Microfluidic Gradient Generator Research Articles

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.

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.

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.

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.

Design of Christmas-Tree-like Microfluidic Gradient Generators for Cell-Based Studies

Microfluidic gradient generators (MGGs) provide a platform for investigating how cells respond to a concentration gradient or different concentrations of a specific chemical. Among these MGGs, those based on Christmas-tree-like structures possess advantages of precise control over the concentration gradient profile. However, in designing these devices, the lengths of channels are often not well considered so that flow rates across downstream outlets may not be uniform. If these outlets are used to culture cells, such non-uniformity will lead to different fluidic shear stresses in these culture chambers. As a result, cells subject to various fluidic stresses may respond differently in aspects of morphology, attachment, alignment and so on. This study reports the rationale for designing Christmas-tree-like MGGs to attain uniform flow rates across all outlets. The simulation results suggest that, to achieve uniform flow rates, the lengths of vertical channels should be as long as possible compared to those of horizontal channels, and modifying the partition of horizontal channels is more effective than elongating the lengths of vertical channels. In addition, PMMA-based microfluidic chips are fabricated to experimentally verify these results. In terms of chemical concentrations, perfect linear gradients are observed in devices with modified horizontal channels. This design rationale will definitely help in constructing optimal MGGs for cell-based applications including chemotherapy, drug resistance and drug screening.

Scalable large-area mesh-structured microfluidic gradient generator for drug testing applications.

Microfluidic concentration gradient generators are useful in drug testing, drug screening, and other cellular applications to avoid manual errors, save time, and labor. However, expensive fabrication techniques make such devices prohibitively costly. Here, in the present work, we developed a microfluidic concentration gradient generator (μCGG) using a recently proposed non-conventional photolithography-less method. In this method, ceramic suspension fluid was shaped into a square mesh by controlling Saffman Taylor instability in a multiport lifted Hele-Shaw cell (MLHSC). Using the shaped ceramic structure as the template, μCGG was prepared by soft lithography. The concentration gradient was characterized and effect of the flow rates was studied using COMSOL simulations. The simulation result was further validated by creating a fluorescein dye (fluorescein isothiocanate) gradient in the fabricated μCGG. To demonstrate the use of this device for drug testing, we created various concentrations of an anticancer drug-curcumin-using the device and determined its inhibitory concentration on cervical cancer cell-line HeLa. We found that the IC50 of curcumin for HeLa matched well with the conventional multi-well drug testing method. This method of μCGG fabrication has multiple advantages over conventional photolithography such as: (i) the channel layout and inlet-outlet arrangements can be changed by simply wiping the ceramic fluid before it solidifies, (ii) it is cost effective, (iii) large area patterning is easily achievable, and (iv) the method is scalable. This technique can be utilized to achieve a broad range of concentration gradient to be used for various biological and non-biological applications.

Machine-Learning-Enabled Design and Manipulation of a Microfluidic Concentration Gradient Generator.

Microfluidics concentration gradient generators have been widely applied in chemical and biological fields. However, the current gradient generators still have some limitations. In this work, we presented a microfluidic concentration gradient generator with its corresponding manipulation process to generate an arbitrary concentration gradient. Machine-learning techniques and interpolation algorithms were implemented to help researchers instantly analyze the current concentration profile of the gradient generator with different inlet configurations. The proposed method has a 93.71% accuracy rate with a 300× acceleration effect compared to the conventional finite element analysis. In addition, our method shows the potential application of the design automation and computer-aided design of microfluidics by leveraging both artificial neural networks and computer science algorithms.

Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose–response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

Advances in Concentration Gradient Generation Approaches in a Microfluidic Device for Toxicity Analysis.

This systematic review aimed to analyze the development and functionality of microfluidic concentration gradient generators (CGGs) for toxicological evaluation of different biological organisms. We searched articles using the keywords: concentration gradient generator, toxicity, and microfluidic device. Only 33 of the 352 articles found were included and examined regarding the fabrication of the microdevices, the characteristics of the CGG, the biological model, and the desired results. The main fabrication method was soft lithography, using polydimethylsiloxane (PDMS) material (91%) and SU-8 as the mold (58.3%). New technologies were applied to minimize shear and bubble problems, reduce costs, and accelerate prototyping. The Christmas tree CGG design and its variations were the most reported in the studies, as well as the convective method of generation (61%). Biological models included bacteria and nematodes for antibiotic screening, microalgae for pollutant toxicity, tumor and normal cells for, primarily, chemotherapy screening, and Zebrafish embryos for drug and metal developmental toxicity. The toxic effects of each concentration generated were evaluated mostly with imaging and microscopy techniques. This study showed an advantage of CGGs over other techniques and their applicability for several biological models. Even with soft lithography, PDMS, and Christmas tree being more popular in their respective categories, current studies aim to apply new technologies and intricate architectures to improve testing effectiveness and reduce common microfluidics problems, allowing for high applicability of toxicity tests in different medical and environmental models.

Modeling-based design specifications for microfluidic gradients generators for biomedical applications

This work proposes a comprehensive approach to optimize the design of microfluidic concentration gradient generators (MGGs) for biomedical applications. Exposing biological systems to controlled gradients enables fast screenings of induced concentration-dependent signals and surpasses several limitations of conventional cell culture techniques. The MGG working principle is the formation of diffusion-driven concentration gradients between a source and a sink, both connected to a cell culture chamber through an array of microchannels. The devices were modeled with Comsol Multiphysics®, in a combined fluid-dynamic and mass-transport study allowing prediction of the internal fields and guiding design optimization. Ideal MGGs must ensure fast transients (< 1 h) to reach a steady gradient. To identify the key features determining the device performance, we analyzed design specifications and operating parameters including: the shape of the source and sink (width 1–2 mm), the number and length of the microchannels (17–34, and 2.5–5 mm), the flow rate (5–10 μl/min). Our results prove that the formation and shape of the gradient are strongly affected by the device geometry, and mostly by the microchannels length. In addition, higher flow rates lead to the generation of stagnation zones and increase the gradient steepness. The model optimized MGG was fabricated and proved successful in the generation of stable concentration gradients over cell monolayers inside the culture chamber.

Emergence of Resistant Escherichia coli Mutants in Microfluidic On-Chip Antibiotic Gradients

Spatiotemporal structures and heterogeneities are common in natural habitats, yet their role in the evolution of antibiotic resistance is still to be uncovered. We applied a microfluidic gradient generator device to study the emergence of resistant bacteria in spatial ciprofloxacin gradients. We observed biofilm formation in regions with sub-inhibitory concentrations of antibiotics, which quickly expanded into the high antibiotic regions. In the absence of an explicit structure of the habitat, this multicellular formation led to a spatial structure of the population with local competition and limited migration. Therefore, such structures can function as amplifiers of selection and aid the spread of beneficial mutations. We found that the physical environment itself induces stress-related mutations that later prove beneficial when cells are exposed to antibiotics. This shift in function suggests that exaptation occurs in such experimental scenarios. The above two processes pave the way for the subsequent emergence of highly resistant specific mutations.

Hand-powered vacuum-driven microfluidic gradient generator for high-throughput antimicrobial susceptibility testing

The growth of bacterial resistance to antimicrobials is a serious problem attracting much attention nowadays. To prevent the misuse and abuse of antimicrobials, it is important to carry out antibiotic susceptibility testing (AST) before clinical use. However, conventional AST methods are relatively laborious and time-consuming (18–24 h). Here, we present a hand-powered vacuum-driven microfluidic (HVM) device, in which a syringe is used as the only vacuum source for rapid generating concentration gradient of antibiotics in different chambers. The HVM device can be preassembled with various amounts of antibiotics, lyophilized, and stored for ready-to-use. Bacterial samples can be loaded into the HVM device through a simple suction step. With the assistance of Alamar Blue, the AST assay and the minimum inhibitory concentration (MIC) of different antibiotics can be investigated by comparing the growth results of bacteria in different culture chambers. In addition, a parallel HVM device was proposed, in which eight AST assays can be performed simultaneously. The results of MIC of three commonly used antibiotics against E. coli K-12 in our HVM device were consistent with those obtained by traditional method while the detection time was shortened to less than 8 h. We believe that our platform is high-throughput, cost-efficient, easy to use, and suitable for POCT applications.

A 3D-printed microfluidic gradient generator with integrated photonic silicon sensors for rapid antimicrobial susceptibility testing.

With antimicrobial resistance becoming a major threat to healthcare settings around the world, there is a paramount need for rapid point-of-care antimicrobial susceptibility testing (AST) diagnostics. Unfortunately, most currently available clinical AST tools are lengthy, laborious, or are simply inappropriate for point-of-care testing. Herein, we design a 3D-printed microfluidic gradient generator that automatically produces two-fold dilution series of clinically relevant antimicrobials. We first establish the compatibility of these generators for classical AST (i.e., broth microdilution) and then extend their application to include a complete on-chip label-free and phenotypic AST. This is accomplished by the integration of photonic silicon chips, which provide a preferential surface for microbial colonization and allow optical tracking of bacterial behavior and growth at a solid-liquid interface in real-time by phase shift reflectometric interference spectroscopic measurements (PRISM). Using Escherichia coli and ciprofloxacin as a model pathogen-drug combination, we successfully determine the minimum inhibitory concentration within less than 90 minutes. This gradient generator-based PRISM assay provides an integrated AST device that is viable for convenient point-of-care testing and offers a promising and most importantly, rapid alternative to current clinical practices, which extend to 8-24 h.

Modular 3D In Vitro Artery-Mimicking Multichannel System for Recapitulating Vascular Stenosis and Inflammation.

Inflammation and the immune response in atherosclerosis are complex processes involving local hemodynamics, the interaction of dysfunctional cells, and various pathological environments. Here, a modular multichannel system that mimics the human artery to demonstrate stenosis and inflammation and to study physical and chemical effects on biomimetic artery models is presented. Smooth muscle cells and endothelial cells were cocultured in the wrinkled surface in vivo-like circular channels to recapitulate the artery. An artery-mimicking multichannel module comprised four channels for the fabrication of coculture models and assigned various conditions for analysis to each model simultaneously. The manipulation became reproducible and stable through modularization, and each module could be replaced according to analytical purposes. A chamber module for culture was replaced with a microfluidic concentration gradient generator (CGG) module to achieve the cellular state of inflamed lesions by providing tumor necrosis factor (TNF)-α, in addition to the stenosis structure by tuning the channel geometry. Different TNF-α doses were administered in each channel by the CGG module to create functional inflammation models under various conditions. Through the tunable channel geometry and the microfluidic interfacing, this system has the potential to be used for further comprehensive research on vascular diseases such as atherosclerosis and thrombosis.

Complete experimental and theoretical characterization of nonlinear concentration gradient generator microfluidic device for analytical purposes.

Microfluidic devices that generate stable concentration gradients are efficient instruments for automated calibration for analytical and bioanalytical systems. However, little attention has been paid tothe development of reusable microfluidic concentration gradient generators, which can be useful for a range of species through mathematical characterization. In this work, we develop a microfluidic device based on three steps of serial dilution that were able to generate nonlinear concentration gradient for dyes and biomolecules. The microfluidic device was described mathematically, statistically and was suitable for reusable analytical and bioanalytical analysis. The device reproducibility was assessed by experimental tests, which have shown the same gradient concentration profile for different dyes and statistical reproducibility with 95% confidence interval for bovine serum albumin (BSA). Moreover, the experimental data converged well withthose obtained by computational fluid dynamics simulation. Applicability was verified by coupling the microfluidic device to a surface plasmon resonance (SPR) biosensor, based on nanohole arrays with sensitivity of 358.7 nm RIU-1 determined by white-light SPR excitation exposed to different D-(+)-glucose aqueous solutions with 1.3361-1.4035 refractive index interval. The transmission light intensities obtained by the array of images allowed to quantify a pseudo-unknown BSA sample (160 µg mL-1) at 138 µg mL-1. The SPR analysis has been validated in parallel by fluorescence emissions, which showed a concentration of 154.8 ± 16.6 µg mL-1.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

Development of a Flow-free Gradient Generator Using a Self-Adhesive Thiol-acrylate Microfluidic Resin/Hydrogel (TAMR/H) Hybrid System.

Microfluidic gradient generators have been used to study cellular migration, growth, and drug response in numerous biological systems. One type of device combines a hydrogel and polydimethylsiloxane (PDMS) to generate “flow-free” gradients; however, their requirements for either negative flow or external clamps to maintain fluid-tight seals between the two layers have restricted their utility among broader applications. In this work, a two-layer, flow-free microfluidic gradient generator was developed using thiol-ene chemistry. Both rigid thiol-acrylate microfluidic resin (TAMR) and diffusive thiol-acrylate hydrogel (H) layers were synthesized from commercially available monomers at room temperature and pressure using a base-catalyzed Michael addition. The device consisted of three parallel microfluidic channels negatively imprinted in TAMR layered on top of the thiol-acrylate hydrogel to facilitate orthogonal diffusion of chemicals to the direction of flow. Upon contact, these two layers formed fluid-tight channels without any external pressure due to a strong adhesive interaction between the two layers. The diffusion of molecules through the TAMR/H system was confirmed both experimentally (using fluorescent microscopy) and computationally (using COMSOL). The performance of the TAMR/H system was compared to a conventional PDMS/agarose device with a similar geometry by studying the chemorepulsive response of a motile strain of GFP-expressing Escherichia coli. Population-based analysis confirmed a similar migratory response of both wild-type and mutant E. coli in both of the microfluidic devices. This confirmed that the TAMR/H hybrid system is a viable alternative to traditional PDMS-based microfluidic gradient generators and can be used for several different applications.

A simple, low cost and reusable microfluidic gradient strategy and its application in modeling cancer invasion

Microfluidic chemical gradient generators enable precise spatiotemporal control of chemotactic signals to study cellular behavior with high resolution and reliability. However, time and cost consuming preparation steps for cell adhesion in microchannels as well as requirement of pumping facilities usually complicate the application of the microfluidic assays. Here, we introduce a simple strategy for preparation of a reusable and stand-alone microfluidic gradient generator to study cellular behavior. Polydimethylsiloxane (PDMS) is directly mounted on the commercial polystyrene-based cell culture surfaces by manipulating the PDMS curing time to optimize bonding strength. The stand-alone strategy not only offers pumpless application of this microfluidic device but also ensures minimal fluidic pressure and consequently a leakage-free system. Elimination of any surface treatment or coating significantly facilitates the preparation of the microfluidic assay and offers a detachable PDMS microchip which can be reused following to a simple cleaning and sterilization step. The chemotactic signal in our microchip is further characterized using numerical and experimental evaluations and it is demonstrated that the device can generate both linear and polynomial signals. Finally, the feasibility of the strategy in deciphering cellular behavior is demonstrated by exploring cancer cell migration and invasion in response to chemical stimuli. The introduced strategy can significantly decrease the complexity of the microfluidic chemotaxis assays and increase their throughput for various cellular and molecular studies.