Flow-focusing Microfluidic Chip Research Articles

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor

BackgroundSingle-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides.ResultsThe water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.ConclusionsWe established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Sensitivity improvement of subharmonic-based pressure measurement using phospholipid-coated monodisperse microbubbles

The use of the subharmonic signal from microbubbles exposed to ultrasound is a promising safe and cost-effective approach for the non-invasive measurement of blood pressure. Achieving a high sensitivity of the subharmonic amplitude to the ambient overpressure is crucial for clinical applications. However, currently used microbubbles have a wide size distribution and diverse shell properties. This causes uncertainty in the response of the subharmonic amplitude to changes in ambient pressure, which limits the sensitivity. The aim of this study was to use monodisperse microbubbles to improve the sensitivity of subharmonic-based pressure measurements. With the same shell materials and gas core, we used a flow-focusing microfluidic chip and a mechanical agitation method to fabricate monodisperse (∼2.45-µm mean radius and 4.7 % polydisperse index) and polydisperse microbubbles (∼1.51-µm mean radius and 48.4 % polydisperse index), respectively. We varied the ultrasound parameters (i.e., the frequency, peak negative pressure (PNP) and pulse length), and found that there was an optimal excitation frequency (2.8 MHz) for achieving maximal subharmonic emission for monodisperse microbubbles, but not for polydisperse microbubbles. Three distinct regimes (occurrence, growth, and saturation) were identified in the response of the subharmonic amplitude to increasing PNP for both monodisperse and polydisperse microbubbles. For the polydisperse microbubbles, the subharmonic amplitude decreased either monotonically or non-monotonically with ambient overpressure, depending on the PNP. By contrast, for the monodisperse microbubbles, there was only a monotonic decrease at all PNPs. The maximum sensitivity (1.18 dB/kPa, R2 = 0.97) of the subharmonic amplitude to ambient overpressure for the monodisperse microbubbles was ∼6.5 times higher than that for the polydisperse microbubbles (0.18 dB/kPa, R2 = 0.88). These results show that monodisperse microbubbles can achieve a more consistent response of the subharmonic signal to changes in ambient overpressure and greatly improve the measurement sensitivity.

Characterization of helium microplasma generated in a flow focusing microfluidic device

Non-thermal microplasmas produced in a microchannel have several potential applications in analytical chemistry, environmental sensing, and surface modification of microfluidic chips for biomedical and lab-on-chip devices. This paper investigates the properties of an atmospheric pressure helium microplasma excited in a polydimethylsiloxane flow focusing microfluidic chip. The influence of input parameters such as applied voltage and gas flow rate on discharge characteristics is investigated in detail. Electron excitation and molecular rotational temperatures are determined with the Boltzmann plot technique. The rotational temperature from the N2+ emission band was calculated in the range of 348–417 K. Electron density and temperature are determined using the well-known plasma diagnostic technique of Stark broadening. The emission lines of hydrogen Balmer (Hα) and neutral helium (501, 667, and 728 nm) are selected to measure the parameters of Stark broadening. The electron density and electron temperature were found to be in the range 0.7×1016–3.39×1016 cm−3 and 10 800–12 493 K, respectively. The evaluated discharge parameters validate the non-thermal equilibrium state of the microplasma. The electrical diagnostics of plasma were performed by monitoring the signals of high voltage and current of the discharge. Moreover, the plasma modified surface (hydrophobic to hydrophilic) was verified by successfully utilizing the microchannel to form an oil-in-water micro-emulsion.

Microfluidic Synthesis of Indomethacin-Loaded PLGA Microparticles Optimized by Machine Learning.

Several attempts have been made to encapsulate indomethacin (IND), to control its sustained release and reduce its side effects. To develop a successful formulation, drug release from a polymeric matrix and subsequent biodegradation need to be achieved. In this study, we focus on combining microfluidic and artificial intelligence (AI) technologies, alongside using biomaterials, to generate drug-loaded polymeric microparticles (MPs). Our strategy is based on using Poly (D,L-lactide-co-glycolide) (PLGA) as a biodegradable polymer for the generation of a controlled drug delivery vehicle, with IND as an example of a poorly soluble drug, a 3D flow focusing microfluidic chip as a simple device synthesis particle, and machine learning using artificial neural networks (ANNs) as an in silico tool to generate and predict size-tunable PLGA MPs. The influence of different polymer concentrations and the flow rates of dispersed and continuous phases on PLGA droplet size prediction in a microfluidic platform were assessed. Subsequently, the developed ANN model was utilized as a quick guide to generate PLGA MPs at a desired size. After conditions optimization, IND-loaded PLGA MPs were produced, and showed larger droplet sizes than blank MPs. Further, the proposed microfluidic system is capable of producing monodisperse particles with a well-controllable shape and size. IND-loaded-PLGA MPs exhibited acceptable drug loading and encapsulation efficiency (7.79 and 62.35%, respectively) and showed sustained release, reaching approximately 80% within 9 days. Hence, combining modern technologies of machine learning and microfluidics with biomaterials can be applied to many pharmaceutical applications, as a quick, low cost, and reproducible strategy.

Formation of alginate microspheres prepared by optimized microfluidics parameters for high encapsulation of bioactive molecules

Drug delivery systems such as microspheres have shown potential in releasing biologicals effectively for tissue engineering applications. Microfluidic systems are especially attractive for generating microspheres as they produce microspheres of controlled-size and in low volumes, using micro-emulsion processes. However, the flow rate dependency on the encapsulation of molecules at a microscale is poorly understood. In particular, the flow rate and pressure parameters might influence the droplet formation and drug encapsulation efficiency. We evaluated the parameters within a two-reagent flow focusing microfluidic chip under continuous formation of hydrogel particles using a flourinated oil and an ionic crosslinkable alginate hydrogel. Fluorescein isothiocyanate-dextran sulfate (FITC-dextran sulfate MW: 40 kDa) was used to evaluate the variation of the encapsulation efficiency with the flow parameters, optimizing droplets and microsphere formation. The ideal flow rates allowing for maximum encapsulation efficiency, were utilised to form bioactive microspheres by delivering transforming growth factor beta-3 (TGFβ-3) in cell culture media. Finally, we evaluated the potential of microfluidic-formed microspheres to be included within biological environments. The biocompatibility of the microspheres was tested over 28 days using adult human mesenchymal stem cells (hMSCs). The release profile of the growth factors from microspheres showed a sustained release in media, after an initial burst, up to 30 days. The metabolic activity of the cells cultured in the presence of the microspheres was similar to controls, supporting the biocompatibility of this approach. The fine-tuned parameters for alginate hydrogel to form microspheres have potential in encapsulating and preserving functional structure of bioactive agents for future tissue engineering applications.

Microfluidic preparation of polymer-lipid Janus microparticles with staged drug release property

This work demonstrated a microfluidic preparation process for novel Janus microparticles with individual drug release properties in each compartment. A flow-focusing microfluidic chip was designed to produce oil-in-water droplets from a mixed solution of poly(lactic-co-glycolic acid) and a triglyceride type lipid. Based on solvent evaporation-induced phase separation, droplets evolved and were solidified into Janus particles, each of which had a polymer compartment and a lipid compartment. The ratio of the two compartments in a particle can be discretionarily regulated, and the particle structure can also be flexibly altered to Janus-patchy, triple, quadruple or core-shell type. Phase transition of the chosen lipid from solid to liquid would occur under physiological temperature, which was applied for rapid release of the loaded drug. The polymer compartment would undergo a slow degradation process in physiological environment, facilitating sustained drug release. Paclitaxel was loaded into Janus particles during preparation, and staged release was achieved, leading to a combination of rapid and sustained release, which is highly desired in target drug delivery. This study would start the application of hybrid Janus particles of polymer-lipid type with novel release kinetics in drug delivery systems.

Visualization study on solid-core encapsulation behaviors of double emulsion in a flow-focusing microchannel

A flow-focusing microfluidic chip based on assembled capillary method is developed and the solid-core encapsulation behaviors of double emulsion in a flow-focusing microchannel are visually observed via high-speed microscopic imaging system. The experimental results identify the encapsulation of single, double and triple cores in a flow-focusing microchannel arising from the competition among the inertial force of inner phase, the shear force of outer phase fluid, and the interfacial tension. In addition, the solid-core encapsulation of double emulsion in flow-focusing microchannel is either in the stable state or in the transition state, depending on the flow rates of inner and outer phase fluid. The stable microencapsulation includes the stable single-core, double-cores and triple-cores encapsulation states, while the transition microencapsulation includes the single-double-cores and double-triple-cores transition encapsulation state. Irrespective of encapsulation state, the solid-core encapsulation process can be classified into four stages, including entering, neck stretch, neck shrinking and breakup.

Silica microcarriers produced from a microfluidics-generated emulsion

Fabrication of micrometric and submicrometric capsules capable of carrying bioactive substances is important for many biological applications. Here we show the results of our work on creating water-in-oil emulsion and encasing the water droplets in silica shell with microfluidic techniques. The emulsion was generated in a polydimethylsiloxane (PDMS) flow-focusing microfluidic chip. TWEEN 80 and lecithin were used as surfactants to stabilize the emulsion. Tetraethylorthosilicate (TEOS) and 3-aminopropyl trimethoxysilane (APTMOS) were then added to the emulsion to form silica shell around water droplets via hydrolysis. We obtained silica microcapsules of about 2 μm in diameter and studied them using confocal fluorescent microscopy. This method allows encapsulating a wide range of sensitive substances including genetic material, enzymes, proteins for intracellular delivery and other biomedical applications.

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 µm to 80 µm in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Micropipette-powered droplet based microfluidics.

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

Digital analysis with droplet-based microfluidic for the ultrasensitive detection of β-gal and AFP

In this study, we presented a digital analysis with droplet-based microfluidic, which was applied for the detection of β-galactosidase (β-Gal) and AFP. The β-Gal was quantified according to the Poisson Distribution in digital analysis with droplets containing β-Gal and fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG). In our method, the lowest concentration of β-Gal we could accurately detect was 5 fM. We found that the digital detection could be applied in quantifying protein biomarkers, take AFP for model sample. The AFP was detected through indirect detection with excess streptavidin-conjugated β-galactosidase (SA-β-gal) as signal tag. To be specific, firstly SA-β-gal was conjugated to biotin-labeled AFP antibody, and then the unbounded signal tag in the reaction solution was taken out for digital detection with droplet-based microfluidic. As a result, the AFP could be also indirectly quantified at an fM level through subtraction. This experimental result demonstrated that the indirect detection with droplet-based digital analysis was able to detect biomarkers at a low level with a few samples on an ordinary flow-focusing microfluidic chip.

The kinetic study of solidification PEGDA microparticles in flow-focusing microfluidic chip

Polyethylene glycol diacrylate (PEGDA) microparticles (MPs) have a great potential in a wider range of applications including cell carriers, drug delivery agents, and multiplexing assays. Among existing MP fabricating methods, only the microfluidic droplet-based methods provide both morphology complexity and substance diversity. This work is concerned with the production PEGDA-based droplets with the photoinitiator (Irgacure 2959) in a flow-focusing microfluidic generator. The kinetics of MPs solidification in mineral oil with various common surfactants (Abil EM 180, Span 80) were investigated. Also, the Young’s modulus of solid PEGDA were obtained.

Synthesis of Gold Nanoparticles Using the Interface of an Emulsion Droplet.

A facile and rapid method for synthesizing single crystal gold spherical or platelet (nonspherical) particles is reported. The reaction takes place at the interface of two immiscible liquids where the reducing agent decamethylferrocene (DmFc) was initially added to hexane and gold chloride (AuCl4-) to an aqueous phase. The reaction is spontaneous at room temperature, leading to the creation of Au nanoparticles (AuNP). A flow focusing microfluidic chip was used to create emulsion droplets, allowing the same reaction to take place within a series of microreactors. The technique allows the number of droplets, their diameter, and even the concentration of reactants in both phases to be controlled. The size and shape of the AuNP are dependent upon the concentration of the reactants and the size of the droplets. By tuning the reaction parameters, the synthesized nanoparticles vary from nanometer to micrometer sized spheres or platelets. The surfactant used to stabilize the emulsion was also shown to influence the particle shape. Finally, the addition of other nanoparticles within the droplet allows for core@shell particles to be readily formed, and we believe this could be a versatile platform for the large scale production of core@shell particles.