cardiomyoblast H9c2. The 170 mm Hg pressure increased the cellular area and the expression of atrial natriuretic peptide. With the same device, we demonstrated that the effects of hydraulic pressure on the cardiomyoblast could be reduced by the inhibitor of focal adhesion kinase. This mechanical–chemical antagonism could lead to a potential therapeutic strategy of hypertension-induced cardiac hypertrophy.     

Hydrodynamic self-focusing in a parallel microfluidic device through cross-filtration

The flow focusing is a fundamental prior step in order to sort, analyze, and detect particles or cells. The standard hydrodynamic approach requires two fluids to be injected into the microfluidic device: one containing the sample and the other one, called the sheath fluid, allows squeezing the sample fluid into a narrow stream. The major drawback of this approach is the high complexity of the layout for microfluidic devices when parallel streams are required. In this work, we present a novel parallelized microfluidic device that enables hydrodynamic focusing in each microchannel using a single feed flow. At each of the parallel channels, a cross-filter region is present that allows removing fluid from the sample fluid. This fluid is used to create local sheath fluids that hydrodynamically pinch the sample fluid. The great advantage of the proposed device is that, since only one inlet is needed, multiple parallel micro-channels can be easily introduced into the design. In the paper, the design method is described and the numerical simulations performed to define the optimal design are summarized. Moreover, the operational functionality of devices tested by using both polystyrene beads and Acute Lymphoid Leukemia cells are shown.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

Gold nanoparticle-assisted single base-pair mismatch discrimination on a microfluidic microarray device

Two simple gold nanoparticle (GNP)-based DNA analysis methods using a microfluidic device are presented. In the first method, probe DNA molecules are immobilized on the surface of a self-assembled submonolayer of GNPs. The hybridization efficiency of the target oligonulceotides was improved due to nanoscale spacing between probe molecules. In the second method, target DNA molecules, oligonulceotides or polymerase chain reaction (PCR) amplicons, are first bound to GNPs and then hybridized to the immobilized probe DNA on a glass slide. With the aid of GNPs, we have successfully discriminated, at room temperature, between two PCR amplicons (derived from closely related fungal pathogens, Botrytis cinerea and Botrytis squamosa) with one base-pair difference. DNA analysis on the microfluidic chip avoids the use of large sample volumes, and only a small amount of oligonucelotides (8 fmol) or PCR products (3 ng), was needed in the experiment. The whole procedure was accomplished at room temperature in 1 h, and apparatus for high temperature stringency was not required.     

Simulation guided design of a microfluidic device for electrophoretic stretching of DNA

We have used Brownian dynamics-finite element method (BD-FEM) to guide the optimization of a microfluidic device designed to stretch DNA for gene mapping. The original design was proposed in our previous study [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011)] for demonstrating a new pre-conditioning strategy to facilitate DNA stretching through a microcontraction using electrophoresis. In this study, we examine the efficiency of the original device for stretching DNA with different sizes ranging from 48.5 kbp (λ-DNA) to 166 kbp (T4-DNA). The efficiency of the device is found to deteriorate with increasing DNA molecular weight. The cause of the efficiency loss is determined by BD-FEM, and a modified design is proposed by drawing an analogy between an electric field and a potential flow. The modified device does not only regain the efficiency for stretching large DNA but also outperforms the original device for stretching small DNA.     

Rapid separation of bacteriorhodopsin using a laminar-flow extraction system in a microfluidic device

A protein separation technology using the microfluidic device was developed for the more rapid and effective analysis of target protein. This microfluidic separation system was carried out using the aqueous two-phase system (ATPS) and the ionic liquid two-phase system (ILTPS) for purification method of the protein sample, and the three-flow desalting system was used for the removal of salts from the sucrose-rich sample. Partitioning of the protein sample was observed in ATPS or ILTPS with the various pHs. The microdialysis system was applied to remove small molecules, such as sucrose and salts in the microfluidic channel with the different flow rates of buffer phase. A complex purification method, which combines microdialysis and ATPS or ILTPS, was carried out for the effective purification of bacteriorhodopsin (BR) from the purple membrane of Halobacterium salinarium, which was then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and matrix-assisted laser desorption∕ionization time-of-flight. Furthermore, we were able to make a stable three-phase flow controlling the flow rate in the microfluidic channel. Our complex purification methods were successful in purifying and recovering the BR to its required value.     

A microfluidic device for simultaneous electrical and mechanical measurements on single cells

This paper presents a microfluidic device for simultaneous mechanical and electrical characterization of single cells. The device performs two types of cellular characterization (impedance spectroscopy and micropipette aspiration) on a single chip to enable cell electrical and mechanical characterization. To investigate the performance of the device design, electrical and mechanical properties of MC-3T3 osteoblast cells were measured. Based on electrical models, membrane capacitance of MC-3T3 cells was determined to be 3.39±1.23 and 2.99±0.82 pF at the aspiration pressure of 50 and 100 Pa, respectively. Cytoplasm resistance values were 110.1±37.7 kΩ (50 Pa) and 145.2±44.3 kΩ (100 Pa). Aspiration length of cells was found to be 0.813±0.351 μm at 50 Pa and 1.771±0.623 μm at 100 Pa. Quantified Young's modulus values were 377±189 Pa at 50 Pa and 344±156 Pa at 100 Pa. Experimental results demonstrate the device's capability for characterizing both electrical and mechanical properties of single cells.     

Assessment of the inhibition of Dengue virus infection by carrageenan via real-time monitoring of cellular oxygen consumption rates within a microfluidic device

A microfluidic device combined with a light modulation system was developed to assess the inhibitory effect of carrageenan on Dengue virus (DENV) infection via real-time monitoring of cellular oxygen consumption rates (OCRs). Measuring cellular OCRs, which can reflect cellular metabolic activity, enabled us to monitor the process of viral infection in real time and to rapidly determine the antiviral activity of potential drugs/chemical compounds. The time variation of the cellular OCR of single cells that were infected in situ by DENV at different multiplicity of infection (m.o.i.) values was first successfully measured within a microfluidic device. The influence of the timing of carrageenan treatment on DENV infection was then examined by real-time monitoring of cellular OCRs in three groups. Cells that were pre-treated with carrageenan and then infected with DENV served as a pre-treatment group, cells to which carrageenan was added simultaneously with DENV served as a virucide group, and cells that were pre-infected with DENV and then treated with carrageenan served as a post-treatment group. By monitoring cellular OCRs, we could rapidly evaluate the inhibitory effect of carrageenan on DENV infection, obtaining a result within 7 h and showing that carrageenan had strong and effective anti-DENV activity in the three groups. In particular, a strong inhibitory effect was observed in the virucide group. Moreover, once the virus enters host cells in the post-treatment group, the immediate treatment with carrageenan for the infected cells has higher efficiency of antiviral activity. Our proposed platform enables to perform time-course or dose-response measurements of changes in cellular metabolic activity caused by diseases, chemical compounds, and drugs via monitoring of the cellular OCR, with rapid and real-time detection. This approach provides the potential to study a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.     

Bile canaliculi formation by aligning rat primary hepatocytes in a microfluidic device

In this study, we propose a microfluidic cell culture device mimicking the microscopic structure in liver tissue called hepatic cords. The cell culture area of the device was designed to align hepatocytes in two lines in a similar way to hepatic cords. Thanks to the structural design together with a cell seeding procedure, rat primary hepatocytes were successfully aligned in two lines and cultured under perfusion condition. It is shown that aligned hepatocytes gradually self-organize and form bile canaliculi along the hepatic cord-like structure. The present technique to culture hepatocytes with functional bile canaliculi could be used as an alternative to animal testing in the field of drug discovery and toxicological studies, and also be beneficial to tissue engineering applications.     

An inexpensive microfluidic device for three-dimensional hydrodynamic focusing in imaging flow cytometry

We present design, characterization, and testing of an inexpensive, sheath-flow based microfluidic device for three-dimensional (3D) hydrodynamic focusing of cells in imaging flow cytometry. In contrast to other 3D sheathing devices, our device hydrodynamically focuses the cells in a single-file near the bottom wall of the microchannel that allows imaging cells with high magnification and low working distance objectives, without the need for small device dimensions. The relatively large dimensions of the microchannels enable easy fabrication using less-precise fabrication techniques, and the simplicity of the device design avoids the need for tedious alignment of various layers. We have characterized the performance of the device with 3D numerical simulations and validated these simulations with experiments of hydrodynamic focusing of a fluorescently dyed sample fluid. The simulations show that the width and the height of the 3D focused sample stream can be controlled independently by varying the heights of main and side channels of the device, and the flow rates of sample and sheath fluids. Based on simulations, we also provide useful guidelines for choosing the device dimensions and flow rates for focusing cells of a particular size. Thereafter, we demonstrate the applicability of our device for imaging a large number of RBCs using brightfield microscopy. We also discuss the choice of the region of interest and camera frame rate so as to image each cell individually in our device. The design of our microfluidic device makes it equally applicable for imaging cells of different sizes using various other imaging techniques such as phase-contrast and fluorescence microscopy.     

A microfluidic platform for real-time and in situ monitoring of virus infection process

Microfluidic chip is a promising platform for studying virus behaviors at the cell level. However, only a few chip-based studies on virus infection have been reported. Here, a three-layer microfluidic chip with low shear stress was designed to monitor the infection process of a recombinant Pseudorabies virus (GFP-PrV) in real time and in situ, which could express green fluorescent protein during the genome replication. The infection and proliferation characteristics of GFP-PrV were measured by monitoring the fluorescence intensity of GFP and determining the one-step growth curve. It was found that the infection behaviors of GFP-PrV in the host cells could hardly be influenced by the microenvironment in the microfluidic chip. Furthermore, the results of drug inhibition assays on the microfluidic chip with a tree-like concentration gradient generator showed that one of the infection pathways of GFP-PrV in the host cells was microtubule-dependent. This work established a promising microfluidic platform for the research on virus infection.     

Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.     

Hybrid capillary-inserted microfluidic device for sheathless particle focusing and separation in viscoelastic flow

A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.     

Covalently immobilized biomolecule gradient on hydrogel surface using a gradient generating microfluidic device for a quantitative mesenchymal stem cell study

Precisely controlling the spatial distribution of biomolecules on biomaterial surface is important for directing cellular activities in the controlled cell microenvironment. This paper describes a polydimethylsiloxane (PDMS) gradient-generating microfluidic device to immobilize the gradient of cellular adhesive Arg-Gly-Asp (RGD) peptide on poly (ethylene glycol) (PEG) hydrogel. Hydrogels are formed by exposing the mixture of PEG diacrylate (PEGDA), acryloyl-PEG-RGD, and photo-initiator with ultraviolet light. The microfluidic chip was simulated by a fluid dynamic model for the biomolecule diffusion process and gradient generation. PEG hydrogel covalently immobilized with RGD peptide gradient was fabricated in this microfluidic device by photo-polymerization. Bone marrow derived rat mesenchymal stem cells (MSCs) were then cultured on the surface of RGD gradient PEG hydrogel. Cell adhesion of rat MSCs on PEG hydrogel with various RGD gradients were then qualitatively and quantitatively analyzed by immunostaining method. MSCs cultured on PEG hydrogel surface with RGD gradient showed a grated fashion for cell adhesion and spreading that was proportional to RGD concentration. It was also found that 0.107–0.143 mM was the critical RGD concentration range for MSCs maximum adhesion on PEG hydrogel.     

Fabrication of a gel particle array in a microfluidic device for bioassays of protein and glucose in human urine samples

This paper describes a simple method for fabricating a series of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures inside microfluidic channels as probe for proteins and glucose. In order to demonstrate the feasibility of this newly developed system, bovine serum albumin (BSA) was chosen as a model protein. PEG microcolumns were used for the parallel detection of multiple components. Using tetrabromophenol blue (TBPB) and the horseradish peroxidase/glucose oxidase reaction system, bovine serum albumin (BSA) and glucose in human urine were detected by color changes. The color changes for BSA within a concentration range of 1-150 μM, and glucose within a range of 50 mM-2 M could be directly distinguished by eyes or precisely identified by optical microscope. To show the practicability of the gel particle array, protein and glucose concentrations of real human urine samples were determined, resulting in a good correlation with hospital analysis. Notably, only a 5 μL sample was needed for a parallel measurement of both analytes. Conveniently, no special readout equipment or power source was required during the diagnosis process, which is promising for an application in rapid point-of-care diagnosis.     

Release monitoring of single cells on a microfluidic device coupled with fluorescence microscopy and electrochemistry

A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis.     

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 μM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.     

Continuous removal of glycerol from frozen-thawed red blood cells in a microfluidic membrane device

Cryopreservation of human red blood cells (RBCs) in the presence of 40% glycerol allows a shelf-life of 10 years, as opposed to only 6 weeks for refrigerated RBCs. Nonetheless, cryopreserved blood is rarely used in clinical therapy, in part because of the requirement for a time-consuming (∼1 h) post-thaw wash process to remove glycerol before the product can be used for transfusion. The current deglycerolization process involves a series of saline washes in an automated centrifuge, which gradually removes glycerol from the cells in order to prevent osmotic damage. We recently demonstrated that glycerol can be extracted in as little as 3 min without excessive osmotic damage if the composition of the extracellular solution is precisely controlled. Here, we explore the potential for carrying out rapid glycerol extraction using a membrane-based microfluidic device, with the ultimate goal of enabling inline washing of cryopreserved blood. To assist in experimental design and device optimization, we developed a mass transfer model that allows prediction of glycerol removal, as well as the resulting cell volume changes. Experimental measurements of solution composition and hemolysis at the device outlet are in reasonable agreement with model predictions, and our results demonstrate that it is possible to reduce the glycerol concentration by more than 50% in a single device without excessive hemolysis. Based on these promising results, we present a design for a multistage process that is predicted to safely remove glycerol from cryopreserved blood in less than 3 min.     

Handling of artificial membranes using electrowetting-actuated droplets on a microfluidic device combined with integrated pA-measurements

Artificial membranes, as a controllable environment, are an essential tool to study membrane proteins. Electrophysiology provides information about the ion transport mechanism across a membrane at the single-protein level. Unfortunately, high-throughput studies and screening are not accessible to electrophysiology because it is a set of not automated and technically delicate methods. Therefore, it is necessary to automate and parallelize electrophysiology measurement in artificial membranes. Here, we present a first step toward this goal: the fabrication and characterization of a microfluidic device integrating electrophysiology measurements and the handling of an artificial membrane which includes its formation, its displacement and the separation of its leaflets using electrowetting actuation of sub-μL droplets. To validate this device, we recorded the insertion of a model porin, α-hemolysin.