Dynamic tissue model in vitro and its application for assessment of microplastics-induced toxicity to air-blood barrier (ABB).

The replication of the hominine physiological environment was identified as an effectual strategy to develop the physiological model in vitro to perform the intuitionistic assessment of toxicity of contaminations. Herein, we proposed a dynamic interface strategy that accurately mimicked the blood flow and shear stress in human capillaries to subtly evaluate the physiological damages. To proof the concept, the dynamic air-blood barrier (ABB) model in vitro was developed by the dynamic interface strategy and was utilized to assess the toxicity of polyethylene terephthalate microplastics (PET-MPs). The developed dynamic ABB model was compared with the static ABB model developed by the conventional Transwell® system and the animal model, then the performance of the dynamic ABB model in evaluation of the PET-MPs induced pulmonary damage via replicating the hominine ABB. The experimental data revealed that the developed dynamic ABB model in vitro effectively mimicked the physiological structure and barrier functions of human ABB, in which more sophisticated physiological microenvironment enabled the distinguishment of the toxicities of PET-MPs in different sizes and different concentrations comparing with the static ABB model constructed on Transwell® systems. Furthermore, the consistent physiological and biochemical characters adopted dynamic ABB model could be achieved in a quick manner referring with that of the mouse model in the evaluation of the microplastics-induced pulmonary damage. The proposed dynamic interface strategy supplied a general approach to develop the hominine physiological environment in vitro and exhibited a potential to develop the ABB model in vitro to evaluate the hazards of inhaled airborne pollutants.

Localized hydrodynamic flow confinement assisted nanowire sensor for ultrasensitive protein detection.

Affordable methods for ultra-sensitive biomarkers detection may improve the standard of living in resource-constrained countries. Nanowire biosensor is preponderant in ultra-sensitive protein detection. However, current strategies for nanowire sensor (NWS) fabrication often require sophisticated instruments, being inaccessible in less-resourced laboratories. Herein, we circumvent this challenge by developing a simple methodology, localized hydrodynamic flow confinement assisted nanowire sensor fabrication, enable the detection with limits of detection (LOD) for IgA and IgG measurement were 0.089 fg/mL and 0.93 fg/mL, respectively, demonstrating a 10-fold increase in detection sensitivity compared with the published NWS. Noteworthy, an X-Y positioner combined with a homemade microchemical pen (MCP) for tunable chemical deposition were sufficient to complete the fabrication of the nanowire biosensor without other expensive and demanding equipment. Overall, a particularly accessible, competitive, and low-cost approach of nanowire sensor fabrication for ultra-sensitive protein detection was developed, which could widely facilitate the application of nanowire biosensors. Besides, the nanowire sensor can also be employed to detect other analytes of interest by the use of different stimuli-responsive biosystems.

Microsphere amplified fluorescence and its application in sensing.

The far-field fluorescence amplification, the intense fluorescence emission addresses the great potential in sensitive detection to large biomolecules, was seriously ignored for the failure in amplifying the weak fluorescence excepting the electromagnetic field (EM) induced fluorescence amplification on the metallic surfaces. Here, a microsphere in hundreds of micrometers was adopted to proceed with the fluorescence amplification via building up a local dielectric surrounding for fluorophore. The wide range of contribution-angle fluorescence could be efficiently restricted within the microsphere by facilitating the energy of reflection restraining and declining the energy of refraction decaying and the intense fluorescence emission confined within the microsphere could be directly observed. The proposed microsphere amplified fluorescence was demonstrated to induce about 2600 times of improved sensitivity in the detection of the fluorescent resorufin referring that of the original resorufin solution through the laser induced fluorescence (LIF). Furthermore, the limit of detection (LOD) of human IgA was successfully obtained to 3.25 fM through the microsphere in 47.7 pL when the microsphere amplified fluorescence was utilized in the fluoroimmunoassay. We believe the microsphere amplified fluorescence would be a potential strategy to implement the sensitive fluorescence sensing.

Mechanism of Budded Virus Envelope Fusion into a Planar Bilayer Lipid Membrane on a SiO2 Substrate.

Artificial planar bilayer lipid membranes (BLMs) are simple models of cellular systems under physically and chemically controlled conditions, and they have been used to investigate membrane protein activity. Baculovirus-budded virus (BV) systems can express recombinant membrane proteins. In this study, aiming for membrane protein reconstitution, we examined the fusion of BVs containing recombinant membrane proteins into artificial planar BLMs on a Si microwell substrate. BV fusion with the BLMs depended on the pH of the solution, and it was enhanced at lower pH. Based on fluorescence recovery after photobleaching (FRAP) measurement, the fusion state of BVs was evaluated, and full fusion at low pH was confirmed. The fluorescent labeling the membrane proteins was also observed in the freestanding part of the BLMs as well as in the supported part. These results demonstrate the effectiveness of BLMs as a platform to examine detailed fusion dynamics of BVs. Furthermore, this study revealed that the fusion of BVs is a promising method for reconstituting membrane proteins to artificial freestanding BLMs for the development of biodevices with which we can examine membrane protein activity.

Regioselective fabrication of gold nanowires using open-space laminar flow for attomolar protein detection.

Gold nanowires are expected to be applied to biosensing due to their advantages, such as high stability and biocompatibility. However, it is still inconvenient to fabricate a single gold nanowire at a precise position, and without a special demanding environment. In this study, we present an open-space laminar flow approach for fabricating a single gold nanowire at a precise position under normal conditions. The fabricated gold nanowire demonstrated excellent biosensing of IgA with an extremely low limit of detection (1 aM).

In Situ Single-Cell Stimulation and Real-Time Electrochemical Detection of Lactate Response Using a Microfluidic Probe.

Metabolism of a single cell, even within the same organization, differs from other cells by orders of magnitude. Single-cell analysis provides key information for early diagnosis of cancer as well as drug screening. Any slight change in the microenvironment may affect the state of a single cell. Timely and effective cell monitoring is conducive to better understand the behavior of single cells. The immediate response of a single cell described in this study is a liquid transfer-based approach for real-time electrochemical detection. The cell was in situ stimulated by continuous flow with glucose, and lactate secreted from the cell would diffuse into the microflow. The microflow was aspirated into the detection channel where lactate was then decomposed by coupled enzyme reactions and detected by an electrode. This work provides a novel approach for detecting lactate response from a single cell by noninvasive measurements, and the position resolution of the microfluidic probe reaches the level of a single cell and permits individual heterogeneity in cells to be explored in the diagnosis and treatment of cancer as well as in many other situations.

Observation of intracellular protein localization area in a single neuron using gold nanoparticles with a scanning electron microscope.

The localization areas of intracellular proteins in rat cortical neurons were visualized using a scanning electron microscope (SEM) coupled with a focused ion beam (FIB) system. To obtain a clear contrast in the SEM images, gold nanoparticles (GNPs) were bound to specific intracellular proteins by antigen-antibody reactions. By obtaining a cross section of the desired location of the neurons by FIB milling under the SEM imaging condition, it was possible to observe the proteins inside the cells as clear bright spots. When a neuron was stained with anti-tau and anti-histone H1 antibodies, the bright spots were localized in the cross section of the axon and the nucleus, respectively. It was confirmed that targeted proteins in a single neuron on a substrate could be successfully identified. The development of FIB/SEM observation with immunological GNP staining will offer important information for the stable growth of neurons on various substrate structures, since the elongation and turning of axons on the substrates are activated by the redistribution of intracellular proteins.

Validation of wearable textile electrodes for ECG monitoring.

A highly conductive textile was woven from nano-fibers coated with the PEDOT-PSS polymer. The aim of this study was to assess the usefulness of textile electrodes for ECG recording as a smart garment. Electrode textile pads and lead wires were sewn to the lining of sportswear and their tolerability to repeated washings were tested up to 150 times. The electrical conductivity of the textile electrode remained functional for up to 50 machine washes. To assess the level of motion artifacts or noise during the daily monitoring of ECG, a single lead ECG with conventional or textile electrodes was recorded during supine rest, seated rest, upright trunk rotation (i.e., twisting), and stepping movement in 66 healthy adults. A Holter system was used for data storage and analysis. ECG patterns of P, QRS, and T waves were comparable between the conventional and textile electrodes. However, the signal-to-artifact-and/or-noise ratio (SAR) during twisting was larger in the textile electrodes than in the conventional electrodes. No skin irritation was seen in the textile electrodes. The single lead textile electrodes embedded in an inner garment were usable for continuous and/or repeated ECG monitoring in daily life except during vigorous trunk movement.

Regulation of neuritogenesis in hippocampal neurons using stiffness of extracellular microenvironment.

The mechanosensitivity of neurons in the central nervous system (CNS) is an interesting issue as regards understanding neuronal development and designing compliant materials as neural interfaces between neurons and external devices for treating CNS injuries and disorders. Although neurite initiation from a cell body is known to be the first step towards forming a functional nervous network during development or regeneration, less is known about how the mechanical properties of the extracellular microenvironment affect neuritogenesis. Here, we investigated the filamentous actin (F-actin) cytoskeletal structures of neurons, which are a key factor in neuritogenesis, on gel substrates with a stiffness-controlled substrate, to reveal the relationship between substrate stiffness and neuritogenesis. We found that neuritogenesis was significantly suppressed on a gel substrate with an elastic modulus higher than the stiffness of in vivo brain. Fluorescent images of the F-actin cytoskeletal structures showed that the F-actin organization depended on the substrate stiffness. Circumferential actin meshworks and arcs were formed at the edge of the cell body on the stiff gel substrates unlike with soft substrates. The suppression of F-actin cytoskeleton formation improved neuritogenesis. The results indicate that the organization of neuronal F-actin cytoskeletons is strongly regulated by the mechanical properties of the surrounding environment, and the mechanically-induced F-actin cytoskeletons regulate neuritogenesis.

Scanning Electron Microscopy Observation of Interface Between Single Neurons and Conductive Surfaces.

Interfaces between single neurons and conductive substrates were investigated using focused ion beam (FIB) milling and subsequent scanning electron microscopy (SEM) observation. The interfaces play an important role in controlling neuronal growth when we fabricate neuron-nanostructure integrated devices. Cross sectional images of cultivated neurons obtained with an FIB/SEM dual system show the clear affinity of the neurons for the substrates. Very few neurons attached themselves to indium tin oxide (ITO) and this repulsion yielded a wide interspace at the neuron-ITO interface. A neuron-gold interface exhibited partial adhesion. On the other hand, a neuron-titanium interface showed good adhesion and small interspaces were observed. These results are consistent with an assessment made using fluorescence microscopy. We expect the much higher spatial resolution of SEM images to provide us with more detailed information. Our study shows that the interface between a single neuron and a substrate offers useful information as regards improving surface properties and establishing neuron-nanostructure integrated devices.

Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy.

Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes.

Reconstitution of homomeric GluA2(flop) receptors in supported lipid membranes: functional and structural properties.

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2(flop) receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain.

AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers.

BACKGROUND: Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment. METHODS: Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate. RESULTS: Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements. CONCLUSIONS: Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment. GENERAL SIGNIFICANCE: AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.

Effect of Mg2+ on neural activity of rat cortical and hippocampal neurons in vitro.

Mg2+ plays an important role in biological functions, similar to that of Ca2+. In terms of neural activity, it is well known that Mg2+ blocks the NMDA receptor. However, the relationship between Mg2+ and neural function has not been well understood. We have investigated the effect of low extracellular Mg2+ concentration ([Mg2+]o) on neural activity in rat cortical and hippocampal neurons by using microelectrode array (MEA) measurements and glutamate measurements, with an enzyme modified MEA-based multi-array sensor. In this study, we investigated the effects of low [Mg2+]o on intracellular Ca2+ concentration ([Ca2+]i) using a confocal laser microscope and a flow cytometer with a fluorescence probe. The results indicate that low [Mg2+]o has an effect on neural activity. The responses of cortical and hippocampal neurons to low [Mg2+]o differed in the developmental period. The results suggest that hippocampal neurons are more sensitive to [Mg2+ than cortical neurons. The glutamate receptor distributions in the cortex and hippocampus may be different. Further investigation is required to understand the mechanisms of the Mg2+ effect on neural activity.

CA2: the most vulnerable sector to bicuculline exposure in rat hippocampal slice cultures.

The vulnerability of the CA2 sector to chronic exposure to bicuculline was investigated in rat hippocampal slice cultures. Selective neuronal cell death was observed only in the CA2 sector after exposure to 6 microM bicuculline for 12 h, but the effect of the cell toxicity extended to the CA3 sector after 24 h. The effect was increased by adding 20 microM roscovitine but was reduced by adding 200 nM omega-agatoxin IVA. Bicuculline also induced a calcium influx into neuronal cells mainly in the CA2 sector. These results suggest that CA2 is the most vulnerable sector to bicuculline exposure in hippocampal slice cultures, and that neuronal cell death in the CA2 sector involves the P/Q-type voltage-dependent calcium channel.

[Effect of magnesium on neural activities in vitro].

It has been well known that magnesium ion (Mg(2+)) plays an important role in biological functions, especially in neural activities and functions. However, not so many researches have been carried to this subject. Here we investigated the Mg(2+)effect on neuronal electrical activities together with NMDA receptor and synaptic glutamate release by using Multi-Electrode Array (MEA) and Enzyme modified MEA-based multi-array sensor.

[Real-time detection of neurotransmitter release and its spatial distribution].

Neurotransmitters have been well known as information carriers for a long time. Recently, some of the research indicated their neurotoxicity, while some indicated their neurotrophic actions. It is very important to understand the role of neurotransmitters. Glutamate is one of the most important excitatory neurotransmitter in the brain. We developed a novel measurement method for glutamate. The method we describe here is based on the enzyme-mediated electrochemical detection. Glutamate oxidase and horseradish peroxidase were deposited together with polymer-mediator on the electrode. We applied this idea on ITO multi-array electrode and developed a 64 channel multi-array sensor. The sensor permits us to detect glutamate release from multiple regions simultaneously in real time. As it is possible to illustrate the distribution of glutamate release, the sensor could be used not only in the pharmacological field, but also in medical treatment in the near future.

A system for MEA-based multisite stimulation.

The capability for multisite stimulation is one of the biggest potential advantages of microelectrode arrays (MEAs). There remain, however, several technical problems which have hindered the development of a practical stimulation system. An important design goal is to allow programmable multisite stimulation, which produces minimal interference with simultaneous extracellular and patch or whole cell clamp recording. Here, we describe a multisite stimulation and recording system with novel interface circuit modules, in which preamplifiers and transistor transistor logic-driven solid-state switching devices are integrated. This integration permits PC-controlled remote switching of each substrate electrode. This allows not only flexible selection of stimulation sites, but also rapid switching of the selected sites between stimulation and recording, within 1.2 ms. This allowed almost continuous monitoring of extracellular signals at all the substrate-embedded electrodes, including those used for stimulation. In addition, the vibration-free solid-state switching made it possible to record whole-cell synaptic currents in one neuron, evoked from multiple sites in the network. We have used this system to visualize spatial propagation patterns of evoked responses in cultured networks of cortical neurons. This MEA-based stimulation system is a useful tool for studying neuronal signal processing in biological neuronal networks, as well as the process of synaptic integration within single neurons.

Electrochemical monitoring of glutamate release at multiple positions in a rat hippocampal slice.

The continuous monitoring of the distribution of glutamate (Glu), a neurotransmitter released at synaptic terminals, is important in terms of understanding the signal transfer mechanism in the brain. In this study, we monitored the concentration of Glu released at multiple positions in a hippocampal slice continuously, and obtained an approximate Glu distribution by using our electrochemical glutamate sensor array. After confirming our sensor's high sensitivity to Glu, we placed a slice on the array, and measured the currents at selected electrodes in the array. When we stimulated a specific position in the slice electrically, the glutamate concentration increased in different areas after several tens of seconds. The presence of glutamate receptor blockers suppressed these increases. This suggests that the electrical signal was transferred along with neurons through synapses and stimulated the Glu release. Our multichannel glutamate sensor should be a powerful tool to determining the distribution of real-time glutamate non-invasively for the studies using biological samples.