Showing papers in "Lab on a Chip in 2009"
TL;DR: A theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells, and all of the recovered cells were the active strain.
Abstract: We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s−1. To validate the system, mixtures of E. colicells, expressing either the reporter enzyme β-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of ∼300 droplets s−1. The false positive error rate of the sorter at this throughput was <1 in 104 droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. colicells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density (∼1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.
911 citations
TL;DR: A spiral lab-on-a-chip (LOC) for size-dependent focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture is demonstrated for the first time.
Abstract: In this work we report on a simple inertial microfluidic device that achieves continuous multi-particle separation using the principle of Dean-coupled inertial migration in spiral microchannels. The dominant inertial forces coupled with the Dean rotational force due to the curvilinear microchannel geometry cause particles to occupy a single equilibrium position near the inner microchannel wall. The position at which particles equilibrate is dependent on the ratio of the inertial lift to Dean drag forces. Using this concept, we demonstrate, for the first time, a spiral lab-on-a-chip (LOC) for size-dependant focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture. The individual particle streams can be collected with an appropriately designed outlet system. To demonstrate this principle, a 5-loop Archimedean spiral microchannel with a fixed width of 500 µm and a height of 130 µm was used to simultaneously and continuously separate 10 µm, 15 µm, and 20 µm polystyrene particles. The device exhibited 90% separation efficiency. The versatility of the device was demonstrated by separating neuroblastoma and glioma cells with 80% efficiency and high relative viability (>90%). The achieved throughput of ∼1 million cells/min is substantially higher than the sorting rates reported by other microscale sorting methods and is comparable to the rates obtained with commercial macroscale flow cytomerty techniques. The simple planar structure and high throughput offered by this passive microfluidic approach make it attractive for LOC devices in biomedical and environmental applications.
650 citations
TL;DR: In this paper, an active patterning technique named acoustic tweezers is presented that utilizes standing surface acoustic wave (SSAW) to manipulate and pattern cells and microparticles.
Abstract: Here we present an active patterning technique named “acoustic tweezers” that utilizes standing surface acoustic wave (SSAW) to manipulate and pattern cells and microparticles. This technique is capable of patterning cells and microparticles regardless of shape, size, charge or polarity. Its power intensity, approximately 5 × 105 times lower than that of optical tweezers, compares favorably with those of other active patterning methods. Flow cytometry studies have revealed it to be non-invasive. The aforementioned advantages, along with this technique's simple design and ability to be miniaturized, render the “acoustic tweezers” technique a promising tool for various applications in biology, chemistry, engineering, and materials science.
612 citations
TL;DR: Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules from serum-containing media into the polymer bulk.
Abstract: Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e.estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable viaMALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogenvia ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.
611 citations
TL;DR: A new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition is developed and demonstrated to study cellular morphogenesis both qualitatively and quantitatively.
Abstract: Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.
512 citations
TL;DR: The particle separation method presented here is simple and versatile, capable of separating virtually all kinds of particles (regardless of charge/polarization or optical properties) with high separation efficiency and low power consumption.
Abstract: This work introduces a method of continuous particle separation through standing surface acoustic wave (SSAW)-induced acoustophoresis in a microfluidic channel. Using this SSAW-based method, particles in a continous laminar flow can be separated based on their volume, density and compressibility. In this work, a mixture of particles of equal density but dissimilar volumes was injected into a microchannel through two side inlets, sandwiching a deonized water sheath flow injected through a central inlet. A one-dimensional SSAW generated by two parallel interdigital transducers (IDTs) was established across the channel, with the channel spanning a single SSAW pressure node located at the channel center. Application of the SSAW induced larger axial acoustic forces on the particles of larger volume, repositioning them closer to the wave pressure node at the center of the channel. Thus particles were laterally moved to different regions of the channel cross-section based on particle volume. The particle separation method presented here is simple and versatile, capable of separating virtually all kinds of particles (regardless of charge/polarization or optical properties) with high separation efficiency and low power consumption.
480 citations
TL;DR: Basic trends and aspects of microsystems as they relate to continuous-flow microchemical synthesis are reviewed and the major challenges and outlook related to these topics are summarized.
Abstract: Microchemical systems have evolved rapidly over the last decade with extensive chemistry applications. Such systems enable discovery and development of synthetic routes while simultaneously providing increased understanding of underlying pathways and kinetics. We review basic trends and aspects of microsystems as they relate to continuous-flow microchemical synthesis. Key literature reviews are summarized and principles governing different microchemical operations discussed. Current trends and limitations of microfabrication, micromixing, chemical synthesis in microreactors, continuous-flow separations, multi-step synthesis, and integration of analytics are delineated. We conclude by summarizing the major challenges and outlook related to these topics.
469 citations
TL;DR: A microfluidic single cell impedance cytometer that performs a white blood cell differential count and showed 95% correlation against commercial blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.
Abstract: Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.
423 citations
TL;DR: Comparison of experimental results demonstrated that the microCCA was able to reproduce the metabolism of Tegafur to 5-FU in the liver and consequent death of cells by5-FU, while the cultures in a 96-well microtiter plate were unable to do so.
Abstract: A microfluidic device with 3-D hydrogel cell cultures has been developed to test the cytotoxicity of anti-cancer drugs while reproducing multi-organ interactions. In this device, a micro cell culture analog (µCCA), cells embedded in 3-D hydrogels are cultured in separate chambers representing the liver, tumor, and marrow, which are connected by channels mimicking blood flow. While the microfluidic network provides a platform for mimicking the pharmacokinetic and pharmacodynamic profiles of a drug in humans, the 3-D hydrogel provides a more physiologically realistic environment to mimic the tissue than monolayer culture. Colon cancer cells (HCT-116) and hepatoma cells (HepG2/C3A) were encapsulated in Matrigel and cultured in the tumor and the liver chamber in a µCCA, respectively. Myeloblasts (Kasumi-1) were encapsulated in alginate in the marrow chamber; a stiffer hydrogel was necessary to prevent cell migration out of the matrix. The cytotoxic effect of Tegafur, an oral prodrug of 5-fluorouracil (5-FU), on each cell line was tested using the µCCA with cell-embedded hydrogel. The comparison of experimental results using a 96-well microtiter plate and a µCCA demonstrated that the µCCA was able to reproduce the metabolism of Tegafur to 5-FU in the liver and consequent death of cells by 5-FU, while the cultures in a 96-well microtiter plate were unable to do so. The µCCA utilizing 3-D hydrogel cell cultures has potential as a platform for pharmacokinetic-based drug screening in a more physiologically realistic environment.
412 citations
TL;DR: A microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity and its values correlate well with the reported in vivo LD(50) values.
Abstract: We have developed a microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity. The 3D HepaTox Chip is based on multiplexed microfluidic channels where a 3D microenvironment is engineered in each channel to maintain the hepatocytes' synthetic and metabolic functions. The multiplexed channels allow for simultaneous administration of multiple drug doses to functional primary hepatocytes while an incorporated concentration gradient generator enables the in vitro dose-dependent drug responses to predict in vivo hepatotoxicity. The IC50 values of 5 model drugs derived from the dose-dependent on-chip testing correlate well with the reported in vivo LD50 values. The 3D HepaTox Chip can be integrated with on-chip sensors and actuators as the next generation cell-based on-chip drug testing platform.
392 citations
TL;DR: This method allows individual drops to be directed along separate microchannel paths at high volume flow rates, which is useful for droplet sorting.
Abstract: We direct the motion of droplets in microfluidic channels using a surface acoustic wave device. This method allows individual drops to be directed along separate microchannel paths at high volume flow rates, which is useful for droplet sorting.
TL;DR: This review discusses the state of the art of the optical whole-field velocity measurement technique micro-scale Particle Image Velocimetry (microPIV), a useful tool for fundamental research of microfluidic applications in life science, lab-on-a-chip, biomedical research, micro chemical engineering, analytical chemistry and other related fields of research.
Abstract: In this review we discuss the state of the art of the optical whole-field velocity measurement technique micro-scale Particle Image Velocimetry (µPIV). µPIV is a useful tool for fundamental research of microfluidics as well as for the detailed characterization and optimization of microfluidic applications in life science, lab-on-a-chip, biomedical research, micro chemical engineering, analytical chemistry and other related fields of research. An in depth description of the µPIV method is presented and compared to other flow visualization and measurement methods. An overview of the most relevant applications is given on the topics of near-wall flow, electrokinetic flow, biological flow, mixing, two-phase flow, turbulence transition and complex fluid dynamic problems. Current trends and applications are critically reviewed. Guidelines for the implementation and application are also discussed.
TL;DR: A novel microfluidic device is described that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps and allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment.
Abstract: Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell–cell and cell–matrix interactions.
TL;DR: The design, fabrication and use of a single-layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in an array format controlled entirely by liquid flow is described.
Abstract: We describe the design, fabrication and use of a single-layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in an array format controlled entirely by liquid flow. Aqueous picoliter droplets are trapped en masse and optically monitored for extended periods of time. Such an array-based approach is used to characterize droplet shrinkage, aggregation of encapsulated E. colicells and enzymatic reactions. We also demonstrate that trapped droplets may be recovered from the microfluidic array for further processing.
TL;DR: Due to this technique's simple design, excellent mixing performance, and fast mixing speed (a few milliseconds), this single-bubble-based acoustic micromixer may prove useful for many biochemical studies and applications.
Abstract: We present ultra-fast homogeneous mixing inside a microfluidic channelvia single-bubble-based acoustic streaming. The device operates by trapping an air bubble within a “horse-shoe” structure located between two laminar flows inside a microchannel. Acoustic waves excite the trapped air bubble at its resonance frequency, resulting in acoustic streaming, which disrupts the laminar flows and triggers the two fluids to mix. Due to this technique's simple design, excellent mixing performance, and fast mixing speed (a few milliseconds), our single-bubble-based acoustic micromixer may prove useful for many biochemical studies and applications.
TL;DR: The work presented here may spur the adoption of fluorescence immunoassays using capillary driven microfluidics and PDMS substrates for point-of-care diagnostics.
Abstract: Point-of-care diagnostics will strongly benefit from miniaturization based on microfluidics because microfluidics integrate functions that can together preserve valuable samples and reagents, increase sensitivity of a test, and accelerate mass transport limited reactions. But a main challenge is to incorporate reagents into microfluidics and to make microfluidics simple to use. Here, we integrate microfluidic functional elements, some of which were developed earlier, and reagents such as detection antibodies (dAbs), capture antibodies (cAbs) and analyte molecules for making one-step immunoassays: the integrated device only requires the addition of sample to trigger a cascade of events powered by capillary forces for effecting a sandwich immunoassay that is read using a fluorescence microscope. The microfluidic elements comprise a sample collector, delay valves, flow resistors, a deposition zone for dAbs, a reaction chamber sealed with a polydimethylsiloxane (PDMS) substrate, and a capillary pump and vents. Parameters for depositing 3.6 nL of a solution of dAb on the chip using an inkjet are optimized and the PDMS substrate is patterned with analytes, which provide a positive control, and cAbs. Various storage conditions of the patterned PDMS are investigated for up to 6 months revealing that storage with a desiccant preserved at least 51% of the activity of the cAbs. C-reactive protein (CRP), a general inflammation and cardiac marker, is detected using this one-step chip using only 5 µL of human serum by measuring fluorescent signals from 30 × 100 µm2 areas of the PDMS substrate in the wet reaction chamber. The one-step chip can detect CRP at a concentration of 10 ng mL−1 in less than 3 min and below 1 ng mL−1 within 14 min. The work presented here may spur the adoption of fluorescence immunoassays using capillary driven microfluidics and PDMS substrates for point-of-care diagnostics.
TL;DR: The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation.
Abstract: We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-µFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, the multi-channel 3D-µFCCS was designed for culturing different 3D cellular aggregates simultaneously to mimic multiple organs in the body. Four human cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-β1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-β1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-µFCCS could be potentially used to supplement or even replace animal models in drug screening.
TL;DR: A chip-based electrophysiological platform enabling the study of micro- and macro-circuitry in in-vitro neuronal preparations and how this approach enables neuronal signals to be acquired for investigating neuronal activity from single cells and microcircuits to large scale neuronal networks is presented.
Abstract: This paper presents a chip-based electrophysiological platform enabling the study of micro- and macro-circuitry in in-vitro neuronal preparations. The approach is based on a 64 × 64 microelectrode array device providing extracellular electrophysiological activity recordings with high spatial (21 µm of electrode separation) and temporal resolution (from 0.13 ms for 4096 microelectrodes down to 8 µs for 64 microelectrodes). Applied to in-vitro neuronal preparations, we show how this approach enables neuronal signals to be acquired for investigating neuronal activity from single cells and microcircuits to large scale neuronal networks. The main elements of the platform are the metallic microelectrode array (MEA) implemented in Complementary Metal Oxide Semiconductor (CMOS) technology similar to a light imager, the in-pixel integrated low-noise amplifiers (11 µVrms) and the high-speed random addressing logic. The chip is combined with a real-time acquisition system providing the capability to record at 7.8 kHz/electrode the whole array and to process the acquired signals.
TL;DR: A portable, disc-based, and fully automated enzyme-linked immuno-sorbent assay (ELISA) system is developed to test infectious diseases from whole blood and the operation time was dramatically reduced from over 2 hours to less than 30 minutes.
Abstract: A portable, disc-based, and fully automated enzyme-linked immuno-sorbent assay (ELISA) system is developed to test infectious diseases from whole blood. The innovative laser irradiated ferrowax microvalves and centrifugal microfluidics were utilized for the full integration of microbead-based suspension ELISA assays on a disc starting from whole blood. The concentrations of the antigen and the antibody of Hepatitis B virus (HBV), HBsAg and Anti-HBs respectively, were measured using the lab-on-a-disc (LOD). All the necessary reagents are preloaded on the disc and the total process of the plasma separation, incubation with target specific antigen or antibody coated microbeads, multiple steps of washing, enzyme reaction with substrates, and the absorbance detection could be finished within 30 minutes. Compared to the conventional ELISA, the operation time was dramatically reduced from over 2 hours to less than 30 minutes while the limit of detection was kept similar; e.g. the limit of detection of Anti-HBs tests were 8.6 mIU mL−1 and 10 mIU mL−1 for the disc-based and the conventional ELISA respectively.
TL;DR: In this article, a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array that has ∼2 µm pixel size is recorded.
Abstract: We experimentally illustrate a lensfree holographic imaging platform to perform on-chip cytometry. By controlling the spatial coherence of the illumination source, we record a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array that has ∼2 µm pixel size. The recorded holographic image is then processed by using a custom developed decision algorithm for matching the detected hologram texture to existing library images for on-chip characterization and counting of a heterogeneous solution of interest. The holographic diffraction signature of any microscopic object is significantly different from the classical diffraction pattern of the same object. It improves the signal to noise ratio and the signature uniformity of the cell patterns; and also exhibits much better sensitivity for on-chip imaging of weakly scattering phase objects such as small bacteria or cells. We verify significantly improved performance of this holographic on-chip cytometry approach by automatically characterizing heterogeneous solutions of red blood cells, yeast cells, E. coli and various sized micro-particles without the use of any lenses or microscope objectives. This lensless on-chip holography platform will especially be useful for point-of-care cytometry and diagnostics applications involving e.g., infectious diseases such as HIV or malaria.
TL;DR: A simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter - to picoliter-scale aqueous drops suspended in inert carrier oil enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery.
Abstract: We present a simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter- to picoliter-scale aqueous drops suspended in inert carrier oil. This device enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery.
TL;DR: It is demonstrated that the optomagnetic technology enables high-sensitivity one-step assays in blood serum/plasma and whole saliva and is expected to open a new paradigm in biosensing.
Abstract: The realization of biomolecular detection assays for diagnostic purposes is technologically very challenging because such tests demand full integration for ease of use and need to deliver a high analytical performance with cost-effective use of materials. In this article an optomagnetic immunoassay technology is described based on nanoparticles that are magnetically actuated and optically detected in a stationary sample fluid. The dynamic control of nanoparticles by magnetic fields impacts the key immunoassay process steps, giving unprecedented speed, assay control and seamless integration of the total test. The optical detection yields sensitive and multiplexed assays in a low-cost disposable cartridge. We demonstrate that the optomagnetic technology enables high-sensitivity one-step assays in blood serum/plasma and whole saliva. Drugs of abuse are detected at sub-nanogram per millilitre levels in a total assay time of 1 min, and the cardiac marker troponin I is detected at sub-picomole per litre concentrations in a few minutes. The optomagnetic technology is fundamentally suited for high-performance integrated testing and is expected to open a new paradigm in biosensing.
TL;DR: This work presents a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light.
Abstract: We present a simple and environmentally friendly process for cell patterning on glass covered with an ultrathin layer of poly-L-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light. The patterned substrates are stable for months in the lab atmosphere before incubation with proteins. Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells seeded on fibronectin/fibrinogen–Alexa 488 patterns were constrained for days on the deep UV exposed regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of micro-patterned microplates in 96-well format.
TL;DR: A new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device is developed, robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems.
Abstract: We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behaviour was studied in detail using 9.9 and 1.0 µm particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm2. The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Rep), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 108/mL), using a sample flow rate of up to 18 µL/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.
TL;DR: A new microfluidic method for focusing microparticles through the combined use of inertial lift forces and turbulent secondary flows generated in a topographically patterned microchannel is developed, which offers continuous, high-throughput performance and simple operation.
Abstract: We developed a new microfluidic method for focusing microparticles through the combined use of inertial lift forces and turbulent secondary flows generated in a topographically patterned microchannel. The mechanism of particle focusing is based on the hydrodynamic inertial forces exerted on particles migrating along a non-circular microchannel, i.e.tubular pinch effect and wall effect, which induce particle movement away from walls and along a specific lateral position in the microchannel. With the extraordinary geometry of multi-orifice microchannel, an ordered and focused particle distribution was achieved at central or side regions according to a particle Reynolds number (Rep) range. The focusing of particles was controlled by the particle Reynolds number, microchannel length, and volume fraction of particles in suspension. This method will be beneficial in particle focusing processes in a microfluidic device since it offers continuous, high-throughput performance and simple operation.
TL;DR: It is found that the microfluidic drifting technique can be effectively applied to three-dimensionally focus microparticles with density and size equivalent to those of human CD4+ T lymphocytes.
Abstract: In this work, we demonstrate an on-chip microfluidic flow cytometry system based on a three-dimensional (3D) hydrodynamic focusing technique, microfluidic drifting. By inducing Dean flow in a curved microfluidic channel, microfluidic drifting can be used to hydrodynamically focus cells or particles in the vertical direction and enables the 3D hydrodynamic focusing in a single-layer planar microfluidic device. Through theoretical calculation, numerical simulation, and experimental characterization, we found that the microfluidic drifting technique can be effectively applied to three-dimensionally focus microparticles with density and size equivalent to those of human CD4+ T lymphocytes. In addition, we developed a flow cytometry platform by integrating the 3D focusing device with a laser-induced fluorescence (LIF) detection system. The system was shown to provide effective high-throughput flow cytometry measurements at a rate of greater than 1700 cells s−1.
TL;DR: De deformable particles that are close packed are used to insert a controllable number of particles into every drop to provide a simple, flexible means of efficiently encapsulating a controLLableNumber of particles per drop.
Abstract: Loading drops with discrete objects, such as particles and cells, is often necessary when performing chemical and biological assays in microfluidic devices. However, random loading techniques are inefficient, yielding a majority of empty and unusable drops. We use deformable particles that are close packed to insert a controllable number of particles into every drop. This provides a simple, flexible means of efficiently encapsulating a controllable number of particles per drop.
TL;DR: The ability to uncouple the enzymatic assay from in vitro translation greatly extends the range of activities of in vitro translated proteins that can potentially be screened in droplet-based microfluidic systems and opens up the possibility of performing a wide range of other new (bio)chemical reactions in droplets.
Abstract: Microdroplets in water-in-oil emulsions can be used as microreactors with volumes 10(3) to 10(9) times smaller than the smallest working volumes in a microtitre plate well (1-2 microL). However, many reactions and assays require multiple steps where new reagents are added at defined times, to start, modify or terminate a reaction. The most flexible way to add new reagents to pre-formed droplets is by controlled, pairwise droplet fusion. We describe a droplet-based microfluidic system capable of performing multiple operations, including pairwise droplet fusion, to analyze complex and sequential multi-step reactions. It is exemplified by performing a series of six on-chip and two off-chip operations which enable the coupled in vitro transcription and translation of cotA laccase genes in droplets and, after performing a controlled fusion with droplets containing laccase assay reagents, the end-point and kinetic analysis of the catalytic activity of the translated protein. In vitro translation and the laccase assay must be performed sequentially as the conditions for the laccase assay are not compatible with in vitro translation. Droplet fusion was performed by electrocoalescence at a rate of approximately 3000 fusion events per second and nearly 90% of droplets were fused one-to-one (one droplet containing in vitro translated laccase fused to one droplet containing the reagents for the laccase assay). The ability to uncouple the enzymatic assay from in vitro translation greatly extends the range of activities of in vitro translated proteins that can potentially be screened in droplet-based microfluidic systems. Furthermore, the system also opens up the possibility of performing a wide range of other new (bio)chemical reactions in droplets.
TL;DR: These results provide the first proof-of-principle that a multiplexed micromagnetic-microfluidic separation system can be used to cleanse pathogens from flowing human blood at a rate and separation efficiency that is relevant for clinical applications.
Abstract: Sepsis is a lethal disease caused by a systemic microbial infection that spreads via the bloodstream to overwhelm the body's defenses. Current therapeutic approaches are often suboptimal, in part, because they do not fully eliminate the pathogen, and hence the source of deadly toxins. Here we describe an extracorporeal blood cleansing device to selectively remove pathogens from contaminated blood and thereby enhance the patient's response to antibiotic therapy. Immunomagnetic microbeads were modified to create magnetic opsonins that were used to cleanse flowing human whole blood of Candida albicans fungi, a leading cause of sepsis-related deaths. The micromagnetic–microfluidic blood cleansing device generates magnetic field gradients across vertically stacked channels to enable continuous and high throughput separation of fungi from flowing whole blood. A multiplexed version of the device containing four parallel channels achieved over 80% clearance of fungi from contaminated blood at a flow rate of 20 mL/h in a single pass, a rate 1000 times faster than a previously described prototype micromagnetic–microfluidic cell separation system. These results provide the first proof-of-principle that a multiplexed micromagnetic–microfluidic separation system can be used to cleanse pathogens from flowing human blood at a rate and separation efficiency that is relevant for clinical applications.
TL;DR: Integrated microvalves have been used to precisely and flexibly control the generation, size, composition of individual droplets and fusion of different droplets in microfluidic devices.
Abstract: Integrated microvalves have been used to precisely and flexibly control the generation, size, composition of individual droplets and fusion of different droplets in microfluidic devices.