Six logical steps that connect the introduction of the Nernst equation to its clinical application.

The Nernst equation is key to understanding the electrophysiology of the cell membrane and the pathophysiology of K+ imbalances (i.e., hyperkalemia and hypokalemia). However, in our experience teaching medical students over the years, many students struggle to make connections between a brief introduction of the Nernst equation and its clinical application to K+ imbalances. This article aims to connect the introduction of the equation to its clinical application to understand K+ imbalances using six logical steps with detailed visual illustrations that make the connection explicit and cohesive. In addition, we highlight a few common areas related to the six steps that are often overlooked by both teachers and students. Students who are able to thoroughly demonstrate an understanding of all the six steps highlighted in this article will achieve mastery of this topic.NEW & NOTEWORTHY This article fills the gaps in teaching about the Nernst equation, which is important in medical physiology. Six logical steps are presented that connect the introduction of the equation to its clinical applications to hyperkalemia and hypokalemia, two conditions that can be life-threatening if left untreated. Only when students know how to apply the equation will their learning transition from surface to mastery.

Microfluidic devices for neutrophil chemotaxis studies.

Neutrophil chemotaxis plays a vital role in human immune system. Compared with traditional cell migration assays, the emergence of microfluidics provides a new research platform of cell chemotaxis study due to the advantages of visualization, precise control of chemical gradient, and small consumption of reagents. A series of microfluidic devices have been fabricated to study the behavior of neutrophils exposed on controlled, stable, and complex profiles of chemical concentration gradients. In addition, microfluidic technology offers a promising way to integrate the other functions, such as cell culture, separation and analysis into a single chip. Therefore, an overview of recent developments in microfluidic-based neutrophil chemotaxis studies is presented. Meanwhile, the strength and drawbacks of these devices are compared.

Selective Autonomous Molecular Transport and Collection by Hydrogel-Embedded Supramolecular Chemical Gradients.

Selective transport and concentration of molecules to specified regions on a substrate both enhances the potential to detect such molecules and provides a path to spatially localize such molecules prior to initiation of subsequent chemical reactions. Here, we first embed radially symmetric α-, ß-, and γ-cyclodextrin gradients in a hydrogel matrix. Driven by host-guest interactions between the cyclodextrins and the target molecule, we observe these gradients can serve to direct 2D molecular transport. Using xanthene dyes and organophosphates as target molecules, we found the transport metrics, e.g., selectivity, rate, and concentration limits, are strongly dependent on the specific cyclodextrin forming the gradient. In all cases, as the concentrating power of the gradient increased, the rate of target concentration slowed, which we hypothesize is because stronger interactions between the target and the cyclodextrin decrease the rate of target diffusion. The concentration enhancement for the nerve agent simulant 4-methylumbelliferyl phosphate (15.8) is the greatest when the gradient is formed using ß-cyclodextrin while directed concentration of cyanomethyl phosphonate, a smaller non-aromatic organophosphate, is observed only for the smaller α-CD. To provide a near real-time read-out of the concentration of the analyte, we used an array of IR resonant metallic nanoantennas tuned to a specific IR absorption band of the analyte to enhance the IR signal generated by the analyte.

A hybrid actuated microrobot using an electromagnetic field and flagellated bacteria for tumor-targeting therapy.

In this paper, we propose a new concept for a hybrid actuated microrobot for tumor-targeting therapy. For drug delivery in tumor therapy, various electromagnetic actuated microrobot systems have been studied. In addition, bacteria-based microrobot (so-called bacteriobot), which use tumor targeting and the therapeutic function of the bacteria, has also been proposed for solid tumor therapy. Compared with bacteriobot, electromagnetic actuated microrobot has larger driving force and locomotive controllability due to their position recognition and magnetic field control. However, because electromagnetic actuated microrobot does not have self-tumor targeting, they need to be controlled by an external magnetic field. In contrast, the bacteriobot uses tumor targeting and the bacteria's own motility, and can exhibit self-targeting performance at solid tumors. However, because the propulsion forces of the bacteria are too small, it is very difficult for bacteriobot to track a tumor in a vessel with a large bloodstream. Therefore, we propose a hybrid actuated microrobot combined with electromagnetic actuation in large blood vessels with a macro range and bacterial actuation in small vessels with a micro range. In addition, the proposed microrobot consists of biodegradable and biocompatible microbeads in which the drugs and magnetic particles can be encapsulated; the bacteria can be attached to the surface of the microbeads and propel the microrobot. We carried out macro-manipulation of the hybrid actuated microrobot along a desired path through electromagnetic field control and the micro-manipulation of the hybrid actuated microrobot toward a chemical attractant through the chemotaxis of the bacteria. For the validation of the hybrid actuation of the microrobot, we fabricated a hydrogel microfluidic channel that can generate a chemical gradient. Finally, we evaluated the motility performance of the hybrid actuated microrobot in the hydrogel microfluidic channel. We expect that the hybrid actuated microrobot will be utilized for tumor targeting and therapy in future.

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.

Recent Developments in Bacterial Chemotaxis Analysis Based on the Microfluidic System.

Bacterial motility in response to chemicals, also called bacterial chemotaxis, is a critical ability to search for the optimal environment to ensure the survival of bacterial species. Recent advances in microbiology have allowed the engineering of bacterial chemotactic properties. Conventional methods for characterizing bacterial motility are not able to fully monitor chemotactic behavior. Developments in microfluidic technology have enabled the designing of new experimental protocols in which spatiotemporal control of the cellular microenvironment can be achieved, and in which bacterial motility can be precisely and quantitatively measured and compared. This review provides an overview of recent developments of and new insights into microfluidic systems for chemotaxis assay.

Multi-scale design of the chela of the hermit crab Coenobita brevimanus.

The chela of the hermit crab protects its body against the attack from predators. Yet, a deep understanding of this mechanical defense is still lacking. Here, we investigate the chela of hermit crab, Coenobita brevimanus, and establish the relationships between the microstructures, chemical compositions and mechanical properties to gain insights into its biomechanical functions. We find that the chela is a multi-layered shell composed of five different layers with distinct features of the microstructures and chemical compositions, conferring different mechanical properties. Especially, an increase of the calcium carbonate content towards the layer furthest from the exterior, unlike the chemical gradients of many crustacean exoskeletons, provides a strong resistance to deformation. Nanoindentation measurements reveal that the overall gradient of the elastic modulus and hardness in the cross-section displays a sandwich profile, i.e., a soft core clamped by two stiff surface layers. Further mechanics modeling demonstrates that the high curvature and stiff innermost sublayer enhance the structural rigidity of the chela. In conjunction with the experimental observations, dynamic finite element analysis maps the time-spatial distribution of principal stress and indicates that fiber bridging might be the major mechanism against crack propagation at microscale. The lessons gained from the study of this multiphase biological composite could provide important insights into the design and fabrication of bioinspired materials for structural applications. STATEMENT OF SIGNIFICANCE: Multiple hierarchical structures have been discovered in a variety of exoskeletons. They are naturally designed to maintain the structural integrity and act as a protective layer for the animals. However, each kind of the hierarchical structures has its unique topology, chemical gradients as well as mechanical properties. We find that the chela is multi-layered shell composed of five different layers with distinct features of the microstructures and chemical compositions, conferring different mechanical properties. Especially, a large amount of helicoidal organic fibrils form highly organized 3D woven matrix in the innermost layer, providing a strong mechanical resistance to avoid catastrophic failure. The overall gradient of the elastic modulus and hardness in the cross-section display a sandwich profile, effectively minimizing the stress concentration and deformation. The lessons gained from the multiscale design strategy of the chela provide important insights into the design and fabrication of bioinspired materials.

Optimized Hierarchical Structure and Chemical Gradients Promote the Biomechanical Functions of the Spike of Mantis Shrimps.

The tail spike of the mantis shrimp is the appendage for counteracting the enemy from behind. Here, we investigate the correlations between the chemical compositions, the microstructures, and the mechanical properties of the spike. We find that the spike is a hollow beam with a varying cross section along the length. The cross section comprises four different layers with distinct features of microstructures and chemical compositions. The local mechanical properties of these layers correlate well with the microstructures and chemical compositions, a combination of which effectively restricts the crack propagation while maximizing the release of strain energy during deformation. Finite element analysis and mechanics modeling demonstrate that the optimized structure of the spike confines the mechanical damage in the region near the tip and prevents catastrophic breakage at the base. Furthermore, we use a 3D printing technique to fabricate multiple hollow cylindrical samples consisting of biomimetic microstructures of the spike and confirm that the combination of the Bouligand structure with radially oriented parallel sheets greatly improves the toughness and strength during compression tests. The multiscale design strategy of the spike revealed here is expected to be of great interest for the development of novel bioinspired materials.

A combined structural and wettability gradient surface for directional droplet transport and efficient fog collection.

HYPOTHESIS: The droplet manipulation behavior is affected by chemical structural driving force (including the superposition of electric, magnetic, optical and thermal fields), which directly determine transportation velocity. A lot of research has focused on a single driving force that induces the directional transportation behavior, which affects its performance. EXPERIMENTS: A simple method for preparing wettability gradient conical copper needles (WGCCN) combining structural gradient and chemical gradient was formulated. The effect of droplet volume and tilt angles on droplet transport velocity was systematically studied. The process of droplet transport was revealed through theoretical model and mechanical analysis. Finally, the application of WGCCN and its array model in fog collection were explored. FINDINGS: A continuous chemical gradient in the conical structure gradient induces the droplet directional transportation, and the transportation velocity depends on the droplet volume. In addition, under the cooperation effect of multiple driving force, the droplet can still be transported in a directional orientation even if it is tilted at a certain angle. The simple droplet manipulation behavior portends that the droplets directional transport behavior can be applied in microfluidic manipulation by cooperation of effective multiple driving force with satisfactory results.

Patterning Neuroepithelial Cell Sheet via a Sustained Chemical Gradient Generated by Localized Passive Diffusion Devices.

Recent advances in human pluripotent stem cells (hPSCs)-derived in vitro models open a new avenue for studying early stage human development. While current approaches leverage the self-organizing capability of hPSCs, it remains unclear whether extrinsic morphogen gradients are sufficient to pattern neuroectoderm tissues in vitro. While microfluidics or hydrogel-based approaches to generate chemical gradients are well-established, these systems either require continuous pumping or encapsulating cells in gels, making it difficult for adaptation in standard biology laboratories and downstream analysis. In this work, we report a new device design that leverages localized passive diffusion, or LPaD for short, to generate a stable chemical gradient in an open environment. As LPaD is operated simply by media changing, common issues for microfluidic systems such as leakage, bubble formation, and contamination can be avoided. The device contains a slit carved in a film filled with solid gelatin and connected to a static aqueous morphogen reservoir. Concentration gradients generated by the device were visualized via DAPI fluorescent intensity and were found to be stable for up to 168 h. Using this device, we successfully induced cellular response of Madin-Darby canine kidney (MDCK) cells to the concentration gradient of a small-molecule drug, cytochalasin D. Furthermore, we efficiently patterned the dorsal-ventral axis of hPSC-derived forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal gradient generated by the LPaD devices. Together, LPaD devices are powerful tools to control the local chemical microenvironment for engineering organotypic structures in vitro.

Surface, Interface, and Bulk Electronic and Chemical Properties of Complete Perovskite Solar Cells: Tapered Cross-Section Photoelectron Spectroscopy, a Novel Solution.

The surface, interface, and bulk properties are a few of the most critical factors that influence the performance of perovskite solar cells. The photoelectron spectroscopy (PES) is used as a technique to analyze these properties. However, the information depth of PES is limited to 10-20 nm, which makes it not suitable to study the complete devices, which have a thickness of ∼1 µm. Here, we introduce a novel and simple technique of PES on a tapered cross section (TCS-PES). It provides both lateral and vertical resolutions compared to the conventional PES so that it is suitable to study a complete perovskite solar cell. It offers many benefits over conventional PES methods such as the chemical composition in the micrometer scale from the surface to the bulk and the electronic properties at the multiple interfaces. The chemical natures of different layers of the perovskite-based solar cells [(FAPbI3)0.85(MAPbBr3)0.15] can be identified precisely for the first time using the TCS-PES method. We found that the perovskite layer has higher iodine concentration at the Spiro/perovskite interface and higher bromine concentration at the TiO2/perovskite interface. UPS measurements on the tapered cross section revealed that the perovskite is n-type, and the solar cell studied here is a p-n-n structure type device. The unique possibilities to analyze the complete solar cell by XPS and UPS allow us to estimate the band bending in a working solar cell. Moreover, this technique can further be used to study the device under operating conditions, and it can be applied in other solid-state devices like solid electrolyte Li-ion batteries, LEDs, or photoelectrodes.

Emergent spatiotemporal dynamics of the actomyosin network in the presence of chemical gradients.

We used particle-based computer simulations to study the emergent properties of the actomyosin cytoskeleton. Our model accounted for biophysical interactions between filamentous actin and non-muscle myosin II and was motivated by recent experiments demonstrating that spatial regulation of myosin activity is required for fibroblasts responding to spatial gradients of platelet derived growth factor (PDGF) to undergo chemotaxis. Our simulations revealed the spontaneous formation of actin asters, consistent with the punctate actin structures observed in chemotacting fibroblasts. We performed a systematic analysis of model parameters to identify biochemical steps in myosin activity that significantly affect aster formation and performed simulations in which model parameter values vary spatially to investigate how the model responds to chemical gradients. Interestingly, spatial variations in motor stiffness generated time-dependent behavior of the actomyosin network, in which actin asters continued to spontaneously form and dissociate in different regions of the gradient. Our results should serve as a guide for future experimental investigations.

pH sensing by lipids in membranes: The fundamentals of pH-driven migration, polarization and deformations of lipid bilayer assemblies.

Most biological molecules contain acido-basic groups that modulate their structure and interactions. A consequence is that pH gradients, local heterogeneities and dynamic variations are used by cells and organisms to drive or regulate specific biological functions including energetic metabolism, vesicular traffic, migration and spatial patterning of tissues in development. While the direct or regulatory role of pH in protein function is well documented, the role of hydrogen and hydroxyl ions in modulating the properties of lipid assemblies such as bilayer membranes is only beginning to be understood. Here, we review approaches using artificial lipid vesicles that have been instrumental in providing an understanding of the influence of pH gradients and local variations on membrane vectorial motional processes: migration, membrane curvature effects promoting global or local deformations, crowding generation by segregative polarization processes. In the case of pH induced local deformations, an extensive theoretical framework is given and an application to a specific biological issue, namely the structure and stability of mitochondrial cristae, is described. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Yeast chemotropism: A paradigm shift in chemical gradient sensing.

The ability of cells to direct their movement and growth in response to shallow chemical gradients is essential in the life cycles of all eukaryotic organisms. The signaling mechanisms underlying directional sensing in chemotactic cells have been well studied; however, relatively little is known about how chemotropic cells interpret chemical gradients. Recent studies of chemotropism in budding and fission yeast have revealed 2 quite different mechanisms-biased wandering of the polarity complex, and differential internalization of the receptor and G protein. Each of these mechanisms has been proposed to play a key role in decoding mating pheromone gradients. Here we explore how they may work together as 2 essential components of one gradient sensing machine.

Microfluidic Mixing and Analog On-Chip Concentration Control Using Fluidic Dielectrophoresis.

Microfluidic platforms capable of complex on-chip processing and liquid handling enable a wide variety of sensing, cellular, and material-related applications across a spectrum of disciplines in engineering and biology. However, there is a general lack of available active microscale mixing methods capable of dynamically controlling on-chip solute concentrations in real-time. Hence, multiple microfluidic fluid handling steps are often needed for applications that require buffers at varying on-chip concentrations. Here, we present a novel electrokinetic method for actively mixing laminar fluids and controlling on-chip concentrations in microfluidic channels using fluidic dielectrophoresis. Using a microfluidic channel junction, we co-flow three electrolyte streams side-by-side so that two outer conductive streams enclose a low conductive central stream. The tri-laminar flow is driven through an array of electrodes where the outer streams are electrokinetically deflected and forced to mix with the central flow field. This newly mixed central flow is then sent continuously downstream to serve as a concentration boundary condition for a microfluidic gradient chamber. We demonstrate that by actively mixing the upstream fluids, a variable concentration gradient can be formed dynamically downstream with single a fixed inlet concentration. This novel mixing approach offers a useful method for producing variable on-chip concentrations from a single inlet source.

Integration of PCL and PLA in a monolithic porous scaffold for interface tissue engineering.

A novel bi-layered multiphasic scaffold (BLS) have been fabricated for the first time by combining melt mixing, compression molding and particulate leaching. One layer has been composed by polylactic acid (PLA) presenting pore size in the range of 90-110µm while the other layer has been made of polycaprolactone (PCL) with pores ranging from 5 to 40µm. The different chemo-physical properties of the two biopolymers combined with the tunable pore architecture permitted to realize monolithic functionally graded scaffolds engineered to be potentially used for interface tissues regenerations. BLS have been characterized from a morphological and a mechanical point of view. In particular, mechanical tests have been carried out both in air and immersing the specimens in phosphate buffered saline (PBS) solution at 37°C, in order to evaluate the elastic modulus and the interlayer adhesion strength. Fibroblasts and osteoblasts have been cultured and co-cultured in order to investigate the cells permeation trough the different layers. The results indicate that the presented method is appropriate for the preparation of multiphasic porous scaffolds with tunable morphological and mechanical characteristics. Furthermore, the cells seeded were found to grow with a different trend trough the different layers thus demonstrating that the presented device has good potential to be used in interface tissue regeneration applications.

Quantitative analysis of Caenorhabditis elegans chemotaxis using a microfluidic device.

Caenorhabditis elegans, one of the widely studied model organisms, sense external chemical cues and perform relative chemotaxis behaviors through its simple chemosensory neuronal system. To study the mechanism underlying chemosensory behavior, a rapid and reliable method for quantitatively analyzing the worms' behaviors is essential. In this work, we demonstrated a microfluidic approach for investigating chemotaxis responses of worms to chemical gradients. The flow-based microfluidic chip was consisted of circular tree-like microchannels, which was able to generate eight flow streams containing stepwise chemical concentrations without the difference in flow velocity. Worms' upstream swimming into microchannels with various concentrations was monitored for quantitative analysis of the chemotaxis behavior. By using this microfluidic chip, the attractive and repellent responses of C. elegans to NaCl were successfully quantified within several minutes. The results demonstrated the wild type-like repellent responses and severely impaired attractive responses in grk-2 mutant animals with defects in calcium influx. In addition, the chemotaxis analysis of the third stage larvae revealed that its gustatory response was different from that in the adult stage. Thus, our microfluidic method provided a useful platform for studying the chemosensory behaviors of C. elegans and screening of chemosensation-related chemical drugs.

Hydrogel patterning by diffusion through the matrix and subsequent light-triggered chemical immobilization.

A novel approach to hyaluronic acid (HA) hydrogel with a chemical gradient of the matrix-linked bisphosphonate (BP) groups is presented. The method consists of two steps, including initial generation of physical gradient patterns of BPs by diffusion of BP acrylamide reagent into HA matrix carrying thiol groups and subsequent chemical immobilization of the BP groups by UV light-triggered thiol-ene addition reaction. This gradient hydrogel permits spatial three-dimensional regulation of secondary interactions of different molecules with the polymer matrix. In particular, graded amounts of cytochrome c (cyt c) were reversibly absorbed in the hydrogel, thus enabling the subsequent spatially controlled release of the therapeutic protein. The obtained patterned hydrogel acts also as a unique reactor in which peroxidase-catalyzed oxidation of a substrate is determined by spatial position of the enzyme (cyt c) in the matrix resulting in a range of product concentrations. As an example, matrix template-assisted oxidation of 3,3',5,5'-tetarmethylbenzydine (TMB) in the presence of H2O2 occurs simultaneously at different rates within the gradient hydrogel. Moreover, calcium binding to the gradient HABP hydrogel reflects the pattern of immobilized BP groups eventually leading to the graded biomineralization of the matrix. This approach opens new possibilities for use of hydrogels as dynamic models for biologic three-dimensional structures such as extracellular matrix.

Surface Chemical Gradient Affects the Differentiation of Human Adipose-Derived Stem Cells via ERK1/2 Signaling Pathway.

To understand the role of surface chemistry on cell behavior and the associated molecular mechanisms, we developed and utilized a surface chemical gradient of amine functional groups by carefully adjusting the gas composition of 1,7-octadiene (OD) and allylamine (AA) of the plasma phase above a moving substrate. The chemical gradient surface used in the present work shows an increasing N/C ratio and wettability from the OD side toward the AA side with no change in surface topography. Under standard culture conditions (with serum), human adipose-derived stem cells (hASCs) adhesion and spreading area increased toward the AA side of the gradient. However, there were no differences in cell behavior in the absence of serum. These results, supported by the trends in proteins adsorption on the gradient surface, demonstrated that surface chemistry affects the response of hASCs through cell-adhesive serum proteins, rather than interacting directly with the cells. The expression of p-ERK and the osteogenic differentiation increased toward the AA side of the gradient, while adipogenic differentiation decreased in the same direction; however, when the activation of ERK1/2 was blocked by PD98059, the levels of osteogenic or adipogenic differentiation on different regions of the chemical gradient were the same. This indicates that ERK1/2 may be an important downstream signaling pathway of surface chemistry directed stem cell fate.

Patterning gradients.

Surface-chemical and -morphological gradients can be extremely useful in cell-biological research as high-throughput screening tools-for example, exposing a given set of cells to many different surface conditions at once, under identical ambient conditions, in order to monitor cell behavior such as proliferation or specific gene expression. They can also be used to investigate the effects of gradients themselves on cell behavior, such as migration. A number of simple, reliable techniques for both chemical- and morphological-gradient fabrication have been developed in our laboratories and are described in detail in the following.