Recent Developments in Nanosensors for Imaging Applications in Biological Systems.

Sensors are key tools for monitoring the dynamic changes of biomolecules and biofunctions that encode valuable information that helps us understand underlying biological processes of fundamental importance. Because of their distinctive size-dependent physicochemical properties, materials with nanometer scales have recently emerged as promising candidates for biological sensing applications by offering unique insights into real-time changes of key physiological parameters. This review focuses on recent advances in imaging-based nanosensor developments and applications categorized by their signal transduction mechanisms, namely, fluorescence, plasmonics, MRI, and photoacoustics. We further discuss the synergy created by multimodal nanosensors in which sensor components work based on two or more signal transduction mechanisms.

Imaging Sodium Flux during Action Potentials in Neurons with Fluorescent Nanosensors and Transparent Microelectrodes.

Sodium flux plays a pivotal role in neurobiological processes including initiation of action potentials and regulation of neuronal cell excitability. However, unlike the wide range of fluorescent calcium indicators used extensively for cellular studies, the choice of sodium probes remains limited. We have previously demonstrated optode-based nanosensors (OBNs) for detecting sodium ions with advantageous modular properties such as tunable physiological sensing range, full reversibility, and superb selectivity against key physiological interfering ion potassium. (1) Motivated by bridging the gap between the great interest in sodium imaging of neuronal cell activity as an alternative to patch clamp and limited choices of optical sodium indicators, in this Letter we report the application of nanosensors capable of detecting intracellular sodium flux in isolated rat dorsal root ganglion neurons during electrical stimulation using transparent microelectrodes. Taking advantage of the ratiometric detection scheme offered by this fluorescent modular sensing platform, we performed dual color imaging of the sensor to monitor the intracellular sodium currents underlying trains of action potentials in real time. The combination of nanosensors and microelectrodes for monitoring neuronal sodium dynamics is a novel tool for investigating the regulatory role of sodium ions involved during neural activities.

Optical Probes for Neurobiological Sensing and Imaging.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

A method for estimating intracellular ion concentration using optical nanosensors and ratiometric imaging.

Optical nanoparticle (NP)-based sensors have been widely implemented as tools for detection of targeted ions and biomolecules. The NP sensing platform offer a modular design that can incorporate different sensing components for greater target specificity and the ability to tune the dynamic range, as well as encapsulation of multiple dyes to generate a ratiometric signal with varying spectra. Despite these advantages, demonstrating quantitative ion imaging for intracellular measurement still possess a major challenge. Here, we describe fundamentals that enable intracellular validation of this approach using ion-selective nanosensors for investigating calcium (Ca2+) as a model ion. While conventional indicators can improve individual aspects of indicator performance such as Kd, wavelength, and ratiometric measurements, the use of NP sensors can achieve combined benefits of addressing these issues simultaneously. The nanosensor incorporates highly calcium-selective ionophores and two fluorescence indicators that act as signal transducers to facilitate quantitative ratiometric imaging. For intracellular Ca2+ application, the sensors are fine-tuned to physiological sensing range, and live-cell imaging and quantification are demonstrated in HeLa cells loaded with nanosensors and their responsiveness to carbachol-evoked store release (~400 nM). The current nanosensor design thus provides a promising sensing platform for real-time detection and optical determination of intracellular ions.

In Vivo Biosensing: Progress and Perspectives.

In vivo biosensors are emerging as powerful tools in biomedical research and diagnostic medicine. Distinct from "labels" or "imaging", in vivo biosensors are designed for continuous and long-term monitoring of target analytes in real biological systems and should be selective, sensitive, reversible and biocompatible. Due to the challenges associated with meeting all of the analytical requirements, we found relatively few reports of research groups demonstrating devices that meet the strict definition in vivo. However, we identified several case studies and a range of emerging materials likely to lead to significant developments in the field.

Monitoring the size and lateral dynamics of ErbB1 enriched membrane domains through live cell plasmon coupling microscopy.

To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60-250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of D = |0.0054±0.0064| µm(2)/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions.

Optical sizing of immunolabel clusters through multispectral plasmon coupling microscopy.

The wavelength dependent scattering cross sections of self-assembled silver nanoparticle clusters of known size (n) were measured on five different wavelength channels between 427 and 510 nm through correlation of multispectral imaging and scanning electron microscopy. A multivariate statistical analysis of the spectral response of this training set provided a correlation between spectral response and cluster size and enabled a classification of new measurements into four distinct nanoparticle association levels (I1-I4) whose compositions were dominated by monomers (I1), dimers (I2), trimers and tetramers (I3), and larger clusters (I4), respectively. One potential application of the optical sizing approach is to map association levels of silver immunolabels on cellular surfaces. We demonstrate the feasibility of this approach using silver immunolabels targeted at the epidermal growth factor receptor on A431 cells in a proof of principle experiment. The ability to measure immunolabel association levels on subcellular length scales in an optical microscope provides new opportunities for experimentally assessing receptor density distributions on living cells in solution.

Insights from a nanoparticle minuet: two-dimensional membrane profiling through silver plasmon ruler tracking.

Individual pairs of polymer-tethered silver nanoparticles, so-called silver plasmon rulers, enable distance and orientation measurements on the nanoscale. The reduced linear dichroism and the spectrum of the light scattered from individual plasmon rulers encode information about their orientation and average interparticle separation, respectively. We took advantage of the gain in information silver plasmon rulers offer as probes in optical tracking and analyzed the translational and rotational motions as well as the extension of individual silver plasmon rulers diffusing on the plasma membrane of lysed HeLa cells. Consistent with a compartmentalization of the cell surface on the length scales of the plasmon rulers, most rulers were either immobilized or performed a confined lateral diffusion. Structural details of a plasmon ruler's confinement region became accessible utilizing the orientation and interparticle separation dependent optical response of the plasmon rulers. This approach, which we refer to as polarization-resolved plasmon coupling microscopy, enabled a detailed structural characterization of individual membrane compartments and provided a quantitative metrics to characterize the structural lateral heterogeneity of cell membranes on submicrometer length scales. In combination with adequate tracking methods, the "dance" performed by membrane confined dimers of flexibly linked noble metal nanoparticles revealed deep insight into the underlying membrane morphology.

Resolving sub-diffraction limit encounters in nanoparticle tracking using live cell plasmon coupling microscopy.

We use plasmon coupling between individual gold nanoparticle labels to monitor subdiffraction limit distances in live cell nanoparticle tracking experiments. While the resolving power of our optical microscope is limited to approximately 500 nm, we improve this by more than an order of magnitude by detecting plasmon coupling between individual gold nanoparticle labels using a ratiometric detection scheme. We apply this plasmon coupling microscopy to resolve the interparticle separations during individual encounters of gold nanoparticle labeled fibronectin-integrin complexes in living HeLa cells.

In2O3 hollow microspheres: synthesis from designed In(OH)3 precursors and applications in gas sensors and photocatalysis.

In this work, well-shaped In(OH)3 hollow microspheres have been successfully prepared via a novel surfactant-free vesicle-template-interface route in the "formamide-resorcinol-water" system, in which spontaneous vesicles were formed under hydrothermal conditions and NH3 from the hydrolysis of formamide acted as the OH- provider. Morphological and structural characterizations indicate that the shells of as-prepared In(OH)3 hollow microspheres were constructed by numerous nanocubes about 80 nm in size. As desired, In2O3 hollow microspheres were obtained from annealing the designed In(OH)3 precursors, and the as-obtained In2O3 hollow microspheres performed well as a gas-sensing material in response to both ethanol and formaldehyde gases and as a photocatalyst for photocatalytic degradation of rhodamine B. The facile preparation method and the improved properties derived from special microstructures are significant in the synthesis and future applications of functional nanomaterials.

Low-temperature synthesis of alpha-MnO2 hollow urchins and their application in rechargeable Li+ batteries.

Novel alpha-MnO2 hollow urchins were synthesized on a large scale by a facile and efficient low-temperature (60 degrees C) mild reduction route, without templates or surfactants in the system. The formation mechanism for the hollow urchins was proved to be the Ostwald ripening process by tracking the crystallization and morphology of the product at different reaction stages. The as-prepared hollow-urchin sample has a high Brunauer-Emmett-Teller surface area of 132 m(2)/g and a mesoporous structure, which were expected to help improve the electrochemical property in Li+ batteries. When the alpha-MnO2 hollow urchins were used as the cathode material in Li batteries, they performed better than the other alpha-MnO2 samples (solid urchins and dispersed nanorods), indicating that the electrochemical performance of the electrode material is sensitive to its morphology. This synthetic procedure is straightforward and inexpensive and thus facilitates mass production of alpha-MnO2 hollow urchins.

Vanadium pentoxide nanobelts and nanorolls: from controllable synthesis to investigation of their electrochemical properties and photocatalytic activities.

Uniform V(2)O(5)· xH(2)O nanobelts with high aspect ratios and ultra-long V(2)O(5)· xH(2)O nanorolls with novel scroll-like structures were synthesized on a large scale by a simple hydrothermal growth route using NH(4)VO(3) as the raw material in the presence of different acids at 180 °C for 24 h. Their morphologies were observed by scanning electron microscopy (SEM). X-ray powder diffraction measurement and thermal gravimetric analysis revealed the composition of nanobelts and nanorolls to be V(2)O(5)·0.9H(2)O and V(2)O(5)·0.6H(2)O, respectively. The possible mechanisms of formation of the nanobelts and nanorolls were schematically elucidated based on the layered structure of vanadium pentoxide. In addition, corresponding anhydrous V(2)O(5) nanostructures with better crystallinity were obtained by calcining the precursors of V(2)O(5)·0.9H(2)O nanobelts or V(2)O(5)·0.6H(2)O nanorolls. Furthermore, we have investigated the electrochemical intercalation properties with Li(+) and the photocatalytic activities of the synthesized V(2)O(5)·0.9H(2)O nanobelts, V(2)O(5)·0.6H(2)O nanorolls and their corresponding post-annealing products. It was observed that the morphologies and compositions of the synthesized products had an evident influence on the electrochemical intercalation properties with Li(+) and photocatalytic activities.