On-chip microfluidic biosensor using superparamagnetic microparticles

In this paper, an integrated solution towards an on-chip microfluidic biosensor using the magnetically induced motion of functionalized superparamagnetic microparticles (SMPs) is presented. The concept of the proposed method is that the induced velocity on SMPs in suspension, while imposed to a magnetic field gradient, is inversely proportional to their volume. Specifically, a velocity variation of suspended functionalized SMPs inside a detection microchannel with respect to a reference velocity, specified in a parallel reference microchannel, indicates an increase in their non-magnetic volume. This volumetric increase of the SMPs is caused by the binding of organic compounds (e.g., biomolecules) to their functionalized surface. The new compounds with the increased non-magnetic volume are called loaded SMPs (LSMPs). The magnetic force required for the manipulation of the SMPs and LSMPs is produced by current currying conducting microstructures, driven by a programmable microcontroller. Experiments were carried out as a proof of concept. A promising decrease in the velocity of the LSMPs in comparison to that of the SMPs was measured. Thus, it is the velocity variation which determines the presence of the organic compounds in the sample fluid.     

On-chip antibody immobilization for on-demand and rapid immunoassay on a microfluidic chip

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.     

On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips containing 32 porous flow channels of 235 μm depth and 25 μm width and (ii) polystyrene Poros™ beads with a particle size of 20 μm. The immobilized enzymes recycle L-glutamate by oxidation to 2-oxoglutarate followed by the transfer of an amino group from D-4-hydroxyphenylglycine to 2-oxoglutarate. The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD⁺) to NADH, which was monitored by fluorescence detection (ε_ex=340 nm, ε_em=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1–50.0 mM. Second, to be automatically determined, sequential injection analysis (SIA) with the bead-based system was investigated. The bead-based system was evaluated by both flow injection analysis and SIA modes, where good reproducibility for L-glutamate calibrations was obtained (relative standard deviation of 3.3% and 6.6%, respectively). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining. The bead-based system demonstrated high selectivity, where L-glutamate recoveries were between 91% and 108% in the presence of six other L-amino acids tested.     

A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia

Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the host''s vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells.     

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.     

Optimizing design and fabrication of microfluidic devices for cell cultures: An effective approach to control cell microenvironment in three dimensions

The effects of gradients of bioactive molecules on the cell microenvironment are crucial in several biological processes, such as chemotaxis, angiogenesis, and tumor progression. The elucidation of the basic mechanisms regulating cell responses to gradients requires a tight control of the spatio-temporal features of such gradients. Microfluidics integrating 3D gels are useful tools to fulfill this requirement. However, even tiny flaws in the design or in the fabrication process may severely impair microenvironmental control, thus leading to inconsistent results. Here, we report a sequence of actions aimed at the design and fabrication of a reliable and robust microfluidic device integrated with collagen gel for cell culturing in 3D, subjected to a predetermined gradient of biomolecular signals. In particular, we developed a simple and effective solution to the frequently occurring technical problems of gas bubble formation and 3D matrix collapsing or detaching from the walls. The device here proposed, in Polydimethylsiloxane, was designed to improve the stability of the cell-laden hydrogel, where bubble deprived conditioning media flow laterally to the gel. We report the correct procedure to fill the device with the cell populated gel avoiding the entrapment of gas bubbles, yet maintaining cell viability. Numerical simulations and experiments with fluorescent probes demonstrated the establishment and stability of a concentration gradient across the gel. Finally, chemotaxis experiments of human Mesenchymal Stem Cells under the effects of Bone Morphogenetic Protein-2 gradients were performed in order to demonstrate the efficacy of the system in controlling cell microenvironment. The proposed procedure is sufficiently versatile and simple to be used also for different device geometries or experimental setups.     

Membrane-integrated microfluidic device for high-resolution live cell imaging

The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane     

A novel microfluidic platform for studying mammalian cell chemotaxis in different oxygen environments under zero-flow conditions

The cell''s micro-environment plays an important role in various physiological and pathological phenomena. To better investigate in vivo cellular behaviors, researchers have expended great effort in building controlled in vitro biophysical and biochemical environments. Because a cell''s gaseous environment affects properties such as its division, metastasis, and differentiation, we developed a zero-flow based platform for studying mammalian cell chemotaxis behavior in different oxygen environments. This platform can construct a linear range of oxygen tensions within one chip (i.e., from 1.4% to 3.6% or 5.5% to 14.5%). To study cell chemotaxis behavior under varying oxygen environments, the chemical gradient direction is established perpendicularly to oxygen change within an observation area. Because the observation area is not subject to flow, shear force is of no concern. In addition, water flow around the cell chambers greatly reduces evaporation and makes long-term microscope imaging possible. In this study, we precisely measure the chemotaxis velocity of MCF-7 human breast cancer cells under different oxygen tension conditions towards CXCL12, which is a stromal cell-derived factor. We find that cell migration rates are not equivalent, even under two close oxygen tensions. We also observed that cells move faster towards high concentrations of chemoattractant when the oxygen tension is below 3% due to the increased expression of HIF-1 (hypoxia-inducible factor 1), which promotes a transition to the amoeboid rather than mesenchymal mode of movement. Our experiments demonstrate that this new microfluidic platform is useful for the quantitative study of mammalian cell chemotaxis under different oxygen conditions in the absence of shear force. We also shed light on the study of chemotaxis under other gaseous environments.     

On-chip microelectrode impedance analysis of mammalian cell viability during biomanufacturing

The characterization of cell viability is a challenging task in applied biotechnology, as no clear definition of cell death exists. Cell death is accompanied with a change in the electrical properties of the membrane as well as the cell interior. Therefore, changes in the physiology of cells can be characterized by monitoring of their dielectric properties. We correlated the dielectric properties of industrially used mammalian cells, sedimented over interdigitated microelectrodes, to the AC signal response across the chip. The voltage waveforms across the electrodes were processed to obtain the circuit impedance, which was used to quantify the changes in cell viability. We observed an initial decrease in impedance, after which it remained nearly constant. The results were compared with data from the dye exclusion viability test, the cell specific oxygen uptake rate, and the online viable cell density data from capacitance probes. The microelectrode technique was found to be sensitive to physiological changes taking place inside the cells before their membrane integrity is compromised. Such accurate determination of the metabolic status during this initial period, which turned out to be less well captured in the dye exclusion tests, may be essential for several biotechnology operations.     

Gravity-oriented microfluidic device for uniform and massive cell spheroid formation

We propose a simple method for forming massive and uniform three-dimensional (3-D) cell spheroids in a multi-level structured microfluidic device by gravitational force. The concept of orienting the device vertically has allowed spheroid formation, long-term perfusion, and retrieval of the cultured spheroids by user-friendly standard pipetting. We have successfully formed, perfused, and retrieved uniform, size-controllable, well-conditioned spheroids of human embryonic kidney 293 cells (HEK 293) in the gravity-oriented microfluidic device. We expect the proposed method will be a useful tool to study in-vitro 3-D cell models for the proliferation, differentiation, and metabolism of embryoid bodies or tumours.     

A microfluidic chip for highly efficient cell capturing and pairing

This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.     

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate^TM (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte''s functions, Matrigel^TM-embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes'' responses to external stimuli.     

On-chip actuation transmitter for enhancing the dynamic response of cell manipulation using a macro-scale pump

An on-chip actuation transmitter for achieving fast and accurate cell manipulation is proposed. Instead of manipulating cell position by a directly connected macro-scale pump, polydimethylsiloxane deformation is used as a medium to transmit the actuation generated from the pump to control the cell position. This actuation transmitter has three main advantages. First, the dynamic response of cell manipulation is faster than the conventional method with direct flow control based on both the theoretical modeling and experimental results. The cell can be manipulated in a simple harmonic motion up to 130 Hz by the proposed actuation transmitter as opposed to 90 Hz by direct flow control. Second, there is no need to fill the syringe pump with the sample solution because the actuation transmitter physically separates the fluids between the pump and the cell flow, and consequently, only a very small quantity of the sample is required (<1 μl). In addition, such fluid separation makes it easy to keep the experiment platform sterilized because there is no direct fluid exchange between the sample and fluid inside the pump. Third, the fabrication process is simple because of the single-layer design, making it convenient to implement the actuation transmitter in different microfluidic applications. The proposed actuation transmitter is implemented in a lab-on-a-chip system for red blood cell (RBC) evaluation, where the extensibility of red blood cells is evaluated by manipulating the cells through a constriction channel at a constant velocity. The application shows a successful example of implementing the proposed transmitter.     

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.     

Streamline based design guideline for deterministic microfluidic hydrodynamic single cell traps

A prerequisite for single cell study is the capture and isolation of individual cells. In microfluidic devices, cell capture is often achieved by means of trapping. While many microfluidic trapping techniques exist, hydrodynamic methods are particularly attractive due to their simplicity and scalability. However, current design guidelines for single cell hydrodynamic traps predominantly rely on flow resistance manipulation or qualitative streamline analysis without considering the target particle size. This lack of quantitative design criteria from first principles often leads to non-optimal probabilistic trapping. In this work, we describe an analytical design guideline for deterministic single cell hydrodynamic trapping through the optimization of streamline distributions under laminar flow with cell size as a key parameter. Using this guideline, we demonstrate an example design which can achieve 100% capture efficiency for a given particle size. Finite element modelling was used to determine the design parameters necessary for optimal trapping. The simulation results were subsequently confirmed with on-chip microbead and white blood cell trapping experiments.     

A laminar flow microfluidic fuel cell for detection of hexavalent chromium concentration

An electrochemical hexavalent chromium concentration sensor based on a microfluidic fuel cell is presented. The correlation between current density and chromium concentration is established in this report. Three related operation parameters are investigated, including pH values, temperature, and external resistance on the sensor performance. The results show that the current density increases with increasing temperature and the sensor produces a maximum regression coefficient at the catholyte pH value of 1.0. Moreover, it is found that the external resistance has a great influence on the linearity and current densities of the microfluidic sensor. Owing to the membraneless structure and the steady co-laminar flow inside the microchannel, the microfluidic sensor exhibits short response time to hexavalent chromium concentration. The laminar flow fuel cell sensor provides a new and simple method for detecting hexavalent chromium concentration in the industrial wastewater.     

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.     

A microfluidic device enabling high-efficiency single cell trapping

Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm² scale area, as a promising tool to pattern large-scale single cells on specific substrates and facilitate on-chip cellular assay at the single cell level.     

Manufacturing and wetting low-cost microfluidic cell separation devices

Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min⁻¹ while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm.     

Vascular smooth muscle cell culture in microfluidic devices

This paper presents a microfluidic device enabling culture of vascular smooth muscle cells (VSMCs) where extracellular matrix coating, VSMC seeding, culture, and immunostaining are demonstrated in a tubing-free manner. By optimizing droplet volume differences between inlets and outlets of micro channels, VSMCs were evenly seeded into microfluidic devices. Furthermore, the effects of extracellular matrix (e.g., collagen, poly-l-Lysine (PLL), and fibronectin) on VSMC proliferation and phenotype expression were explored. As a platform technology, this microfluidic device may function as a new VSMC culture model enabling VSMC studies.