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.     

Microfluidic platform for the study of intercellular communication via soluble factor-cell and cell-cell paracrine signaling

Diffusion of autocrine and paracrine signaling molecules allows cells to communicate in the absence of physical contact. This chemical-based, long-range communication serves crucial roles in tissue function, activation of the immune system, and other physiological functions. Despite its importance, few in vitro methods to study cell-cell signaling through paracrine factors are available today. Here, we report the design and validation of a microfluidic platform that enables (i) soluble molecule-cell and/or (ii) cell-cell paracrine signaling. In the microfluidic platform, multiple cell populations can be introduced into parallel channels. The channels are separated by arrays of posts allowing diffusion of paracrine molecules between cell populations. A computational analysis was performed to aid design of the microfluidic platform. Specifically, it revealed that channel spacing affects both spatial and temporal distribution of signaling molecules, while the initial concentration of the signaling molecule mainly affects the concentration of the signaling molecules excreted by the cells. To validate the microfluidic platform, a model system composed of the signaling molecule lipopolysaccharide, mouse macrophages, and engineered human embryonic kidney cells was introduced into the platform. Upon diffusion from the first channel to the second channel, lipopolysaccharide activates the macrophages which begin to produce TNF-α. The TNF-α diffuses from the second channel to the third channel to stimulate the kidney cells, which express green fluorescent protein (GFP) in response. By increasing the initial lipopolysaccharide concentration an increase in fluorescent response was recorded, demonstrating the ability to quantify intercellular communication between 3D cellular constructs using the microfluidic platform reported here. Overall, these studies provide a detailed analysis on how concentration of the initial signaling molecules, spatiotemporal dynamics, and inter-channel spacing affect intercellular communication.     

Individually addressable multi-chamber electroporation platform with dielectrophoresis and alternating-current-electro-osmosis assisted cell positioning

A multi-functional microfluidic platform was fabricated to demonstrate the feasibility of on-chip electroporation integrated with dielectrophoresis (DEP) and alternating-current-electro-osmosis (ACEO) assisted cell/particle manipulation. A spatial gradient of electroporation parameters was generated within a microchamber array and validated using normal human dermal fibroblast (NHDF) cells and red fluorescent protein-expressing human umbilical vein endothelial cells (RFP-HUVECs) with various fluorescent indicators. The edge of the bottom electrode, coinciding with the microchamber entrance, may act as an on-demand gate, functioning under either positive or negative DEP. In addition, at sufficiently low activation frequencies, ACEO vortices can complement the DEP to contribute to a rapid trapping/alignment of particles. As such, results clearly indicate that the microfluidic platform has the potential to achieve high-throughput screening for electroporation with spatial control and uniformity, assisted by DEP and ACEO manipulation/trapping of particles/cells into individual microchambers.     

Rapid,one-step fabrication and loading of nanoscale 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes in a simple,double flow-focusing microfluidic device

Liposomes are currently well-established as biocompatible delivery vehicles for numerous compounds. However, conventional manufacturing tends to rely on time-consuming processes, costly equipment, unstable reaction parameters, and numerous pre- and post-processing steps. Herein, we demonstrate a microscope-slide-sized alternative: a double flow-focusing microfluidic geometry capable of sub-hour synthesis and controlled loading of tunable liposomes. Using phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine as the bilayer constituent, the effect of varying the dissolved lipid concentration and flow rate ratio on synthesized liposome diameters was investigated and the encapsulation of fluorescent hydrophobic drug model ergost-5,7,9(11),22-tetraen-3β-ol was performed to ascertain the potential of this device as a loading platform.     

Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis

Understanding biomolecular gradients and their role in biological processes is essential for fully comprehending the underlying mechanisms of cells in living tissue. Conventional in vitro gradient-generating methods are unpredictable and difficult to characterize, owing to temporal and spatial fluctuations. The field of microfluidics enables complex user-defined gradients to be generated based on a detailed understanding of fluidic behavior at the μm-scale. By using microfluidic gradients created by flow, it is possible to develop rapid and dynamic stepwise concentration gradients. However, cells exposed to stepwise gradients can be perturbed by signals from neighboring cells exposed to another concentration. Hence, there is a need for a device that generates a stepwise gradient at discrete and isolated locations. Here, we present a microfluidic device for generating a stepwise concentration gradient, which utilizes a microwell slide''s pre-defined compartmentalized structure to physically separate different reagent concentrations. The gradient was generated due to flow resistance in the microchannel configuration of the device, which was designed using hydraulic analogy and theoretically verified by computational fluidic dynamics simulations. The device had two reagent channels and two dilutant channels, leading to eight chambers, each containing 4 microwells. A dose-dependency assay was performed using bovine aortic endothelial cells treated with saponin. High reproducibility between experiments was confirmed by evaluating the number of living cells in a live-dead assay. Our device generates a fully mixed fluid profile using a simple microchannel configuration and could be used in various gradient studies, e.g., screening for cytostatics or antibiotics.     

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.     

Sub-nanomolar detection of thrombin activity on a microfluidic chip

Bioluminescence resonance energy transfer (BRET) is a form of Förster resonance energy transfer. BRET has been shown to support lower limits of detection than fluorescence resonance energy transfer (FRET) but, unlike FRET, has not been widely implemented on microfluidic devices for bioanalytical sensing. We recently reported a microscope-based microfluidic system for BRET-based biosensing, using a hybrid, high quantum-efficiency, form of BRET chemistry. This paper reports the first optical fiber-based system for BRET detection on a microfluidic chip, capable of quantifying photon emissions from the low quantum-efficiency BRET² system. We investigated the effects of varying core diameter and numerical aperture of optical fibers, as well as varying microfluidic channel design and measurement conditions. We optimized the set-up in order to maximize photon counts and minimize the response time. The optimized conditions supported measurement of thrombin activity, with a limit of detection of 20 pM, which is lower than the microscope-based system and more than 20 times lower than concentrations reported to occur in plasma clots.     

A multichannel acoustically driven microfluidic chip to study particle-cell interactions

Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 μm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s⁻¹ and 2.25 s⁻¹ exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.     

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.     

A microfluidic-based neurotoxin concentration gradient for the generation of an in vitro model of Parkinson's disease

In this study, we developed a miniaturized microfluidic-based high-throughput cell toxicity assay to create an in vitro model of Parkinson's disease (PD). In particular, we generated concentration gradients of 6-hydroxydopamine (6-OHDA) to trigger a process of neuronal apoptosis in pheochromocytoma PC12 neuronal cell line. PC12 cells were cultured in a microfluidic channel, and a concentration gradient of 6-OHDA was generated in the channel by using a back and forth movement of the fluid flow. Cellular apoptosis was then analyzed along the channel. The results indicate that at low concentrations of 6-OHDA along the gradient (i.e., approximately less than 260 μM), the neuronal death in the channel was mainly induced by apoptosis, while at higher concentrations, 6-OHDA induced neuronal death mainly through necrosis. Thus, this concentration appears to be useful for creating an in vitro model of PD by inducing the highest level of apoptosis in PC12 cells. As microfluidic systems are advantageous in a range of properties such as throughput and lower use of reagents, they may provide a useful approach for generating in vitro models of disease for drug discovery applications.     

Continuously perfused microbubble array for 3D tumor spheroid model

Multi-cellular tumor spheroids (MCTSs) have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is time-consuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane (PDMS) microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.     

A micropillar array for sample concentration via in-plane evaporation

We present a method to perform sample concentration within a lab-on-a-chip using a microfluidic structure which controls the liquid-gas interface through a micropillar array fabricated in polydimethylsiloxane between microfluidic channels. The microstructure confines the liquid flow and a thermal gradient is used to drive evaporation at the liquid-gas-interface. The evaporation occurs in-plane to the microfluidic device, allowing for precise control of the ambient environment. This method is demonstrated with a sample containing 1 μm, 100 nm fluorescent beads and SYTO-9 labelled Escherichia coli bacteria. Over 100 s, the fluorescent beads and bacteria are concentrated by a factor of 10.     

Droplet microfluidics for kinetic studies of viral fusion

Viral infections remain a major threat to public health. The speed with which viruses are evolving drug-resistant mutations necessitates the further development of antiviral therapies with a large emphasis on drug discovery. To facilitate these efforts, there is a need for robust, high-throughput assays that allow the screening of large libraries of compounds, while enabling access to detailed kinetic data on their antiviral activity. We report here the development of a droplet-based microfluidic platform to probe viral fusion, an early critical step in infection by membrane-enveloped viruses such as HIV, Hepatitis C, and influenza. Using influenza A, we demonstrate the measurement of the kinetics of fusion of virions with target liposomes with sub-second temporal resolution. In analogy with acidification of the endosome that triggers fusion in a cellular context, we acidify the content of aqueous droplets containing virions and liposomes in situ by introducing acid from the dispersed phase and visualize the kinetics of fusion by using fluorescent probes.     

Microfluidic based high throughput synthesis of lipid-polymer hybrid nanoparticles with tunable diameters

Core-shell hybrid nanoparticles (NPs) for drug delivery have attracted numerous attentions due to their enhanced therapeutic efficacy and good biocompatibility. In this work, we fabricate a two-stage microfluidic chip to implement a high-throughput, one-step, and size-tunable synthesis of mono-disperse lipid-poly (lactic-co-glycolic acid) NPs. The size of hybrid NPs is tunable by varying the flow rates inside the two-stage microfluidic chip. To elucidate the mechanism of size-controllable generation of hybrid NPs, we observe the flow field in the microchannel with confocal microscope and perform the simulation by a numerical model. Both the experimental and numerical results indicate an enhanced mixing effect at high flow rate, thus resulting in the assembly of small and mono-disperse hybrid NPs. In vitro experiments show that the large hybrid NPs are more likely to be aggregated in serum and exhibit a lower cellular uptake efficacy than the small ones. This microfluidic chip shows great promise as a robust platform for optimization of nano drug delivery system.     

Circadian rhythms in Neurospora crassa on a polydimethylsiloxane microfluidic device for real-time gas perturbations

Racetubes, a conventional system employing hollow glass tubes, are typically used for monitoring circadian rhythms from the model filamentous fungus, Neurospora crassa. However, a major technical limitation in using a conventional system is that racetubes are not amenable for real-time gas perturbations. In this work, we demonstrate a simple microfluidic device combined with real-time gas perturbations for monitoring circadian rhythms in Neurospora crassa using bioluminescence assays. The developed platform is a useful toolbox for investigating molecular responses under various gas conditions for Neurospora and can also be applied to other microorganisms.     

A microfluidic chip for controlled release of drugs from microcapsules

A new microfluidic device with liquid-droplet merging and droplet storage functions for the controlled release of drugs from microcapsules is reported. A switching channel is designed and integrated within the microfluidic device, facilitating the generation and capturing of uniform droplets by the storage chambers. The drug model is the MnCO₃ microparticle, which is encapsulated by a microcapsule and fabricated using a simple layer-by-layer nanoassembly process. The merging function is used for dynamically adding the control solution into the droplets, which contain drugs within the microcapsules (DWμCs) and water. The storage chambers are used for collecting DWμCs-laden droplets so that the controlled-drug release in specific droplets can be monitored for an extended period of time, which has been experimentally implemented successfully. This technology could offer a promising technical platform for the long-term observation and studies of drug effects on specific cells in a controlled manner, which is especially useful for single cell analysis.     

Droplet-based microfluidic washing module for magnetic particle-based assays

In this paper, we propose a continuous flow droplet-based microfluidic platform for magnetic particle-based assays by employing in-droplet washing. The droplet-based washing was implemented by traversing functionalized magnetic particles across a laterally merged droplet from one side (containing sample and reagent) to the other (containing buffer) by an external magnetic field. Consequently, the magnetic particles were extracted to a parallel-synchronized train of washing buffer droplets, and unbound reagents were left in an original train of sample droplets. To realize the droplet-based washing function, the following four procedures were sequentially carried in a droplet-based microfluidic device: parallel synchronization of two trains of droplets by using a ladder-like channel network; lateral electrocoalescence by an electric field; magnetic particle manipulation by a magnetic field; and asymmetrical splitting of merged droplets. For the stable droplet synchronization and electrocoalescence, we optimized droplet generation conditions by varying the flow rate ratio (or droplet size). Image analysis was carried out to determine the fluorescent intensity of reagents before and after the washing step. As a result, the unbound reagents in sample droplets were significantly removed by more than a factor of 25 in the single washing step, while the magnetic particles were successfully extracted into washing buffer droplets. As a proof-of-principle, we demonstrate a magnetic particle-based immunoassay with streptavidin-coated magnetic particles and fluorescently labelled biotin in the proposed continuous flow droplet-based microfluidic platform.     

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.     

Generation of nitric oxide gradients in microfluidic devices for cell culture using spatially controlled chemical reactions

In this paper, we develop a microfluidic device capable of generating nitric oxide (NO) gradients for cell culture using spatially controlled chemical reactions. NO plays an essential role in various biological activities, including nervous, immune, and cardiovascular systems. The device developed in this paper can control NO gradients without utilizing expensive and hazardous high purity NO gas sources or direct addition of NO donors. Consequently, the device provides an efficient, cost-effective, robust, and stable platform to generate NO gradients for cell culture studies. In the experiments, NO gradients are first characterized using a NO-sensitive fluorescence dye, and cell experiments using aortic smooth muscle cells are conducted. The results demonstrate that the device can alter the intracellular NO concentrations and further affect the Ca²⁺ concentration oscillation for the cells. The device developed in this paper provides a powerful platform for researchers better study the biological roles of NO and its spatial distribution using in vitro cell models with minimal instrumentation.     

Label-free viscosity measurement of complex fluids using reversal flow switching manipulation in a microfluidic channel

The accurate viscosity measurement of complex fluids is essential for characterizing fluidic behaviors in blood vessels and in microfluidic channels of lab-on-a-chip devices. A microfluidic platform that accurately identifies biophysical properties of blood can be used as a promising tool for the early detections of cardiovascular and microcirculation diseases. In this study, a flow-switching phenomenon depending on hydrodynamic balancing in a microfluidic channel was adopted to conduct viscosity measurement of complex fluids with label-free operation. A microfluidic device for demonstrating this proposed method was designed to have two inlets for supplying the test and reference fluids, two side channels in parallel, and a junction channel connected to the midpoint of the two side channels. According to this proposed method, viscosities of various fluids with different phases (aqueous, oil, and blood) in relation to that of reference fluid were accurately determined by measuring the switching flow-rate ratio between the test and reference fluids, when a reverse flow of the test or reference fluid occurs in the junction channel. An analytical viscosity formula was derived to measure the viscosity of a test fluid in relation to that of the corresponding reference fluid using a discrete circuit model for the microfluidic device. The experimental analysis for evaluating the effects of various parameters on the performance of the proposed method revealed that the fluidic resistance ratio (R_JL/R_L, fluidic resistance in the junction channel (R_JL) to fluidic resistance in the side channel (R_L)) strongly affects the measurement accuracy. The microfluidic device with smaller R_JL/R_L values is helpful to measure accurately the viscosity of the test fluid. The proposed method accurately measured the viscosities of various fluids, including single-phase (Glycerin and plasma) and oil-water phase (oil vs. deionized water) fluids, compared with conventional methods. The proposed method was also successfully applied to measure viscosities of blood with varying hematocrits, chemically fixed RBC_S, and channel sizes. Based on these experimental results, the proposed method can be effectively used to measure the viscosities of various fluids easily, without any fluorescent labeling and tedious calibration procedures.