Generation of disk-like hydrogel beads for cell encapsulation and manipulation using a droplet-based microfluidic device

We report a droplet-based microfluidic synthetic technique to generate disk-like hydrogel beads for cell encapsulation and manipulation. Utilizing this microfluidic synthetic technique, the size of the disk-like calcium alginate (CA) hydrogel beads and the number of cells encapsulated in the disk-like CA hydrogel beads could be well controlled by individually adjusting the flow rates of reagents. As a proof-of-concept, we demonstrated that single cell (yeast cell or mammalian cell) could be successfully encapsulated into disk-like CA hydrogel beads with high cell viability. Taking advantage of the flat top/bottom surfaces of disk-like CA hydrogel beads, cell division processes in culture media were clearly observed and recorded at a desired position without rolling and moving. This facile microfluidic chip provides a feasible method for size-controlled disk-like hydrogel beads generation and cell encapsulation. It could be a promising candidate for cell division observation and quantitative biological study in lab-on-a-chip applications.     

Clustering triple microbeads in a dynamic microarray for timing-controllable bead-based reactions

In this study, we developed a dynamic microfluidic device that enables the clustering of three different types of microbeads in a trapping spot and the rearrangement of contacting modes of the clustered microbeads. To achieve these two functions, two features are added to the conventional dynamic microfluidic device. (1) To trap multiple beads, an extended trapping spot with sub-by-pass channels and a valve was employed. (2) To rearrange the clustered microbeads, trapping spots that work only by backward flow were added. The design of the microfluidic device was realized by calculations based on fluidic resistance. By using the designed device, we successfully clustered different types of hydrogel microbeads including target materials, and observed reactions between clustered microbeads. In addition, by rearranging the contacting modes of the clustered microbeads, the reaction could be initiated/terminated at the desired time. We found that this dynamic microfluidic device is applicable to the quantitative analysis of chemical reactions between small amounts of multiple materials.     

Using a cross-flow microfluidic chip for monodisperse UV-photopolymerized microparticles

In this paper, we report a microfluidic chip containing a cross-junction channel for the manipulation of UV-photopolymerized microparticles. Hydrodynamic-focusing is used to form a series of using 365 nm UV light to solidify the hydrogel droplets. We were able to control the size of the hydrogel droplets from 75 to 300 μm in diameter by altering the sample and by changing the flow rate ratio of the mineral oil in the center inlet channel to that of the side inlet channels. We found that the size of the emulsions increases with an increase in average velocity of the dispersed phase flow (polymer solution flow). The size of the emulsions decreases with an average velocity increase of the continuous phase flow (mineral oil flow). Experimental data show that the emulsions are very uniform. The developed microfluidic chip has the advantages of ease of fabrication, low cost, and high throughput. The emulsions generated are very uniform and have good regularity.     

Development of microfluidic devices incorporating non-spherical hydrogel microparticles for protein-based bioassay

This paper describes the fabrication of a microfluidic device for use in protein-based bioassays that effectively incorporates poly(ethylene glycol) (PEG) hydrogel microparticles within a defined region. The microfluidic device is composed of a polymerization chamber and reaction chamber that are serially connected through the microchannel. Various shapes and sizes of hydrogel microparticles were fabricated in the polymerization chamber by photopatterning and moved to the reaction chamber by pressure-driven flow. All of the hydrogel microparticles were retained within the reaction chamber due to an in-chamber integrated microfilter with smaller mesh size than hydrogel microparticles. Hydrogel microparticles were able to encapsulate enzymes without losing their activity, and different concentrations of glucose were detected by sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) inside the microfluidic device using fluorescence method. Importantly, there was a linear correspondence between fluorescence intensity and the glucose concentration over the physiologically important range of 1.00–10.00 mM. D. Choi and E. Jang contributed equally to this work.     

Disk-like hydrogel bead-based immunofluorescence staining toward identification and observation of circulating tumor cells

Assays toward analysis of rare heterogeneous cells among identical specimen raise a significant challenge in many cell biological studies and clinical diagnosis applications. In this work, we report a disk-like hydrogel bead-based stratagem for rare cell researches at single cell level after a facile microfluidic-based particle synthesis approach. Cells of interested can be encapsulated into alginate droplets which are subsequently solidified into disk-like calcium alginate hydrogel beads and the bead size and cell number inside can be precisely controlled. Due to stability, permeability and disk-like shape of calcium alginate beads, cells immobilized in the disk-like beads can be treated with different chemicals with limited mechanical or fluidic operation influences and observed without distortion comparing with conventional methods or droplet microfluidic methods. Identification of circulating tumor cells, related to early-stage cancer diagnosis, is targeted to demonstrate the potential of our technique in rare cell analysis. This hydrogel bead-based stratagem is performed in immunofluorescence staining treatment and observation of cancer cells from normal hematological cells in blood sample. This method would have a great potential in single cell immobilization, manipulations and observation for biochemical cellular assays of rare cells.     

Moving shot,an affordable and high-throughput setup for direct imaging of fast-moving microdroplets

Droplet microfluidics have a great potential in chemical and biomedical applications, due to their high throughput, versatility, and massive parallelism. To enhance their throughput, many devices based on the droplet microfluidics are using a flow-through configuration, in which the droplets are generated, transported, and analyzed in a continuous stream with a high velocity. Direct imaging of moving droplets is often necessary to characterize the spatiotemporal dynamics of the chemical reaction and physical process in the droplets. However, due to the motion blur caused by the movement of the droplets during exposure, an expensive high-speed camera is required for clear imaging, which is cost prohibitive in many applications. In this paper, we are presenting ‘Moving shot’ to demonstrate direct imaging of fast-moving droplets in microfluidic channels at an affordable cost. A microfluidic device is translated at the same velocity but in the opposite direction of moving droplets in it, so that the droplets are stationary with respect to the objective lens. With this approach, we can image fluorescent droplets moving at 0.34 cm s⁻¹ with an exposure time up to 2 s without motion blur. We strongly believe that the proposed technique can enable cost-effective and high-throughput imaging of fast-moving droplets in a microfluidic channel.

     

Modeling and optimization of a multi-enzyme electrokinetically driven multiplexed microchip for simultaneous detection of sugars

A model-based methodology was developed to optimize microfluidic chips for the simultaneous enzymatic quantification of sucrose, d-glucose and d-fructose in a single microfluidic channel with an integrated optical detection system. The assays were based on measuring the change in concentration of the reaction product NADH, which is stoichiometrically related to the concentration of those components via cascade of specific enzymatic reactions. A reduced order mathematical model that combines species transport, enzyme reaction, and electrokinetic bulk flow was developed to describe the operation of the microfluidic device. Using this model, the device was optimized to minimize sensor response time and maximize signal output by manipulating the process conditions such as sample and reagent volume and flow rate. According to this simulation study, all sugars were quantified within 2.5 min in the optimized microchip. A parallel implementation of the assays can further improve the throughput. In addition, the amount of consumed reagents was drastically reduced compared to microplate format assays. The methodology is generic and can easily be adapted to other enzymatic microfluidic chips.     

On-chip polymerase chain reaction microdevice employing a magnetic droplet-manipulation system

An on-chip polymerase chain reaction (PCR) device employing a magnetic beads-droplet-handling system was developed. Actuation with a magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads by handling with a magnet. The device consists of a reaction chamber channel and two magnet-handling channels for the manipulation of micro-droplets containing magnetic beads. Micro-droplets were placed inside a reaction chamber filled with oil and manipulated with a magnet. When a droplet containing NaOH and magnetic beads was manipulated towards a droplet containing phenol red, a color change was observed after fusion. Sample preparation was performed by fusion of droplets containing a forward primer, reverse primer, template DNA and PCR mixture, using a droplet containing magnetic beads. PCR amplification or RT-PCR was also successfully performed, with efficiency comparable to manual methods that use this device by placing it on a thermal cycler for amplification. With a magnetic beads-manipulation step, purification of amplified DNA was also accomplished by using magnetic beads as the carrier. The amplified DNA was captured on streptavidin conjugated magnetic beads using a biotinylated primer, purified by washing and digested for separation of the target DNA.     

A novel microfluidic switch for pH control using Chitosan based hydrogels

We report a novel pH-sensitive hydrogel based micro-valve for metered flow that has applications in a laboratory made “Intelligent valving system”. The hydrogel solution was prepared through Chitosan and poly vinyl alcohol in acetic acid and crystallized using gluteraldehyde as the crosslinking agent in the form of thin wafers and it was found to be very sensitive to pH changes. The pore structure of hydrogel was investigated through Field Emission Scanning Electron Microscopy and thin wafers of the gel were physically placed inside PDMS microchannels. Flow metering in these channels was observed by controlled expansion of the hydrogel plug till complete valving was realized. This valving device was further precisely characterized with micro Particle Image Velocimetry using a solution containing fluorescent polymeric micro beads. The principle advantage of this hydrogel device is the smaller range of pH (varying between pH 3 and 7) over which the valving response is observed.     

On-chip fabrication of magnetic alginate hydrogel microfibers by multilayered pneumatic microvalves

Alginate hydrogel has widespread applications in tissue engineering, cancer therapy, wound management and drug/cell/growth factor delivery due to its biocompatibility, hydrated environment and desirable viscoelastic properties. However, the lack of controllability is still an obstacle for utilizing it in the fabrication of 3D tissue constructs and accurate targeting in mass delivery. Here, we proposed a new method for achieving magnetic alginate hydrogel microfibers by dispersing magnetic nanoparticles in alginate solution and solidifying the magnetic alginate into hydrogel fiber inside microfluidic devices. The microfluidic devices have multilayered pneumatic microvalves with hemicylindrical channels to fully stop the fluids. In the experiments, the magnetic nanoparticles and the alginate solution were mixed and formed a uniform suspension. No aggregation of magnetic nanoparticles was found, which is crucial for flow control inside microfluidic devices. By regulating the flow rates of different solutions with the microvalves inside the microfluidic device, magnetic hydrogel fibers and nonmagnetic hydrogel fibers were fabricated with controlled sizes. The proposed method for fabricating magnetic hydrogel fiber holds great potential for engineering 3D tissue constructs with complex architectures and active drug release.     

A microfluidic sensor based on ferromagnetic resonance induced in magnetic bead labels

This report details preliminary studies towards the development of a microfluidic sensor that exploits ferromagnetic resonance, excited in magnetic bead labels, for signal transduction. The device consists of a microwave circuit in which a slotline and a coplanar waveguide are integrated with a biochemically activated sensor area. The magnetic beads are immobilized in the sensor area by bio-specific reactions. A microwave signal applied to the slotline is coupled to the coplanar waveguide only in the presence of magnetic beads at the functionalized sensor area. Ferromagnetic resonance in the beads further enhances the coupling. This inductive detection technique lends itself to miniaturization, is inexpensive to fabricate and can be adapted for the detection of a wide range of molecules for which bio-specific ligands are available.Experimentally, the variation of the output signal as a function of the location of magnetic beads was studied for the proposed technique. Subsequently, a prototype device was constructed by biotinylation of the sensor area and integration with a microfluidic chip fabricated in polydimethyl siloxane (PDMS). Preliminary experiments were conducted on this prototype using streptavidin-functionalized magnetic beads as labels. It was shown that the magnetic beads, immobilized at the sensor area by streptavidin-biotin linkage, produced a distinct ferromagnetic resonance response easily discernable from the background signal.     

Autonomous magnetically actuated continuous flow microimmunofluorocytometry assay

This article presents a microfluidic device which integrates autonomous serial immunofluorocytometry binding reactions of cytometric beads with fluorescence detection and quantification in a continuous flow environment. The microdevice assay is intended to alleviate the extensive benchwork and large sample volumes used when conducting traditional immunoassays, without requiring complex external controls. The technology is based on the miniaturization and automation of the serial processing steps of an antigen sandwich immunoassay, with integrated fluorescence detection using paramagnetic microbeads. The continuous flow design may enable temporal tracking of time-varying protein concentrations in a continuously infused sample for clinical applications, specifically for monitoring inflammation marker proteins in blood produced during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures. The device operation was first validated via a single incubation device which measured the concentration of a fluorescently labeled biotin molecule using streptavidin-coated paramagnetic cytometric beads. Subsequently, a dual incubation device was tested with samples of the anaphylatoxin complement protein C3a, and was shown to be capable of differentiating between samples at typical systemic concentrations of the protein (1–5 μg/ml), with very low sample usage (<6 μl/h). It is believed that this continuous flow, automated microimmunosensor technology will be a platform for high sample rate immunoassays capable of tracking and more thoroughly characterizing the systemic inflammation process, and may aid in the development of better treatment options for systemic inflammation during and after CPB.     

Screening of peptide specific to cholangiocarcinoma cancer cells using an integrated microfluidic system and phage display technology

Cholangiocarcinoma (CCA) is a cancer of the bile duct with high mortality rate and poor prognosis, owing to the difficulty in the early diagnosis and prognosis. The specific biomarkers or affinity reagents toward CCA cells could be great tools to assist in detection of CCA. However, screening of biomarkers/affinity reagents are generally labor-intensive, time-consuming and requiring large volume of samples and reagents. Therefore, we developed an integrated microfluidic system which could automatically perform selections of biomarkers and affinity reagents using phage display techniques. The experimental results showed that the selection of phage-displayed peptides bound to CCA cells was successfully demonstrated on the integrated microfluidic system using fewer reagents, samples and less time (5.25 h per biopanning round, and continuously performed for only 4 panning rounds). Three oligopeptides were screened, and their specificity and affinity toward CCA cells were characterized. Furthermore, comparing to conventional EpiEnrich beads for cancer cell capture, the screened CCA-specific peptides showed relatively low capture rate over control normal cells. It is envisioned that this microfluidic system may be a powerful tool for screening of biomarkers/affinity reagents in clinical diagnosis and target therapy for CCA.     

Control of serial microfluidic droplet size gradient by step-wise ramping of flow rates

This paper describes a method to control and detect droplet size gradient by step-wise flow rate ramping of water-in-oil droplets in a microfluidic device. The droplets are generated in a cross channel device with two oil inlets and a water inlet. The droplet images are captured and analyzed in a time sequence in order to quantify the droplet generation frequency. It is demonstrated that by controlling the ramping of the oil flow rates it is possible to manipulate the ramping of droplet sizes. Increasing or decreasing of droplet sizes is achieved for a step-wise triangular ramping profile of the oil flow rate. The dynamic behavior of droplets due to the step-wise flow pulses is investigated. Uniform linear size ramping of water-in-oil droplets from 73 to 83 μm in diameter is generated with an oil flow ramping range from 1 to 11 μL/min in a minimum of five steps while water flow rate is held constant at 2 μL/min.     

Cells adhered and cultured on microcantilevers

This paper shows a novel method to cultivate cells on a π-shape microcantilever inside a polydimethylsiloxane microfluidic system. Only one lithography step was needed to precisely align and pattern a poly(2-hydroxyethyl methacrylate) hydrogel microstructure, of size 200 × 200 μm, onto a silicon nitride microcantilever inside the PDMS microfluidic device. Gelatin was used as a sacrificial layer to resolve the issue of the microfluidic and hydrogel microstructure sticking together, successfully releasing the microcantilevers. BHK-21 cells were successfully laden and cultivated on the hydrogel microstructures of microcantilevers for 24 h. The optical system consisted of a He–Ne laser, a charge-coupled device camera, and a position-sensitive detector, which was used to measure the deflections of the microcantilevers due to the laden cells. The deflection increased continually during the cell-laden period. Meanwhile, the deflection increased with increasing cell concentration. By repeating the cell-laden and culture experiment three times, the magnitude and trend of deflection of microcantilevers were almost the same. It demonstrates that the microcantilever-based biochip has adequate stability and provides reliable measurement results for drug screening applications in the future.     

Inertial microfluidics for continuous particle filtration and extraction

In this paper, we describe a simple passive microfluidic device with rectangular microchannel geometry for continuous particle filtration. The design takes advantage of preferential migration of particles in rectangular microchannels based on shear-induced inertial lift forces. These dominant inertial forces cause particles to move laterally and occupy equilibrium positions along the longer vertical microchannel walls. Using this principle, we demonstrate extraction of 590 nm particles from a mixture of 1.9 μm and 590 nm particles in a straight microfluidic channel with rectangular cross-section. Based on the theoretical analysis and experimental data, we describe conditions required for predicting the onset of particle equilibration in square and rectangular microchannels. The microfluidic channel design has a simple planar structure and can be easily integrated with on-chip microfluidic components for filtration and extraction of wide range of particle sizes. The ability to continuously and differentially equilibrate particles of different size without external forces in microchannels is expected to have numerous applications in filtration, cytometry, and bioseparations.     

Intra-droplet acoustic particle focusing: simulations and experimental observations

The aim of this paper is to study resonance conditions for acoustic particle focusing inside droplets in two-phase microfluidic systems. A bulk acoustic wave microfluidic chip was designed and fabricated for focusing microparticles inside aqueous droplets (plugs) surrounded by a continuous oil phase in a 380-μm-wide channel. The quality of the acoustic particle focusing was investigated by considering the influence of the acoustic properties of the continuous phase in relation to the dispersed phase. To simulate the system and study the acoustic radiation force on the particles inside droplets, a simplified 3D model was used. The resonance conditions and focusing quality were studied for two different cases: (1) the dispersed and continuous phases were acoustically mismatched (water droplets in fluorinated oil) and (2) the dispersed and continuous phases were acoustically matched (water droplets in olive oil). Experimentally, we observed poor acoustic particle focusing inside droplets surrounded by fluorinated oil while good focusing was observed in droplets surrounded by olive oil. The experimental results are supported qualitatively by our simulations. These show that the acoustic properties (density and compressibility) of the dispersed and continuous phases must be matched to generate a strong and homogeneous acoustic field inside the droplet that is suitable for high-quality intra-droplet acoustic particle focusing.     

Microfluidic inverse phase ELISA via manipulation of magnetic beads

We report a new technique for conducting immuno-diagnostics on a microfluidic platform. Rather than handling fluid reagents against a stationary solid phase, the platform manipulates analyte-coated magnetic beads through stationary plugs of fluid reagents to detect an antigenic analyte. These isolated but accessible plugs are pre-encapsulated in a microchannel by capillary force. We call this platform microfluidic inverse phase enzyme-linked immunosorbent assay (μIPELISA). μIPELISA has distinctive advantages in the family of microfluidic immunoassay. In particular, it avoids pumping and valving fluid reagents during assaying, thus leading to a lab-on-a-chip format that is free of instrumentation for fluid actuation and control. We use μIPELISA to detect digoxigenin-labeled DNA segments amplified from E. coli O157:H7 by polymerase chain reaction (PCR), and compare its detection capability with that of microplate ELISA. For 0.259 ng μl⁻¹ of digoxigenin-labeled amplicon, μIPELISA is as responsive as the microplate ELISA. Also, we simultaneously conduct μIPELISA in two parallel microchannels.     

Controlled dispensing and mixing of pico- to nanoliter volumes using on-demand droplet-based microfluidics

We present an integrated droplet-on-demand microfluidic platform for dispensing, mixing, incubating, extracting and analyzing by mass spectrometry pico- to nanoliter-sized droplets. All of the functional components are successfully integrated for the first time into a monolithic microdevice. Droplet generation is accomplished using computer-controlled pneumatic valves. Controlled actuation of valves for different aqueous streams enables accurate dosing and rapid mixing of reagents within droplets in either the droplet generation area or in a region of widening channel cross-section. Following incubation, which takes place as droplets travel in the oil stream, the droplet contents are extracted to an aqueous channel for subsequent ionization at an integrated nanoelectrospray emitter. Using the integrated platform, rapid enzymatic digestions of a model protein were carried out in droplets and detected online by nanoelectrospray ionization mass spectrometry.     

Droplet Formation Utilizing Controllable Moving-Wall Structures for Double-Emulsion Applications

The formation of microscale single- and double-emulsion droplets with various sizes is crucial for a variety of industrial applications. In this paper, we report a new microfluidic device which can actively fine-tune the size of single- and double-emulsion droplets in liquids by utilizing controllable moving-wall structures. Moreover, various sizes of external and internal droplets for double emulsions are also successfully formed by using this device. Three pneumatic side chambers are placed at a T-junction and flow-focusing channels to construct the controllable moving-wall structures. When compressed air is applied to the pneumatic side chambers, the controllable moving-wall structures are activated, thus physically changing the width of the microchannels. The size of the internal droplets at the intersection of the T-junction channel is then fine-tuned due to the increase in the shear force. Then, the internal droplets are focused into a narrow stream hydrodynamically and finally chopped into double-emulsion droplets using another pair of moving-wall structures downstream. For single emulsions, oil-in-water droplets can be actively fine-tuned from 50.07 to 21.80 under applied air pressures from 10 to 25 psi with a variation of less than 3.53%. For a water-in-oil single emulsion, droplets range from 50.32 to 14.76 with a variation of less than 4.62% under the same applied air pressures. For double emulsions, the sizes of the external and internal droplets can be fine-tuned with external/internal droplet diameter ratios ranging from 1.69 to 2.75. The development of this microfluidic device is promising for a variety of applications in the pharmaceutical, cosmetics, and food industries.