Concentration Gradient Generator Research Articles

Label-free single-cell antimicrobial susceptibility testing in droplets with concentration gradient generation.

Bacterial communities exhibit significant heterogeneity, resulting in the emergence of specialized phenotypes that can withstand antibiotic exposure. Unfortunately, the existence of subpopulations resistant to antibiotics often goes unnoticed during treatment initiation. Thus, it is crucial to consider the concept of single-cell antibiotic susceptibility testing (AST) to tackle bacterial infections. Nevertheless, its practical application in clinical settings is hindered by its inability to conduct AST efficiently across a wide range of antibiotics and concentrations. This study introduces a droplet-based microfluidic platform designed for rapid single-cell AST by creating an antibiotic concentration gradient. The advantage of a microfluidic platform is achieved by executing bacteria and antibiotic mixing, cell encapsulation, incubation, and enumeration of bacteria in a seamless workflow, facilitating susceptibility testing of each antibiotic. Firstly, we demonstrate the rapid determination of minimum inhibitory concentration (MIC) of several antibiotics with Gram-negative E. coli and Gram-positive S. aureus, which enables us to bypass the time-consuming bacteria cultivation, speeding up the AST in 3 h from 1 to 2 days of conventional methods. Additionally, we assess 10 clinical isolates including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA) against clinically important antibiotics for analyzing the MIC, compared to the gold standard AST method from the United States Clinical and Laboratory Standards Institute (CLSI), which becomes available only after 48 h. Furthermore, by monitoring single cells within individual droplets, we have found a spectrum of resistance levels among genetically identical cells, revealing phenotypic heterogeneity within isogenic populations. This discovery not only advances clinical diagnostics and treatment strategies but also significantly contributes to the field of antibiotic stewardship, underlining the importance of our approach in addressing bacterial resistance.

Engineered microenvironments and pancreatic islet-on-chips for screening sugar substitute and antidiabetic compounds

Recent advancements in the food industry have rekindled interest in the safety of food additives, such as sugar substitutes and food pigments. Consequently, the main purpose of this study was to develop models that can more accurately predict the effects of these additives on the human body. In response to this demand, we have created an innovative pancreas islet-on-a-chip system featuring a concentration gradient generator and a perfusable 3D cell culture array. This setup facilitates the 3D culture of pseudo-islets under stable biochemical and biophysical conditions. When compared to static culture environments, our dynamic environment maintains islet cell viability at over 95 %, resulting in larger cell clusters that exhibit a higher tendency for aggregation up to 30 μm. Furthermore, the expression levels of key factors integral to islet development, namely INS-1, INS-2, and PDX-1, increased by 4.5-fold, 1.9-fold, and 5.8-fold respectively in the dynamic environment. Utilizing this sophisticated pancreas islet-on-a-chip model, we discovered that the consumption of sugar substitutes like erythritol and sucralose for 1 h does not impact insulin secretion levels. In contrast, the administration of glucagon-like peptide 1 (GLP-1), GLP-1 receptor (GLP-1R) agonist exendin-4, curcumin, and a combination therapy group led to a substantial increase in insulin secretion levels (p < 0.01). Such engineered microenvironments and pancreatic islet-on-chips offer a groundbreaking platform for evaluating sugar substitutes and antidiabetic compounds.

Tumor Microenvironment Based on Extracellular Matrix Hydrogels for On-Chip Drug Screening.

Recent advances in three-dimensional (3D) culturing and nanotechnology offer promising pathways to overcome the limitations of drug screening, particularly for tumors like neuroblastoma. In this study, we develop a high-throughput microfluidic chip that integrates a concentration gradient generator (CGG) with a 3D co-culture system, constructing the vascularized microenvironment in tumors by co-culturing neuroblastoma (SY5Y cell line) and human brain microvascular endothelial cells (HBMVECs) within a decellularized extracellular matrix (dECM) hydrogels. The automated platform enhances the simulation of the tumor microenvironment and allows for the precise control of the concentrations of nanomedicines, which is crucial for evaluating therapeutic efficacy. The findings demonstrate that the high-throughput platform can significantly accelerate drug discovery. It efficiently screens and analyzes drug interactions in a biologically relevant setting, potentially revolutionizing the drug screening process.

Integration of concentration gradient generator with competitive PCR in a droplet for rapid nucleic acid quantification

Competitive PCR is an efficient way for the measurement of nucleic acids without requiring a standard curve. It uses a competitor template mimicking the target sequence for direct comparison and quantification during amplification. However, manually preparing various concentrations for competitive PCR is labor-intensive. Additionally, target and competitor DNA bands are often inadequately separated on gels due to their similar sizes. This leads to inaccurate distinction and imprecise measurement of bandwidths and intensities, resulting in high standard deviations in quantification. Thus, this study presents an alternative way to combine droplet competitive PCR with the concentration gradient generator in a microfluidic device that simplifies competitive PCR preparation and provides precise control over reaction conditions and sample handling. Generating a wide range of competitor-to-target ratios in a single experiment enhances statistical reliability and isolating individual reactions in droplets reduces background noise and increases sensitivity. Additionally, it eliminates gel electrophoresis, improves consistency and reproducibility, requires smaller sample and reagent volumes, and allows direct sample quantification through a calibration curve for fast, simple quantification. We envision that our platform can offer a promising alternative to digital PCR, addressing its challenges and driving progress in molecular biology and diagnostics.

Surface Atomic Rearrangement with High Cation Ordering for Ultra‐Stable Single‐Crystal Ni‐Rich Co‐Less Cathode Materials

AbstractIt is crucial to minimize cobalt content in Ni‐rich layered single‐crystal cathodes due to their high price and limited availability, yet it will inevitably lead to cation disordering, capacity degradation, and thermal issues. Herein, to overcome the intrinsic trade‐off between performance and composition of Ni‐rich Co‐less single‐crystal cathodes, a precursor engineering strategy with an epitaxially grown cobalt enrichment on the surface is innovatively proposed. In contrast to traditional coating modifications with random orientation and rigid surface‐bulk boundary, the epitaxially enriched surface cobalt layer on the precursor undergoes rapid interdiffusion with the internal Ni3+ during the optimized sintering process. This interdiffusion eliminates the surface‐bulk boundary, promoting the uniform distribution of cobalt and synergistically addressing the Li/Ni intermixing. Moreover, an enhanced surface Li+ diffusion is obtained, thereby suppressing the Li+ concentration gradient and intragranular cracks generation. Consequently, the modified LiNi0.7Co0.07Mn0.23O2 exhibits impressive cycling stability with increased capacity retention in both coin‐type half‐cells and pouch‐type full‐cells (91% after 1000 cycles), even under the harsh condition of high‐temperature, surpassing the majority of previously reported Ni‐rich cathodes. This work opens new avenues toward the low cost, high energy density, thermal stability, and long cyclic life for Ni‐rich Co‐less cathodes and sheds light on large‐scale commercial production.

Evidence for a transfer-to-trap mechanism of fluorophore concentration quenching in lipid bilayers

It is important to understand the behaviors of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called “fluorescence quenching” occurs when fluorophores are present at high concentrations, but the mechanisms for quenching are debated. Here, we used a technique called “in-membrane electrophoresis” to generate concentration gradients of fluorophores within a supported lipid bilayer, across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behavior of three commonly used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD), and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). FLIM images provided evidence of quenching in regions where the fluorophores accumulated, but the degree of quenching varied between the different fluorophores. The relationship between quenching and concentration was quantified and the “critical radius for trap formation,” representing the relative quenching strength, was calculated as 2.70, 2.02, and 1.14 nm, for BODIPY, TR, and NBD, respectively. The experimental data support the theory that quenching takes place via a “transfer-to-trap” mechanism which proposes, firstly, that excitation energy is transferred between fluorophores and may reach a “trap site,” resulting in immediate energy dissipation, and, secondly, that trap sites are formed in a concentration-dependent manner. Some previous work suggested that quenching occurs only when fluorophores aggregate, or form long-lived dimers, but our data and this theory argue that traps may be “statistical pairs” of fluorophores that exist only transiently. Our findings should inspire future work to assess whether these traps can be charge-transfer states, excited-state dimers, or something else.

A Cautionary Perspective on Hydrogel-Induced Concentration Gradient Generation for Studying Chemotaxis.

The achievement of consistent and static chemical gradients is critically important in the study of diffusion and chemotaxis at the micro- and nanoscales. In this context, a number of groups have reported on hydrogel-based systems for generating concentration gradients. Here, we analyze the behavior of agarose and gelatin-based hydrogels in hybridization chambers of different heights. Our focus is on the issues that are caused by the presence of robust bulk fluid flows in such systems due to the solutes present in the hydrogel and/or the surrounding fluid. We describe the key insights derived from these experiments, offering practical guidelines for establishing gradients using hydrogel-based systems and make the community aware of different variables that can make the experiments nonreproducible and prone to misinterpretations.

A handheld, wide-range pressure pump for portable microfluidic applications

Pumping is an indispensable operation in microfluidic applications. Numerous simple pumps have been developed to improve the portability of microfluidic systems, but they generally have poor controllability, short duration working time, and narrow pressure range, which compromises the functionality of portable microdevices. To address these limitations, we present a novel handheld, wide-range pressure pump that utilizes a disposable mini gas cylinder as a pressure source and a porous PDMS film as a flow-rate regulator. Specifically, by incorporating a set of parallel porous PDMS films with varying porosity, sectional area, and thickness into the pump system, and selectively switching between the branch flow circuits containing these porous PDMS films, we are able to finely regulate the flow rate of air released from the mini gas cylinder. This capability enables precise control of the pumping pressure applied to microfluidic devices over a wide range, thereby governing the fluid flow rate to accommodate different applications. The compact and lightweight nature of our pressure source renders this pump system particularly advantageous for field-portable microfluidic applications. Additionally, we demonstrate its capabilities through the performance of well-established microfluidic experiments, including concentration gradient generation and emulsion droplet production. We firmly believe that the compactness, operational simplicity, and disposability of our pump will greatly facilitate the widespread adoption of microfluidic technologies, particularly in point-of-care diagnostics and resource-limited settings.

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

Independent Concentration Manipulation Using Sidewall-Driven Micromixer.

Lab-on-a-chip technology has been developed to streamline biochemical experiments by providing experimental environments in microscopic areas. Due to the difficulty of mixing chemicals in such small channels, various micromixers have been created. Our proposed sidewall-driven micromixer offers easy fabrication and precise control over mixing concentrations. In our previous study, we successfully generated concentration gradients by simultaneously mixing multiple chambers using a single actuator. However, it is not possible to mix different chemicals in each chamber. In this study, we developed a sidewall-driven micromixer that enables independent mixing in each chamber by installing one actuator per chamber. Experimental results showed that different conditions were achieved in each chamber using both microbead-mixture water and colored water. Thus, this mixer can be used to manipulate concentrations regardless of whether the mixing targets are particles or fluids.

Droplet array with microfluidic concentration gradient (DA-MCG) for 2-dimensional reaction condition screening

Droplet microfluidic technology can use each microdroplet as a microreactor, which has the advantages of low reagent dosage, less cross contamination, and fast reaction time. Combining concentration gradient generation with droplet formation and capture to form a two-dimensional reaction condition screening platform (including reactant concentration and reaction time) is expected to broaden the application range of microfluidic screening. In this work, a microfluidic chip that can dynamically generate and capture microdroplets and form static microdroplet array was designed and fabricated. An optimized Christmas tree structure by adjusting the horizontal channel length ratio was used to generate a chemical concentration gradient while obtaining a uniform outlet flow rate, forming a droplet array with different concentrations. The performance of droplet array with microfluidic concentration gradient (DA-MCG) was verified using sodium fluorescein as a model reagent. The chromogenic reaction of NaOH and phenolphthalein, and luminol chemiluminescence reaction were used to verify the two-dimensional screening ability of DA-MCG. The results indicated that the DA-MCG has the potential to be applied in the field of multi-dimensional drug screening.

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.

Multi-layered Microfluidic Drug Screening Platform Enabling Simultaneous Generation of Linear and Logarithmic Concentration Gradients

Since many microfluidic devices have limited drug dose order of gradients and incorporate 2D cell culture, we here present a multi-layered platform with linear and logarithmic gradients with 3D-cell culturing chambers. By employing Hagen–Poiseuille flow resistance equation and the parallel electric schematics, we determined the appropriate channel dimensions to achieve the desired target concentrations (100%, 50%, 20%, 10%, 5%, 2%, 1%, 0%). To validate the gradient formation against theoretical values, we introduced a solution containing fluorescein into the microfluidic chip. Moreover, cell culturing chambers were spaced out laterally for every 9 mm, aligning with the dimensions with the standard plate reader, providing enhanced usability. Vertical layout of the chip minimized the lateral dimension required for housing various components while maintaining a favorable height for imaging. By preventing the need to use external tubing to connect concentration gradient generator and cell culturing chamber modules, our platform holds promise in facilitating the integration of microfluidics into drug evaluation processes. To demonstrate use of this flexible platform, we tested two chemotherapy drugs against human bladder cancer cells (T24) embedded in 3D fibrin gel and evaluated their cell viability and proliferation rate. IC50 values were extracted for cells exposed to varying doses of cisplatin, gemcitabine, and gemcitabine with a fixed cisplatin dose, confirming the enhanced apoptosis of the bladder cancer cells and the advantages of combination chemotherapy. This simple multi-layered device may accelerate screening of anti-cancer drugs for a specific cell type by extracting optimal dosage for two drugs.

A Multi-Drug Concentration Gradient Mixing Chip: A Novel Platform for High-Throughput Drug Combination Screening.

Combinatorial drug therapy has emerged as a critically important strategy in medical research and patient treatment and involves the use of multiple drugs in concert to achieve a synergistic effect. This approach can enhance therapeutic efficacy while simultaneously mitigating adverse side effects. However, the process of identifying optimal drug combinations, including their compositions and dosages, is often a complex, costly, and time-intensive endeavor. To surmount these hurdles, we propose a novel microfluidic device capable of simultaneously generating multiple drug concentration gradients across an interlinked array of culture chambers. This innovative setup allows for the real-time monitoring of live cell responses. With minimal effort, researchers can now explore the concentration-dependent effects of single-agent and combination drug therapies. Taking neural stem cells (NSCs) as a case study, we examined the impacts of various growth factors-epithelial growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF)-on the differentiation of NSCs. Our findings indicate that an overdose of any single growth factor leads to an upsurge in the proportion of differentiated NSCs. Interestingly, the regulatory effects of these growth factors can be modulated by the introduction of additional growth factors, whether singly or in combination. Notably, a reduced concentration of these additional factors resulted in a decreased number of differentiated NSCs. Our results affirm that the successful application of this microfluidic device for the generation of multi-drug concentration gradients has substantial potential to revolutionize drug combination screening. This advancement promises to streamline the process and accelerate the discovery of effective therapeutic drug combinations.

Deep learning-assisted concentration gradient generation for the study of 3D cell cultures in hydrogel beads of varying stiffness.

The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50Pa to close to 1kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.

Generating Concentration Gradients across Membranes for Molecular Dynamics Simulations of Periodic Systems.

The plasma membrane forms the boundary between a living entity and its environment and acts as a barrier to permeation and flow of substances. Several computational means of calculating permeability have been implemented for molecular dynamics (MD) simulations-based approaches. Except for double bilayer systems, most permeability studies have been performed under equilibrium conditions, in large part due to the challenges associated with creating concentration gradients in simulations utilizing periodic boundary conditions. To enhance the scientific understanding of permeation and complement the existing computational means of characterizing membrane permeability, we developed a non-equilibrium method that enables the generation and maintenance of steady-state gradients in MD simulations. We utilize PBCs advantageously by imposing a directional bias to the motion of permeants so that their crossing of the boundary replenishes the gradient, like a previous study on ions. Under these conditions, a net flow of permeants across membranes may be observed to determine bulk permeability by a direct application of J=PΔc. In the present study, we explore the results of its application to an exemplary O2 and POPC bilayer system, demonstrating accurate and precise permeability measurements. In addition, we illustrate the impact of permeant concentration and the choice of thermostat on the permeability. Moreover, we demonstrate that energetics of permeation can be closely examined by the dissipation of the gradient across the membrane to gain nuanced insights into the thermodynamics of permeability.

Electroacoustic Responsive Cochlea-on-a-Chip.

Organ-on-chips can highly simulate the complex physiological functions of organs, exhibiting broad application prospects in developmental research, disease simulation, as well as new drug research and development. However, there is still less concern about effectively constructing cochlea-on-chips. Here, a novel cochlear organoids-integrated conductive hydrogel biohybrid system with cochlear implant electroacoustic stimulation (EAS) for cochlea-on-a-chip construction and high-throughput drug screening, is presented. Benefiting from the superior biocompatibility and electrical property of conductive hydrogel, together with cochlear implant EAS, the inner ear progenitor cells can proliferate and spontaneously shape into spheres, finally forming cochlear organoids with good cell viability and structurally mature hair cells. By incorporating these progenitor cells-encapsulated hydrogels into a microfluidic-based cochlea-on-a-chip with culture chambers and a concentration gradient generator, a dynamic and high-throughput evaluation of inner ear disease-related drugs is demonstrated. These results indicate that the proposed cochlea-on-a-chip platform has great application potential in organoid cultivation and deafness drug evaluation.

Spontaneous re-arrangement of evaporating suspension into mesh-patterns towards concentration gradient generation on a chip

Spontaneous re-arrangement of evaporating suspension into mesh-patterns towards concentration gradient generation on a chip

High-throughput 3D microfluidic chip for generation of concentration gradients and mixture combinations.

Concentration gradient generation and mixed combinations of multiple solutions are of great value in the field of biomedical research. However, existing concentration gradient generators for single or two-drug solutions cannot simultaneously achieve multiple concentration gradient formations and mixed solution combinations. Furthermore, the whole system was huge, and required expensive auxiliary equipment, which may lead to complex operations. To address this problem, we devised a novel 3D microchannel network design, which is capable of creating all the desired mixture combinations and concentration gradients of given small amounts of the input solutions. As a proof of concept, the device we presented was verified by both colorimetric and fluorescence detection methods to test the efficiency. This can enable the implementation of one to three solutions with no driving pump and facilitate unique multiple types of more concentration gradients and mixture combinations in a single operation. We envision that this will be a promising candidate for the development of simplified methods for screening of the appropriate concentration and combination, such as various drug screening applications.