Functionalized Quartz Nanopipette for Intracellular Superoxide Sensing: A Tool for Monitoring Reactive Oxygen Species Levels in Single Living Cell.

Reactive oxygen species (ROS), including superoxide radical anions, are vital components in numerous biological functions, including cell signaling and immune responses. Since ROS react with other biomolecules and oxidize them quickly, it is essential for cells to have superoxide-scavenging enzymes and other regulating enzymes that can catalyze the dismutation of superoxide radical anions into less damaging molecules. Otherwise, ROS overproduction can cause oxidative damage to DNA, proteins, cells, and tissues, damage that is associated with the pathogenesis of a range of neurodegenerative disorders, age-related diseases, and cancer. Understanding the relationship between superoxide and these disorders can help the development of innovative therapies for combating oxidative stress and degeneration of nerve cells. Although methods to quantify ROS already exist, they are indirect, destructive, ambiguous, and/or cannot provide real-time measurements in single cells. In this paper, we report a technique for sensing superoxide radical anions in single living cells using functionalized nanopipettes. These nanopipettes allow us to enter the cell as we measure intracellular ROS concentrations over time. We observed that these devices provide precise real-time measurements that are accurate and not possible to obtain with other conventional techniques.

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology.

Metabolic and transcriptomic analysis of Huntington's disease model reveal changes in intracellular glucose levels and related genes.

Huntington's Disease (HD) is a neurodegenerative disorder caused by an expansion in a CAG-tri-nucleotide repeat that introduces a poly-glutamine stretch into the huntingtin protein (mHTT). Mutant huntingtin (mHTT) has been associated with several phenotypes including mood disorders and depression. Additionally, HD patients are known to be more susceptible to type II diabetes mellitus (T2DM), and HD mice model develops diabetes. However, the mechanism and pathways that link Huntington's disease and diabetes have not been well established. Understanding the underlying mechanisms can reveal potential targets for drug development in HD. In this study, we investigated the transcriptome of mHTT cell populations alongside intracellular glucose measurements using a functionalized nanopipette. Several genes related to glucose uptake and glucose homeostasis are affected. We observed changes in intracellular glucose concentrations and identified altered transcript levels of certain genes including Sorcs1, Hh-II and Vldlr. Our data suggest that these can be used as markers for HD progression. Sorcs1 may not only have a role in glucose metabolism and trafficking but also in glutamatergic pathways affecting trafficking of synaptic components.

Smartphone Operated Signal Transduction by Ion Nanogating (STING) Amplifier for Nanopore Sensors: Design and Analytical Application.

In this report, we demonstrated a handheld wireless voltage-clamp amplifier for current measurement of nanopore sensors. This amplifier interfaces a sensing probe and connects wirelessly with a computer or smartphone for the required stimulus input, data processing and storage. To test the proposed Signal Transduction by Ion Nanogating (STING) wireless amplifier, in the current study the system was tested with a nano-pH sensor to measure pH of standard buffer solutions and the performance was compared against the commercial voltage-clamp amplifier. To our best knowledge, STING amplifier is the first miniaturized wireless voltage-clamp platform operated with a customized smart-phone application (app).

Single Cell "Glucose Nanosensor" Verifies Elevated Glucose Levels in Individual Cancer Cells.

Because the transition from oxidative phosphorylation to anaerobic glycolytic metabolism is a hallmark of cancer progression, approaches to identify single living cancer cells by their unique glucose metabolic signature would be useful. Here, we present nanopipettes specifically developed to measure glucose levels in single cells with temporal and spatial resolution, and we use this technology to verify the hypothesis that individual cancer cells can indeed display higher intracellular glucose levels. The nanopipettes were functionalized as glucose nanosensors by immobilizing glucose oxidase (GOx) covalently to the tip so that the interaction of glucose with GOx resulted in a catalytic oxidation of ß-d-glucose to d-gluconic acid, which was measured as a change in impedance due to drop in pH of the medium at the nanopipette tip. Calibration studies showed a direct relationship between impedance changes at the tip and glucose concentration in solution. The glucose nanosensor quantified single cell intracellular glucose levels in human fibroblasts and the metastatic breast cancer lines MDA-MB-231 and MCF7 and revealed that the cancer cells expressed reproducible and reliable increases in glucose levels compared to the nonmalignant cells. Nanopipettes allow repeated sampling of the same cell, as cells remain viable during and after measurements. Therefore, nanopipette-based glucose sensors provide an approach to compare changes in glucose levels with changes in proliferative or metastatic state. The platform has great promise for mechanistic investigations, as a diagnostic tool to distinguish cancer cells from nonmalignant cells in heterogeneous tissue biopsies, as well as a tool for monitoring cancer progression in situ.

RECENT DEVELOPMENTS IN ELECTROCHEMICAL SENSORS FOR THE DETECTION OF NEUROTRANSMITTERS FOR APPLICATIONS IN BIOMEDICINE.

Neurotransmitters are important biological molecules that are essential to many neurophysiological processes including memory, cognition, and behavioral states. The development of analytical methodologies to accurately detect neurotransmitters is of great importance in neurological and biological research. Specifically designed microelectrodes or microbiosensors have demonstrated potential for rapid, real-time measurements with high spatial resolution. Such devices can facilitate study of the role and mechanism of action of neurotransmitters and can find potential uses in biomedicine. This paper reviews the current status and recent advances in the development and application of electrochemical sensors for the detection of small-molecule neurotransmitters. Measurement challenges and opportunities of electroanalytical methods to advance study and understanding of neurotransmitters in various biological models and disease conditions are discussed.

Single-cell intracellular nano-pH probes.

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

Alterations of intestinal serotonin following nanoparticle exposure in embryonic zebrafish.

The increased use of engineered nanoparticles (NPs) in manufacturing and consumer products raises concerns about the potential environmental and health implications on the ecosystem and living organisms. Organs initially and more heavily affected by environmental NPs exposure in whole organisms are the skin and digestive system. We investigate the toxic effect of two types of NPs, nickel (Ni) and copper oxide (CuO), on the physiology of the intestine of a living aquatic system, zebrafish embryos. Embryos were exposed to a range of Ni and CuO NP concentrations at different stages of embryonic development. We use changes in the physiological serotonin (5HT) concentrations, determined electrochemically with carbon fiber microelectrodes inserted in the live embryo, to assess this organ dysfunction due to NP exposure. We find that exposure to both Ni and CuO NPs induces changes in the physiological 5HT concentration that varies with the type, exposure period and concentration of NPs, as well as with the developmental stage during which the embryo is exposed. These data suggest that exposure to NPs might alter development and physiological processes in living organisms and provide evidence of the effect of NPs on the physiology of the intestine.

Glutamate oxidase biosensor based on mixed ceria and titania nanoparticles for the detection of glutamate in hypoxic environments.

We report on the design and development of a glutamate oxidase (GmOx) microelectrode for measuring l-glutamic acid (GluA) in oxygen-depleted conditions, which is based on the oxygen storage and release capacity of cerium oxides. To fabricate the biosensor, a nanocomposite of oxygen-rich ceria and titania nanoparticles dispersed within a semi-permeable chitosan membrane was co-immobilized with the enzyme GmOx on the surface of a Pt microelectrode. The oxygen delivery capacity of the ceria nanoparticles embedded in a biocompatible chitosan matrix facilitated enzyme stabilization and operation in oxygen free conditions. GluA was measured by amperometry at a working potential of 0.6 V vs Ag/AgCl. Detection limits of 0.594 µM and 0.493 µM and a sensitivity of 793 pA/µM (RSD 3.49%, n=5) and 395 pA/µM (RSD 2.48%, n=5) were recorded in oxygenated and deoxygenated conditions, with response times of 2s and 5s, respectively. The biosensor had good operational stability and selectivity against common interfering substances. Operation of the biosensor was tested in cerebrospinal fluid. Preliminary in vivo recording in Sprague-Dawley rats to monitor GluA in the cortex during cerebral ischemia and reperfusion demonstrate a potential application of the biosensor in hypoxic conditions. This method provides a solution to ensure functionality of oxidoreductase enzymes in oxygen-free environments.

Effect of cerium oxide nanoparticles on intestinal serotonin in zebrafish.

Cerium oxide nanoparticles or nanoceria are emerging as a new and promising class of nanoparticle technology for biomedical applications. The safe implementation of these particles in clinical applications requires evaluation of their redox properties and reactivity that might cause neurotoxic effects by interacting with redox components of the physiological environment. We report in vitro and in vivo studies to evaluate the impact of nanoceria exposure on serotonin (5-HT), an important neurotransmitter that plays a critical role in various physiological processes including motility and secretion in the digestive system. In vitro studies of 5-HT in the presence of nanoceria using spectroscopic, electrochemical and surface characterization methods demonstrate that nanoceria interacts with 5-HT and forms a surface adsorbed 5-HT-nanoceria complex. Further in vivo studies in live zebrafish embryos indicate depletion of the 5-HT level in the intestine for exposure periods longer than three days. Intestinal 5-HT was assessed quantitatively in live embryos using implantable carbon fiber microelectrodes and the results were compared to immunohistochemistry of the dissected intestine. 20 and 50 ppm nanoparticle exposure decreased the 5-HT level to 20.5 (±1.3) and 5.3 (±1.5) nM respectively as compared to 30.8 (±3.4) nM for unexposed control embryos. The results suggest that internalized nanoceria particles can concentrate 5-HT at the nanoparticle accumulation site depleting it from the surrounding tissue. This finding might have long term implications in the neurophysiology and functional development of organisms exposed to these particles through intended or unintended exposure.

Comparative evaluation of intestinal nitric oxide in embryonic zebrafish exposed to metal oxide nanoparticles.

Nanoparticle (NP) exposure may induce oxidative stress through generation of reactive oxygen and nitrogen species, which can lead to cellular and tissue damage. The digestive system is one of the initial organs affected by NP exposure. Here, it is demonstrated that exposure to metal oxide NPs induces differential changes in zebrafish intestinal NO concentrations. Intestinal NO concentrations are quantified electrochemically with a carbon fiber microelectrode inserted in the intestine of live embryos. Specificity of the electrochemical signals is demonstrated by NO-specific pharmacological manipulations and the results are correlated with the 4,5-diaminofluorescein-diacetate (DAF-FM-DA). NPs are demonstrated to either induce or reduce physiological NO levels depending on their redox reactivity, type and dose. NO level is altered following exposure of zebrafish embryos to CuO and CeO2 NPs at various stages and concentrations. CuO NPs increase NO concentration, suggesting an intestinal oxidative damage. In contrast, low CeO2 NP concentration exposure significantly reduces NO levels, suggesting NO scavenging activity. However, high concentration exposure results in increased NO. Alterations in NO concentration suggest changes in intestinal physiology and oxidative stress, which will ultimately correspond to NPs toxicity. This work also demonstrates the use of electrochemistry to monitor in vivo changes of NO within zebrafish organs.

Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos.

We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6 nM serotonin with a sensitivity of 5.12 nA/µM, a linear range from 2 to 100 nM and a reproducibility of 6.5% for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels.