Microfluidic Generation Research Articles

3D printed microfluidic devices produced by material extrusion: effect of ironing parameters on mixing efficiency and gradient generation

Purpose This study aims to describe the effect of ironing process parameters on mixing efficiency and gradient generation in Y-micromixers and microfluidic gradient generators (MGGs), respectively. Design/methodology/approach Material extrusion (MEX) enables the production of miniaturized devices with the advantage of lower manufacturing costs and higher design freedom. However, surface finishing is the most important drawback when it comes to microfluidic applications where flow splitting is not required. First, the effect of ironing line spacing (LS) and speed (IS) on mixing efficiency in Y-micromixers was experimentally investigated. Then, the best ironing settings were chosen to further study the spatial stability of the normalized concentration gradient in MGGs. Findings Lower ironing LS and IS enhance the microchannel surface smoothness. The best combination of ironing parameters (lowest values of LS and IS) leads to an increase in mixing length of 191% at Q = 10 µL/min and 198% at Q = 20 µL/min, with respect to a similar Y-micromixer geometry where ironing was not performed. These findings were applied in the production of a MGG, showing that the normalized concentration gradient in the crosswise flow direction does not depend on the streamwise position when ironing is performed. Originality/value To the best of the authors’ knowledge, for the first time, the possibility of optimizing ironing parameters to enhance the surface roughness in MEX microfluidic devices has been investigated. Ironing of the channel bottom surface allows to reduce ridges-induced flow convection, thus delaying mixing in Y-micromixers and achieving stable concentration gradient in MGGs.

Facile and versatile PDMS-glass capillary double emulsion formation device coupled with rapid purification toward microfluidic giant liposome generation

The exceptional ability of liposomes to mimic a cellular lipid membrane makes them invaluable tools in biomembrane studies and bottom-up synthetic biology. Microfluidics provides a promising toolkit for creating giant liposomes in a controlled manner. Nevertheless, challenges associated with the microfluidic formation of double emulsions, as precursors to giant liposomes, limit the full exploration of this potential. In this study, we propose a PDMS-glass capillary hybrid device as a facile and versatile tool for the formation of double emulsions which not only eliminates the need for selective surface treatment, a well-known problem with PDMS formation chips, but also provides fabrication simplicity and reusability compared to the glass-capillary formation chips. These advantages make the presented device a versatile tool for forming double emulsions with varying sizes (spanning two orders of magnitude in diameter), shell thickness, number of compartments, and choice of solvents. We achieved robust thin shell double emulsion formation by operating the hybrid chip in double dripping mode without performing hydrophilic/phobic treatment a priori. In addition, as an alternative to the conventional, time-consuming density-based separation method, a tandem separation chip is developed to deliver double emulsions free of any oil droplet contamination in a continuous and rapid manner without any need for operator handling. The applicability of the device was demonstrated by forming giant liposomes using the solvent extraction method. This easy-to-replicate, flexible, and reliable microfluidic platform for the formation and separation of double emulsion templates paves the way for the high-throughput microfluidic generation of giant liposomes and synthetic cells, opening exciting avenues for biomimetic research.The presented giant liposome assembly line features a novel treatment-free hybrid chip for double emulsion formation coupled with a high throughput separation chip for sample purification.

A Microfluidic Design for Quantitative Measurements of Shear Stress-Dependent Adhesion and Motion of Dictyostelium discoideum Cells.

Shear stress plays a crucial role in modulating cell adhesion and signaling. We present a microfluidic shear stress generator used to investigate the adhesion dynamics of Dictyostelium discoideum, an amoeba cell model organism with well-characterized adhesion properties. We applied shear stress and tracked cell adhesion, motility, and detachment using time-lapse videomicroscopy. In the precise shear conditions generated on-chip, our results show cell migration patterns are influenced by shear stress, with cells displaying an adaptive response to shear forces as they alter their adhesion and motility behavior. Additionally, we observed that DH1-10 wild-type D. discoideum cells exhibit stronger adhesion and resistance to shear-induced detachment compared to phg2 adhesion-defective mutant cells. We also highlight the influence of cell density on detachment kinetics.

Thermo-controlled microfluidic generation of monodisperse alginate microspheres based on external gelation.

Droplet-based microfluidic systems have received much attention as promising tools for fabricating monodisperse microspheres of alginate solutions with high accuracy and reproducibility. The immediate and simple ionotropic gelation of alginate, its biocompatibility, and its tunability of mechanical properties make it a favorable hydrogel in the biomedical and tissue engineering fields. In these fields, micron-sized alginate hydrogel spheres have shown high potential as cell vehicles and drug delivery systems. Although on-chip microfluidic gelation of the produced alginate droplets is common, several challenges remain. Complicated chemical and microfabrication processes are required, and the risk of microchannel clogging is high. In the current study, we present an easy-to-use microfluidic external gelation process to produce highly spherical and monodisperse microspheres from very low-concentrated alginate-RGD solution [0.5% (w/v)]. To accomplish this, gelatin, a thermo-sensitive and inexpensive biomaterial, was incorporated into the alginate solution as a sacrificial biomaterial that mediates the off-chip external gelation of the alginate with Ca2+, and avoids droplet coalescence. Utilizing the methodology mentioned above, we successfully generated monodisperse alginate microspheres (AMs) with diameters ranging from 27 μm to 46 μm, with a coefficient of variation of 0.14, from a mixture of Arg-Gly-Asp (RGD)-modified very low viscosity alginate and gelatin. These RGD-AMs were used as microcarriers for human umbilical vein endothelial cells. The described easy-to-use and cost-effective microfluidic off-chip external gelation strategy exhibits comparable advantages to on-chip external gelation and demonstrates superiority over the latter since clogging is impossible.

High-throughput affinity measurements of direct interactions between activation domains and co-activators.

Sequence-specific activation by transcription factors is essential for gene regulation1,2. Key to this are activation domains, which often fall within disordered regions of transcription factors3,4 and recruit co-activators to initiate transcription5. These interactions are difficult to characterize via most experimental techniques because they are typically weak and transient6,7. Consequently, we know very little about whether these interactions are promiscuous or specific, the mechanisms of binding, and how these interactions tune the strength of gene activation. To address these questions, we developed a microfluidic platform for expression and purification of hundreds of activation domains in parallel followed by direct measurement of co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). By applying STAMMPPING to quantify direct interactions between eight co-activators and 204 human activation domains (>1,500 K ds), we provide the first quantitative map of these interactions and reveal 334 novel binding pairs. We find that the metazoan-specific co-activator P300 directly binds >100 activation domains, potentially explaining its widespread recruitment across the genome to influence transcriptional activation. Despite sharing similar molecular properties (e.g. enrichment of negative and hydrophobic residues), activation domains utilize distinct biophysical properties to recruit certain co-activator domains. Co-activator domain affinity and occupancy are well-predicted by analytical models that account for multivalency, and in vitro affinities quantitatively predict activation in cells with an ultrasensitive response. Not only do our results demonstrate the ability to measure affinities between even weak protein-protein interactions in high throughput, but they also provide a necessary resource of over 1,500 activation domain/co-activator affinities which lays the foundation for understanding the molecular basis of transcriptional activation.

A high-throughput flowless microfluidic single and multi-solute concentration gradient generator: Design and parametric study.

Microfluidic concentration gradient generators (μ-CGGs) are critical in various biochemical assays, including cell migration, drug screening, and antimicrobial susceptibility testing. However, current μ-CGGs rely on integration with flow systems, limiting their scalability and widespread adoption owing to limited infrastructure and technical expertise. Hence, there is a need for flowless diffusional gradient generators capable of standalone operation, thereby improving throughput and usability. In this study, we model such a diffusional μ-CGG as an infinite source-sink system to capture two characteristic timescales: (i) gradient generation dictated by the diffusion timescale and (ii) stability determined by the rate of change in reservoir concentrations. Through finite-element simulations, we explored the influence of various geometric parameters such as the channel length, cross-sectional area, node and reservoir volumes, and the solute diffusivity on these timescales, along with experimental confirmation using fluorescent tracer diffusion. Our results show that while the gradient stability strongly depends on the reservoir volumes, diffusion length, and solute diffusion coefficient, they are independent of the node shape or the shape of the channel cross section. However, gradient profiles were found to be the strong functions of the diffusion length, solute diffusivity, and the geometric pattern of the microfluidic grid. Additionally, we showcased the versatility of the design by generating discrete gradient profiles and combinatorial gradients of two and three solutes, thus improving throughput in a wide range of on-chip biological assays. These findings underscore the potential of our microfluidic device as an easy-to-use, inexpensive, efficient, and high-throughput platform for various on-chip biological assays.

A review on the development and application of microfluidic concentration gradient generators

A review on the development and application of microfluidic concentration gradient generators

Atmospheric Pressure Enhanced Self-Sealing Rotation-SlipChip with Programmable Concentration Gradient Generation for Microbiological Applications.

In microbiological research, traditional methods for bacterial screening and antibiotic susceptibility testing are resource-intensive. Microfluidics offers an efficient alternative with rapid results and minimal sample consumption, but the demand for cost-effective, user-friendly platforms persists in communities and hospitals. Inspired by the Magdeburg hemispheres, the strategy adapts to local conditions, leveraging omnipresent atmospheric pressure for self-sealing of Rotation-SlipChip (RSC) equipped with a 3D circular Christmas tree-like microfluidic concentration gradient generator. This innovative approach provides an accessible and adaptable platform for microbiological research and testing in diverse settings. The RSC can avoid leakage concerns during multiple concentration gradient generation, chip-rotating, and final long-term incubation reaction (≥24h). Furtherly, RSC subtypes adapted to different reactions can be fabricated in less than 15min with cost less than $1, the result can be read through designated observational windows by naked-eye. Moreover, the RSC demonstrates its capability for evaluating bacterial biomarker activity, enabling the rapid assessment of β-galactosidase concentration and enzyme activity within 30min, and the limit of detection can be reduced by 10-fold. It also rapidly determines the minimum antibiotic inhibitory concentration and antibiotic combined medications results within 4 h. Overall, these low-cost and user-friendly RSC make them invaluable tools in determinations at previously impractical environment.

ATPSpin: A Single Microfluidic Platform that Produces Diversified ATPS-Alginate Microfibers.

Microfluidic spinning is emerging as a useful technique in the fabrication of alginate fibers, enabling applications in drug screening, disease modeling, and disease diagnostics. In this paper, by capitalizing on the benefits of aqueous two-phase systems (ATPS) to produce diverse alginate fiber forms, we introduce an ATPS-Spinning platform (ATPSpin). This ATPS-enabled method efficiently circumvents the rapid clogging challenges inherent to traditional fiber production techniques by regulating the interaction between alginate and cross-linking agents like Ba2+ ions. By varying system parameters under the guidance of a regime map, our system produces several fiber forms─solid, hollow, and droplet-filled─consistently and reproducibly from a single device. We demonstrate that the resulting alginate fibers possess distinct features, including biocompatibility. We also encapsulate HEK293 cells in the microfibers as a proof-of-concept that this versatile microfluidic fiber generation platform may have utility in tissue engineering and regenerative medicine applications.

A tempo-spatial controllable microfluidic shear-stress generator for in-vitro mimicking of the thrombus

Pathological conditions linked to shear stress have been identified in hematological diseases, cardiovascular diseases, and cancer. These conditions often exhibit significantly elevated shear stress levels, surpassing 1000 dyn/cm2 in severely stenotic arteries. Heightened shear stress can induce mechanical harm to endothelial cells, potentially leading to bleeding and fatal consequences. However, current technology still grapples with limitations, including inadequate flexibility in simulating bodily shear stress environments, limited range of shear stress generation, and spatial and temporal adaptability. Consequently, a comprehensive understanding of the mechanisms underlying the impact of shear stress on physiological and pathological conditions, like thrombosis, remains inadequate. To address these limitations, this study presents a microfluidic-based shear stress generation chip as a proposed solution. The chip achieves a substantial 929-fold variation in shear stress solely by adjusting the degree of constriction in branch channels after PDMS fabrication. Experiments demonstrated that a rapid increase in shear stress up to 1000 dyn/cm2 significantly detached 88.2% cells from the substrate. Long-term exposure (24 h) to shear stress levels below 8.3 dyn/cm2 did not significantly impact cell growth. Furthermore, cells exposed to shear stress levels equal to or greater than 8.3 dyn/cm2 exhibited significant alterations in aspect ratio and orientation, following a normal distribution. This microfluidic chip provides a reliable tool for investigating cellular responses to the wide-ranging shear stress existing in both physiological and pathological flow conditions.Graphical

Fully 3D-printed, nonelectric, spring-powered syringe pump for operating microfluidic devices

Microfluidic devices require a precise and steady flow for fluid control. The high demand for microfluidics in point-of-care testing (POCT) necessitates a portable, low-cost, and electricity-free pumping method for operating microfluidic platforms in low-resource settings. This study presents a fully 3D-printed and portable syringe pump operated by torque from a flat spiral spring. The syringe pump can be operated by manually winding up the spring, and various flow rates can be obtained using different syringe sizes and selecting appropriate gear combinations. It generated a steady flow rate ranging from 1.4 µL/min to 12.0 µL/min with high repeatability. The pump is suitable for the on-site use of microfluidic platforms because it does not require electricity and does not occupy a large space. The 3D printed pump proposed in this study is named precise, rapid-prototyped, nonelectric, torque-driven (PRNTD) pump. The versatility and utility of the pump are demonstrated by generating a linear concentration gradient using a microfluidic gradient generator and microdroplets using a T-channel microfluidic chip. The PRNTD pump can facilitate the on-site application of microfluidic platforms in resource-limited settings and contribute to the widespread use of microfluidic technologies.

Microfluidic technologies for lipid vesicle generation.

Encapsulating biological and non-biological materials in lipid vesicles presents significant potential in both industrial and academic settings. When smaller than 100 nm, lipid vesicles and lipid nanoparticles are ideal vehicles for drug delivery, facilitating the delivery of payloads, improving pharmacokinetics, and reducing the off-target effects of therapeutics. When larger than 1 μm, vesicles are useful as model membranes for biophysical studies, as synthetic cell chassis, as bio-inspired supramolecular devices, and as the basis of protocells to explore the origin of life. As applications of lipid vesicles gain prominence in the fields of nanomedicine, biotechnology, and synthetic biology, there is a demand for advanced technologies for their controlled construction, with microfluidic methods at the forefront of these developments. Compared to conventional bulk methods, emerging microfluidic methods offer advantages such as precise size control, increased production throughput, high encapsulation efficiency, user-defined membrane properties (i.e., lipid composition, vesicular architecture, compartmentalisation, membrane asymmetry, etc.), and potential integration with lab-on-chip manipulation and analysis modules. We provide a review of microfluidic lipid vesicle generation technologies, focusing on recent advances and state-of-the-art techniques. Principal technologies are described, and key research milestones are highlighted. The advantages and limitations of each approach are evaluated, and challenges and opportunities for microfluidic engineering of lipid vesicles to underpin a new generation of therapeutics, vaccines, sensors, and bio-inspired technologies are presented.

Microfluidic generation of bioinspired core–shell structured microfibers for cultured meat

The simulation of traditional meat characteristics is critical to cultured meat production. We propose bioinspired microfibers with solidified alginate shells and porcine muscle stem cell (PMSC) loaded hydrogel cores. The core–shell structure not only provide a spatially confined environment for directional cell alignment, but also enable the design of a soft hydrogel core to facilitate cell migration and mutual fusion. The myogenic differentiation and muscle protein synthesis of PMSCs in microfibers are thus significantly promoted, which even lead to the spontaneous contraction of cellular microfibers. Microfibers are assembled into bulk cultured meat tissues by integrating the microfluidics and 3D printing technologies. Benefiting from the efficient cell myogenesis, the appearance, texture, and protein composition of the constructed cultured meat are similar to those of native pork. By combining multiple microfluidic devices into the printing system, the simultaneous printing of multiple pieces of cultured meat can be realized as well. These features show the broad prospect of the proposed microfibers in efficient and large-scale production of cultured meat with traditional meat characteristics.

Neural-physics multi-fidelity model with active learning and uncertainty quantification for GPU-enabled microfluidic concentration gradient generator design

The microfluidic concentration gradient generator (μCGG) is an important biomedical device to generate concentration gradients (CGs) of biomolecules at the microscale. Nonetheless, determining their operational parameter values to generate complex, user-specific, biologically desired CGs is not trivial. This paper presents a neural-physics multi-fidelity model (NP-MFM) to predict CGs with equivalent accuracy as high-fidelity CFD simulation at ultra-fast computational speed through a novel uncertainty- and distance-based active learning process. The verified NP-MFM, along with the genetic algorithm, is implemented on a GPU platform to search optimal values of operational parameters that generate CGs closely matching user-prescribed profiles. Results show that the NP-MFM is a feasible multi-fidelity modeling approach for rapid and accurate prediction of CGs (with 0.019s/simulation) and can be used for GPU-enabled μCGG design optimization and automation. Furthermore, design CGs generated by the proposed method match user-prescribed CGs very well with an averaged discrepancy less than 0.34.

High throughput microfluidic nanobubble generation by microporous membrane integration and controlled bubble shrinkage

Microfluidics has recently been proposed as a viable method for producing bulk nanobubbles for use in various applications. The portability, compact size, and capacity to precisely control fluids on a small scale are a few of the benefits of microfluidics that may be exploited to create customized bulk nanobubbles. However, despite the potential of microfluidic nanobubble generation, low throughput and limited nanobubble concentration remain challenging for microfluidics. Here, we integrate a microporous silicon membrane into a polydimethylsiloxane microfluidic chip to generate bulk nanobubbles in the 100–140 nm diameter range with a concentration of up to 108 mL−1. We investigate the nanobubble size and morphology using several characterisation techniques, including transmission electron microscopy, resonance mass measurement, dynamic light scattering, and the Tyndall effect. This new nanobubble generation technique can increase nanobubble concentration by ∼ 23 times compared to earlier microfluidic nanobubble generation platforms, which should increase the feasibility of translation to medical applications.

Multi-fidelity surrogate-based optimization for microfluidic concentration gradient generator design

PurposeThis paper aims to present a multi-fidelity surrogate-based optimization (MFSBO) method for computationally accurate and efficient design of microfluidic concentration gradient generators (µCGGs).Design/methodology/approachCokriging-based multi-fidelity surrogate model (MFSM) is constructed to combine data with varying fidelities and computational costs to accelerate the optimization process and improve design accuracy. An adaptive sampling approach based on parallel infill of multiple low-fidelity (LF) samples without notably adding computation burden is developed. The proposed optimization framework is compared with a surrogate-based optimization (SBO) method that relies on data from a single source, and a conventional multi-fidelity adaptive sampling and optimization method in terms of the convergence rate and design accuracy.FindingsThe results demonstrate that proposed MFSBO method allows faster convergence and better designs than SBO for all case studies with 49% more reduction in the objective function value on average. It is also found that parallel infill (MFSBO-4) with four LF samples, enables more robust, efficient and accurate designs than conventional multi-fidelity infill (MFSBO-1) that only adopts one LF sample during each iteration for more complex optimization problems.Originality/valueA MFSM based on cokriging method is constructed to utilize data with varying fidelities, accuracies and computational costs for µCGG design. A parallel infill strategy based on multiple infill criteria is developed to accelerate the convergence and improve the design accuracy of optimization. The proposed methodology is proved to be a feasible method for µCGG design and its computational efficiency is verified.

Microfluidic Generation of Near-Infrared Photothermal Vitexin/ICG Liposome with Amplified Photodynamic Therapy.

Glioma, in which a malignant tumor cell occurs in neural mesenchymal cells, has a rapid progression and poor prognosis, which is still far from desirable in clinical treatments. We developed a lab-on-a-chip (LOC) device for the rapid and efficient preparation of vitexin/indocyanine green (ICG) liposomes. Vitexin could be released from liposome to kill cancer cell, which can potentially improve the glioma therapeutic effect and reduce the treatment time through synergistic photodynamic/photothermal therapies (PDT/PTT). The vitexin/ICG liposome was fabricated via LOC and its physicochemical property and release in vitro were evaluated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and live/dead staining were used to examine the enhanced antitumor effect of vitexin/ICG liposome in cooperation with PDT/PTT, while the related mechanism was explored by flow cytometry and western blot. The results were as follows: (1) The prepared vitexin/ICG liposome was smaller in size, homogenous in particle size distribution with significant low polydispersity index (PDI), and enhanced cumulative release in vitro. (2) We found that the formulated liposome presented strong cancer cell inhibition and suppression of its migration in a dose-dependent manner. (3) Further mechanistic studies showed that liposome combined with near-infrared irradiation could significantly upregulate levels of B cell lymphoma 2-associated X (Bax) protein and decrease B cell lymphoma 2 (Bcl-2) at protein levels. The vitexin/ICG liposomes prepared based on a simple LOC platform can effectively enhance the solubility of insoluble drugs, and the combined effect of PTT/PDT can effectively increase their antitumor effect, which provides a simple and valid method for the clinical translation of liposomes.

Ultralow and Dynamic Flow Field Generator Composed of Microfluidic Peristaltic Pump

Precise control over flow rate and flow pattern is critical for investigating the contribution of shear force in cellular responses, biofluidic transport, and living matter in a flowing environment. Generation and control over the ultralow flow fields at microscale are, however, difficult, due to thermal agitation and fluctuations in the surrounding environment. Herein, pa novel microfluidic flow field generator is proposed, capable of generating stable flow field down to the limit of thermal noise. Integrated with a customized MATLAB program controlling timely open–close of 18 monolithic PDMS valves, the flow generator outperforms the existing methods by both achievable low flow rate, that is, 0.01 nL s−1, temporal resolution of flow rate fluctuation down to 100 ms, and provides complex flow patterns. Moreover, unlike external devices (e.g., the syringe pump), the design can be easily integrated into any fluidic chip and sealed from the disturbance of experimental conditions, for example, the airflow and the movement of connected pipelines. The methodology is, therefore, suitable for biological and biophysical studies requiring a well‐defined flowing environment.

High throughput measurements of direct activation domain-coactivator interactions.

Human gene expression is regulated by over two thousand transcription factors and chromatin regulator proteins. Activation domains (ADs) within these proteins recruit shared co-activators and Pol II to genes to activate transcription. However, for many human ADs we do not know which co-activators they recruit, if ADs bind single co-activators specifically or many promiscuously, and why some ADs are stronger at activating gene expression than others. In previous work, we have measured the effect on gene expression for thousands of human ADs and mutants inside cells. Here, we developed a microfluidic platform for expression and purification of hundreds of these ADs in parallel followed by direct measurement of AD/co-activator binding affinities (STAMMPPING, for Simultaneous Trapping of Affinity Measurements via a Microfluidic Protein-Protein INteraction Generator). STAMMPPING can reliably quantify interaction strengths ranging across two orders of magnitude (Kds from 0.5 to 50 μM). To date, we used STAMMPPING to measure over 3000 interactions between 204 ADs and 15 co-activators, identifying both specific and promiscuous interactions. Affinities between ADs and individual domains of the co-activator P300 identified ADs that bind multiple domains of the same co-activator, suggesting longer lived total interactions can be achieved with individual short-lived interactions. Complementary mutagenesis of ADs revealed that decreasing their strength of binding to co-activators proportionally decreases their ability to activate gene expression in cells. Our systematic quantification of AD/co-activator interactions advances our understanding of how human ADs activate gene expression. Moreover, direct affinity data produced here will be a rich resource for mapping gene regulatory networks and building interpretable predictive models of AD/co-activator binding.

Towards skin-on-a-chip for screening the dermal absorption of cosmetics.

Over the past few decades, there have been increasing global efforts to limit or ban the use of animals for testing cosmetic products. This ambition has been at the heart of international endeavours to develop new in vitro and animal-free approaches for assessing the safety of cosmetics. While several of these new approach methodologies (NAMs) have been approved for assessing different toxicological endpoints in the UK and across the EU, there remains an absence of animal-free methods for screening for dermal absorption; a measure that assesses the degree to which chemical substances can become systemically available through contact with human skin. Here, we identify some of the major technical barriers that have impacted regulatory recognition of an in vitro skin model for this purpose and propose how these could be overcome on-chip using artificial cells engineered from the bottom-up. As part of our future perspective, we suggest how this could be realised using a digital biomanufacturing pipeline that connects the design, microfluidic generation and 3D printing of artificial cells into user-crafted synthetic tissues. We highlight milestone achievements towards this goal, identify future challenges, and suggest how the ability to engineer animal-free skin models could have significant long-term consequences for dermal absorption screening, as well as for other applications.