Aß1-42 Oligomers Induced a Short-Term Increase of Glutamate Release Prior to Its Depletion As Measured by Amperometry on Single Varicosities.

Glutamate (Glu) is a critical neurotransmitter for neuronal communication in the nervous system. In vivo studies have shown that the concentration of Glu is reduced within the brains of those afflicted with Alzheimer's disease (AD), which is also associated with the accumulation of pathogenic amyloid-beta (Aß). However, the effects of Aß peptides on the level of Glu release, as well as how Aß-mediated Glu fluctuation is initiated, remain largely unknown. Here, we fabricated a Glu electrochemical biosensor and in situ quantitatively monitored the release of Glu from a single varicosity of Aß1-42-insulted hippocampal neurons. We found that before the depletion of Glu after 300 min of treatment with Aß1-42, a short-duration (30 min) incubation with Aß1-42 caused a dramatic increase in vesicular Glu release compared to that of a control. Further investigation demonstrated that the density of vesicular glutamate transporter 1 (VGLUT1), which is responsible for transport of Glu into synaptic vesicles, also displayed a significant elevation and then dramatic depletion with the extension of the time of treatment with Aß1-42. These results indicate that at the early stage of AD, Aß1-42 induces excessive Glu release, which may overstimulate the N-methyl-d-aspartic acid (NMDA) receptor, resulting in excitotoxicity and damage to neurons. In this work, the amount of Glu released together with its fluctuations under Aß1-42 oligomers toxicity conditions was monitored for the first time, and such monitoring could provide direct and new insights for current research on Aß1-42-induced abnormalities in neurotransmitter release and neuron functions.

A single nanowire sensor for intracellular glucose detection.

Glucose metabolism plays an important role in cell energy supply, and quantitative detection of the intracellular glucose level is particularly important for understanding many physiological processes. Glucose electrochemical sensors are widely used for blood and extracellular glucose detection. However, intracellular glucose detection cannot be achieved by these sensors owing to their large size and consequent low spatial resolution. Herein, we developed a single nanowire glucose sensor for electrochemical detection of intracellular glucose by depositing Pt nanoparticles (Pt NPs) on a SiC@C nanowire and further immobilizing glucose oxidase (GOD) thereon. Glucose was converted by GOD to an electroactive product H2O2 which was further electro-catalyzed by Pt NPs. The glucose nanowire sensor is endowed with a high sensitivity, high spatial-temporal resolution and enzyme specificity due to its nanoscale size and enzymatic reaction. This allows the real-time monitoring of the intracellular glucose level, and the increase of the intracellular glucose level induced by a novel potential hypoglycemic agent, reinforcing its potential application in lowering the blood glucose level. This work provides a versatile method for the construction of enzyme-modified nanosensors to electrochemically detect intracellular non-electroactive molecules, which is of great benefit for physiological and pathological studies.

Electrochemical Monitoring of ROS/RNS Homeostasis Within Individual Phagolysosomes Inside Single Macrophages.

The existence of a homeostatic mechanism regulating reactive oxygen/nitrogen species (ROS/RNS) amounts inside phagolysosomes has been invoked to account for the efficiency of this process but could not be unambiguously documented. Now, intracellular electrochemical analysis with platinized nanowire electrodes (Pt-NWEs) allowed monitoring ROS/RNS effluxes with sub-millisecond resolution from individual phagolysosomes impacting onto the electrode inserted inside a living macrophage. This shows for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results establish the existence of the long-postulated ROS/RNS homeostasis and allows its kinetics and efficiency to be quantified. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.

Flexible Electrochemical Urea Sensor Based on Surface Molecularly Imprinted Nanotubes for Detection of Human Sweat.

Flexible electrochemical (EC) sensors have shown great prospect in epidermal detection for personal healthcare and disease diagnosis. However, no reports have been seen in flexible device for urea analysis in body fluids. Herein, we developed a flexible wearable EC sensor based on surface molecularly imprinted nanotubes for noninvasive urea monitoring with high selectivity in human sweat. The flexible EC sensor was prepared by electropolymerization of 3,4-ethylenedioxythiophene (EDOT) monomer on the hierarchical network of carbon nanotubes (CNTs) and gold nanotubes (Au NTs) to imprint template molecule urea. This sensor exhibited a good linear response toward physiologically relevant urea levels with negligible interferences from common coexisting species. Bending test revealed that this sensor possessed excellent mechanical tolerance and its EC performance was almost not affected by bending deformation. On-body results of human subjects showed that the flexible platform could distinctly respond to the urea levels in volunteer's sweat after aerobic exercise. The new flexible epidermal EC sensor can provide useful insights into noninvasive monitoring of urea levels in various biofluids, which is promising in the clinical diagnosis of diverse biomedical applications.

Detection of exosomes by ZnO nanowires coated three-dimensional scaffold chip device.

Exosomes as cell-derived vesicles have the potential to be novel biomarkers for noninvasive diagnosis of cancers. However, cost-effective detection of exosomes in routine clinical settings is still challenging. Herein, we present a ZnO nanowires coated three-dimensional (3D) scaffold chip device for effective immunocapture and classically visible and colorimetric detection of exosomes. The chip device is composed of 3D polydimethylsiloxane (PDMS) scaffold skeleton covered by free-standing ZnO nanowire array. The interconnected micropores of 3D scaffold induces the fluid flow with chaotic or vortex feature, and ZnO nanowire array provides large surface area for immobilization of exosome specific antibody as well as size exclusion-like effect for retaining exosomes. These synergistically and significantly enhance the capture of exosomes at a high flow rate. The captured exosomes are detected by horseradish peroxidase (HRP) labeled antibody which can initiate 3,3',5,5'-tetramethylbenzidine (TMB)-based colorimetric sensing. The quantitative readout of exosomes is easily accomplished by UV-vis spectrometry or microplate reader with a linear range of 2.2 × 105 to 2.4 × 107 particles/µL and a minimal detectable concentration of 2.2 × 104 particles/µL. This chip device was applicable to clinical samples where cancer patients demonstrate statistically significant increase in exosomes compared with healthy individuals. Thus, our chip device is cost-effective and easy-to-use, facilitating visible and colorimetric assay with high sensitivity toward clinical applications.

Quantifying orientational regeneration of injured neurons by natural product concentration gradients in a 3D microfluidic device.

Regeneration of injured neurons in complicated three-dimensional (3D) microenvironments is a key approach for treating neurodegenerative diseases. Microfluidics provides a versatile tool to recapitulate cellular microenvironments in vitro, but it still remains a big challenge to construct a microfluidic platform incorporating extracellular matrix (ECM) structures and highly controlled 3D gradients of soluble factors to study the regeneration of injured neurons. In this work, we developed a microfluidic device which can provide multiple adjustable gradients in a 3D ECM to investigate the regeneration of injured central nervous system (CNS) neurons in response to natural small molecules. With interconnecting but independently controlled central channels, asymmetrically designed side channels and a series of microgrooves connecting the central channels, spatially and temporally controlled 3D biochemical gradients can be generated inside collagen hydrogel in the central channels. This allows quantitative analysis of guided axon growth and the orientational regeneration of injured dopaminergic neurons by 3D chemical gradients of three natural molecules. This study demonstrates a promising microfluidic platform for the generation of highly controlled 3D biochemical gradients in an ECM to quantitatively study neuronal responses, thereby potentially facilitating drug screening and optimization of treatment protocols for neurodegenerative diseases.

Real-Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core-Shell Nanowire Electrodes.

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living-cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC-core-shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.