Generation Of Gradient Research Articles

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

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

Multicolumn Nanoflow Liquid Chromatography with Accelerated Offline Gradient Generation for Robust and Sensitive Single-Cell Proteome Profiling.

Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.

Enhanced microfluidic multi-target separation by positive and negative magnetophoresis

We introduce magnetophoresis-based microfluidics for sorting biological targets using positive Magnetophoresis (pM) for magnetically labeled particles and negative Magnetophoresis (nM) for label-free particles. A single, externally magnetized ferromagnetic wire induces repulsive forces and is positioned across the focused sample flow near the main channel's closed end. We analyze magnetic attributes and separation performance under two transverse dual-mode magnetic configurations, examining magnetic fields, hydrodynamics, and forces on microparticles of varying sizes and properties. In pM, the dual-magnet arrangement (DMA) for sorting three distinct particles shows higher magnetic gradient generation and throughput than the single-magnet arrangement (SMA). In nM, the numerical results for SMA sorting of red blood cells (RBCs), white blood cells (WBCs), and prostate cancer cells (PC3-9) demonstrate superior magnetic properties and throughput compared to DMA. Magnetized wire linear movement is a key design parameter, allowing device customization. An automated device for handling more targets can be created by manipulating magnetophoretic repulsion forces. The transverse wire and magnet arrangement accommodate increased channel depth without sacrificing efficiency, yielding higher throughput than other devices. Experimental validation using soft lithography and 3D printing confirms successful sorting and separation, aligning well with numerical results. This demonstrates the successful sorting and separating of injected particles within a hydrodynamically focused sample in all systems. Both numerical and experimental findings indicate a separation accuracy of 100% across various Reynolds numbers. The primary channel dimensions measure 100 µm in height and 200 µm in width. N52 permanent magnets were employed in both numerical simulations and experiments. For numerical simulations, a remanent flux density of 1.48 T was utilized. In the experimental setup, magnets measuring 0.5 × 0.5 × 0.125 inches and 0.5 × 0.5 × 1 inch were employed. The experimental data confirm the device's capability to achieve 100% separation accuracy at a Reynolds number of 3. However, this study did not explore the potential impact of increased flow rates on separation accuracy.

Multicompartmentalized microvascularized tumor-on-a-chip to study tumor-stroma interactions and drug resistance in ovarian cancer.

The majority of ovarian cancer (OC) patients receiving standard of care chemotherapy develop chemoresistance within 5 years. The tumor microenvironment (TME) is a dynamic and influential player in disease progression and therapeutic response. However, there is a lack of models that allow us to elucidate the compartmentalized nature of TME in a controllable, yet physiologically relevant manner and its critical role in modulating drug resistance. We developed a 3D microvascularized multiniche tumor-on-a-chip formed by five chambers (central cancer chamber, flanked by two lateral stromal chambers and two external circulation chambers) to recapitulate OC-TME compartmentalization and study its influence on drug resistance. Stromal chambers included endothelial cells alone or cocultured with normal fibroblasts or cancer-associated fibroblasts (CAF). The tumor-on-a-chip recapitulated spatial TME compartmentalization including vessel-like structure, stromal-mediated extracellular matrix (ECM) remodeling, generation of oxygen gradients, and delayed drug diffusion/penetration from the circulation chamber towards the cancer chamber. The cancer chamber mimicked metastasis-like migration and increased drug resistance to carboplatin/paclitaxel treatment in the presence of CAF when compared to normal fibroblasts. CAF-mediated drug resistance was rescued by ECM targeted therapy. Critically, these results demonstrate that cellular crosstalk recreation and spatial organization through compartmentalization are essential to determining the effect of the compartmentalized OC-TME on drug resistance. Our results present a functionally characterized microvascularized multiniche tumor-on-a-chip able to recapitulate TME compartmentalization influencing drug resistance. This technology holds the potential to guide the design of more effective and targeted therapeutic strategies to overcome chemoresistance in OC.

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.

Interface microstructural evolution, microhardness variation mechanism and 750 °C tensile behavior of the HIPed/FGed PM superalloy plastic bonded joints

In present research, a novel 3rd-generation powder metallurgy (PM) superalloys with different initial microstructures (hot isostatic pressed (HIPed) and fine grained (FGed)) were selected for plastic bonding (PB). Non-uniform deformation of the HIPed/FGed PB joint can be obtained due to the significant difference in flow behavior of the studied PM superalloys. Microstructure observation reveals that interface recrystallization dominates the successful healing of bonding interface. The lower dynamic recrystallization (DRX) degree accompanied with higher dislocation density can be detected in HIPed side, manifests that incomplete grain refinement would reduce the efficiency of interface healing and induce the generation of strain gradient at bonding interface. In addition, the microhardness variation of HIPed/FGed PB joint is fully dependent on the interface microstructural evolution. The average microhardness at the bonding interface about 501.0 HV is greatly associated with the neutralization effect of strain hardening from HIPed side and fine grain strengthening from FGed side. Special attention was focused on the fracture behavior and underlying mechanism of the HIPed/FGed PB joint in 750 °C tensile test. Owing to the grain size difference and huge strain gradient, the dislocations pile-up caused by localized stress concentration at bonding interface should be responsible for the brittle fracture of the HIPed/FGed PB joint. The whole process from damage initiation to final fracture includes the microvoids nucleation at grain boundaries, microvoids grow and coalesce into the microcracks, the extending microcracks aggregate to form main crack and then the brittle fracture occurs.

Ion gradient induced self-powered flexible strain sensor

To cope with energy hazards and reduce frequent battery replacement of strain sensors, various self-powered flexible strain sensors (including piezoelectric, triboelectric and electrochemical types) have developed recent years. However, the piezoelectric and triboelectric strain sensors cannot monitor static strain, and electrochemical strain sensors face the problem of performance degradation. Herein, inspired by ion gradient generators, a self-powered flexible strain sensor based on strain regulated ion gradient strategy is constructed. The sensor consists of elastic yarn coated with carbon nanotubes (CNTs), LiCl-filter paper (FP) and latex tube encapsulation. LiCl-FP with excellent water molecules absorption performance is wrapped on one side of the elastic yarn, forming a high humidity region in the sensor. The part of the elastic yarn that is not covered by LiCl-FP has weak water molecules absorption performance, forming a low humidity region in the sensor. The ions in the sensor move directionally from the high humidity region to the low humidity region, forming an ion gradient and generating voltage/current. The test results indicate that the sensor exhibits a wide strain detection range (0.5%–100%) and good repeatability (1000 times). The strain sensing mechanism can be attributed to the strain regulated ion gradient and the resistance strain effect. In addition, the strain sensing function of the sensor is verified by monitoring respiration. This work opens a new avenue for development of self-powered strain sensor using strain regulated ion gradient.

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.

Hydrodynamic metamaterial redirector for steering fluid flow in pipelines with arbitrary curvatures

Hydrodynamic metamaterial redirector for steering fluid flow in pipelines with arbitrary curvatures

266 Combination Drug Therapy in H3 K27-Altered Diffuse Midline Glioma Enhances Tumor Retention and Treatment Efficacy

INTRODUCTION: Diffuse midline gliomas (DMG) with H3 K27 alterations are uniformly lethal tumors for which no therapy has been found to be effective. One reason for our therapy failure is the blood-brain barrier (BBB), which severely restricts uptake and accumulation of systemically administered drugs in the brain. Convection-enhanced delivery (CED) has emerged as a technique that circumvents the BBB by directly infusing therapeutics into the tumor parenchyma through generation of a mechanical pressure gradient, allowing for homogenous drug distribution at high local concentrations with minimal systemic toxicity. However, efflux transport proteins expressed by the BBB and the tumor, including P-gp and BCRP, rapidly clear the majority of drugs given by CED, severely limiting its therapeutic potential. METHODS: A panel of FDA-approved drugs (ponatinib, topotecan, tretinoin) were evaluated alone and in combination with the known efflux transporter inhibitor everolimus. RESULTS: A high degree of formal synergy was observed in multiple DMG cell lines in vitro, which was attributed to everolimus’ ability to inhibit mTOR signaling and P-gp/BCRP. In vivo, drugs that were expected to penetrate the BBB and be less likely to undergo active efflux persisted at the site of infusion the longest following CED. Concurrent dosing with everolimus further enhanced this effect, which was particularly apparent with the dual P-gp/BCRP substrate ponatinib. CONCLUSIONS: The proclivity of many drugs to be rapidly cleared from the brain by efflux transporters generates a vulnerability that can be exploited for the development of novel therapeutic regimens in DMG, such as with concomitant dosing of P-gp/BCRP-inhibiting drugs when using CED.

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.

Gradient-based surface nuclear magnetic resonance for groundwater investigation

In medical magnetic resonance imaging, spatial localization (imaging) is based upon the application of controlled magnetic field gradients on top of the main magnetic field to spatially modulate the frequency and/or phase of the nuclear magnetic resonance (NMR) signal across the volume of investigation. In this work, we have applied similar physical principles to produce controlled magnetic field gradients during surface NMR-based groundwater investigations. In this approach, a gradient pulse of variable amplitude or duration is applied immediately after the excitation pulse to cause predictable phase encoding of the NMR signal as a function of depth. This approach is also applicable to emerging surface NMR detection methods that use a prepolarization field with fast nonadiabatic turn-off to generate detectable NMR signals from the shallow subsurface. In this case, the gradient pulse is applied after terminating the prepolarization field and provides a heretofore unavailable means of localizing the NMR response as a function of depth. The application of gradients can also be combined with tip-angle-based modulation to yield higher imaging resolution than can be achieved through either gradient- or tip-angle-based imaging alone. We implemented this new gradient-based capability into a surface NMR gradient generation accessory that is compatible with the GMR-Flex instrument and developed surface NMR-specific forward modeling and linear inverse models. We validated the accuracy of this novel gradient-based sNMR technology using computer simulations, experiments using a small pool filled with a discrete layer of bulk water, and field experiments at well-characterized groundwater test sites along Ebey Island, WA, and Larned, KS. The gradient-based sNMR imaging observations were compared with high-resolution direct push NMR results observed at these sites. The results of computer simulations and field experiments indicate improvements in both the detection (signal-to-noise ratio) and spatial resolution of shallow subsurface water content using gradient-based surface NMR, compared with traditional surface NMR imaging methods.

Analysis of the response to cigarette smoke exposure in cell coculture and monoculture based on bionic-lung microfluidic chips

BackgroundIn vitro toxicity assessment studies with various experimental models and exposure modalities frequently generate diverse outcomes. In the prevalent experimental, aerosol pollutants are dissolved in culture medium through capture for exposure to two-dimensional planar cellular models in multiwell plates via immersion. However, this approach can generate restricted and inconclusive experimental data, significantly constraining the applicability of risk assessment outcomes. Herein, the in vitro cocultivation of lung epithelial and/or vascular endothelial cells was performed using self-designed bionic-lung microfluidic chip housing a gas-concentration gradient generator (GCGG) unit. Exposure experiments involving a concentration gradient of cigarette smoke (CS) aerosol were then conducted through an original assembled real-time aerosol exposure system. ResultsTranscriptomic analysis revealed a potential involvement of the cGMP-signaling pathway following online CS aerosol exposure on different cell culture models. Furthermore, distinct responses to different concentrations of CS aerosol exposure on different culture models were highlighted by detecting inflammation- and oxidative stress-related biomarkers (i.e., cell viability, reactive oxygen species, nitric oxide, IL-6, IL-8, TNF-α, GM-CSF, malondialdehyde, and superoxide dismutase). SignificantThe results underscore the importance of improving chip biomimicry while addressing multi-throughput demands, given the substantial influence of the coculture model on cellular responses triggered by CS. Furthermore, the coculture model exhibited a mutually beneficial protective effect on cells at low CS concentrations within the GCGG unit, yet revealed a mutually amplified damaging effect at higher CS concentrations in contrast to the monoculture model.

Membrane Transport, Molecular Machines, and Maxwell's Demon

AbstractThe spontaneous generation of transmembrane gradients is an important fundamental research goal for artificial nanotechnology. The active transport processes that give rise to such gradients directly mirror the famous Maxwell's Demon thought experiment, where a Demon partitions particles between two chambers to generate a nonequilibrium state. Despite these similarities, discussion of Maxwell's Demon is absent in the literature on artificial membrane transport. By contrast, the emergence of rational design principles for nonequilibrium artificial molecular motors can trace its intellectual roots directly to this famous thought experiment. This perspective highlights the links between Maxwell's Demon and nonequilibrium machines, and argues that understanding the implications of this 19th century thought experiment is crucial to the future development of transmembrane active transport processes.

Front Cover: An embedded obstacle type micromixer‐concentration gradient generator based on capillary driven

Front Cover: An embedded obstacle type micromixer‐concentration gradient generator based on capillary driven

Efficient Vision Transformer YOLOv5 for Accurate and Fast Traffic Sign Detection

Accurate and fast detection of traffic sign information is vital for autonomous driving systems. However, the YOLOv5 algorithm faces challenges with low accuracy and slow detection when it is used for traffic sign detection. To address these shortcomings, this paper introduces an accurate and fast traffic sign detection algorithm–YOLOv5-Efficient Vision TransFormer(EfficientViT)). The algorithm focuses on improving both the accuracy and speed of the model by replacing the CSPDarknet backbone of the YOLOv5(s) model with the EfficientViT network. Additionally, the algorithm incorporates the Convolutional Block Attention Module(CBAM) attention mechanism to enhance feature layer information extraction and boost the accuracy of the detection algorithm. To mitigate the adverse effects of low-quality labels on gradient generation and enhance the competitiveness of high-quality anchor frames, a superior gradient gain allocation strategy is employed. Furthermore, the strategy introduces the Wise-IoU (WIoU), a dynamic non-monotonic focusing mechanism for bounding box loss, to further enhance the accuracy and speed of the object detection algorithm. The algorithm’s effectiveness is validated through experiments conducted on the 3L-TT100K traffic sign dataset, showcasing a mean average precision (mAP) of 94.1% in traffic sign detection. This mAP surpasses the performance of the YOLOv5(s) algorithm by 4.76% and outperforms the baseline algorithm. Additionally, the algorithm achieves a detection speed of 62.50 frames per second, which is much better than the baseline algorithm.