Small Dye Molecules Research Articles

Genetically encoded Ca2+ indicators in cardiac myocytes.

Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2. In the context of cardiac myocytes, we provide guidelines for selecting a genetically encoded Ca(2+) indicator. For future developments, we discuss improvements of a broad range of properties, including photophysical properties such as spectral spread and biocompatibility, as well as cellular and in vivo applications.

Robust Nonenzymatic Hybrid Nanoelectrocatalysts for Signal Amplification toward Ultrasensitive Electrochemical Cytosensing

We have discovered that magnetic Fe3O4 nanoparticles exhibit an intrinsic catalytic activity toward the electrochemical reduction of small dye molecules. Metallic nanocages, which act as efficient signal amplifiers, can be attached to the surface of Fe3O4 beads to further enhance the catalytic electrochemical signals. The Fe3O4@nanocage core-satellite hybrid nanoparticles show significantly more robust electrocatalytic activities than the enzymatic peroxidase/H2O2 system. We have further demonstrated that these nonenzymatic nanoelectrocatalysts can be used as signal-amplifying nanoprobes for ultrasensitive electrochemical cytosensing.

Correlative Photoactivated Localization and Scanning Electron Microscopy

The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

Preparation of porous styrenics-based monolithic layers for thin layer chromatography coupled with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection

Monolithic 50μm thin poly(4-methylstyrene-co-chloromethylstyrene-co-divinylbenzene) layers attached to 6.0cm×3.3cm glass plates have been prepared, using a thermally initiated polymerization process. These layers had a well-defined porous structure with a globular morphology demonstrated with SEM images and exhibited superhydrophobic properties characterized with a water contact angle of 157°. They were then used for thin-layer chromatography of peptides and proteins fluorescently labeled with fluorescamine. The spots of individual separated compounds were visualized using UV light, and their identities were confirmed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry. The presence of chloromethylstyrene units in the polymer enabled hypercrosslinking via a Friedel–Crafts alkylation reaction, and led to monoliths with much larger surface areas, which were suitable for separations of small dye molecules.

Single Molecule Studies on Dynamics in Liquid Crystals

Single molecule (SM) methods are able to resolve structure related dynamics of guest molecules in liquid crystals (LC). Highly diluted small dye molecules on the one hand explore structure formation and LC dynamics, on the other hand they report about a distortion caused by the guest molecules. The anisotropic structure of LC materials is used to retrieve specific conformation related properties of larger guest molecules like conjugated polymers. This in particular sheds light on organization mechanisms within biological cells, where large molecules are found in nematic LC surroundings. This review gives a short overview related to the application of highly sensitive SM detection schemes in LC.

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.

Multiple-stimulus-responsive hydrogels of cationic surfactants and azoic salt mixtures

New hydrogels having high water content, ∼96 wt%, composed of cationic surfactants, alkyltrimethylammonium bromides (C n TAB, n = 12, 14, 16, and 18), and a small dye molecule, sodium azobzenzene 4,4′-dicarboxylic acid (AzoNa2), was firstly obtained. The three-dimensional network structures of hydrogels were determined by transmission electron microscopy images, scanning electron microscopy images, 1H nuclear magnetic resonance, and small-angle X-ray scattering measurements. The mechanism of hydrogel formation was also illustrated. The rheological data were obtained to investigate the mechanical strength of hydrogels, which were turned out to be strong mechanical strength (∼104 Pa) materials. We found that the strength of the hydrogel depends on the fiber density, which can be controlled by changing the proportion of the two compounds, concentration of surfactants, temperature, and the chain length of the surfactant. Interestingly, the hydrogels were found to have a multiple-stimulus response property. A reversible thermal, UV–vis, or a chemical response was investigated in the mixtures of cationic surfactants and azoic salt for the first time. These findings may find potential applications such as sensors, actuators, shape memories, and drug delivery systems, etc.

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

BackgroundUncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores.ResultsIn this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly.ConclusionsTaken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process.

In Situ Growth of Metal–Organic Framework Thin Films with Gas Sensing and Molecule Storage Properties

New porous metal-organic framework (MOF) films based on the flexible ligand 1,3,5-tris[4-(carboxyphenyl)oxamethyl]-2,4,6-trimethylbenzene (H3TBTC) were fabricated on α-Al2O3 substrates under solvent thermal conditions. The factors affecting the fabrication of films, such as the temperature of pre-activation and the dosage of the reagents, were investigated. Tuning the subtle factors on film fabrications, a series of MOF thin films with different morphologies and grain sizes were prepared. The morphology and grain size of the films are monitored by scanning electron microscopy (SEM). X-ray diffraction (XRD) and attenuated total reflection infrared (ATR-IR) were also used to characterize the MOF films. The results indicate that the temperature of pre-activation and the dosage of the reagents are the key parameters during the process of film formation. The properties of the films, especially the sensing and sorption behavior, have been studied by an optical digital cameral and ultraviolet-visible (UV-vis) spectra. The evidence shows that the films are sensitive to small organic molecules, such as methanol and pyridine. Meanwhile, the films can adsorb small dye molecules. Thus, the films may have potential applications in either organic vapor sensing or storage of small dye molecules.

Bioprobes Based on AIE Fluorogens

Fluorescent bioprobes are powerful tools for analytical sensing and optical imaging, which allow direct visualization of biological analytes at the molecular level and offer useful insights into complex biological structures and processes. The sensing and imaging sensitivity of a bioprobe is determined by the brightness and contrast of its fluorescence before and after analyte binding. Emission from a fluorophore is often quenched at high concentration or in aggregate state, which is notoriously known as concentration quenching or aggregation-caused quenching (ACQ). The ACQ effect limits the label-to-analyte ratio and forces researchers to use very dilute solutions of fluorophores. It compels many probes to operate in a fluorescence "turn-off" mode with a narrow scope of practical applications. The unique aggregation-induced emission (AIE) process offers a straightforward solution to the ACQ problem. Typical AIE fluorogens are characterized by their propeller-shaped rotorlike structures, which undergo low-frequency torsional motions as isolated molecules and emit very weakly in solutions. Their aggregates show strong fluorescence mainly due to the restriction of their intramolecular rotations in the aggregate state. This fascinating attribute of AIE fluorogens provides a new platform for the development of fluorescence light-up molecules and photostable nanoaggregates for specific analyte detection and imaging. In this Account, we review our recent AIE work to highlight the utility of AIE effect in the development of new fluorescent bioprobes, which allows the use of highly concentrated fluorogens for biosensing and imaging. The simple design and fluorescence turn-on feature of the molecular AIE bioprobes offer direct visualization of specific analytes and biological processes in aqueous media with higher sensitivity and better accuracy than traditional fluorescence turn-off probes. The AIE dot-based bioprobes with different formulations and surface functionalities show advanced features over quantum dots and small molecule dyes, such as large absorptivity, high luminosity, excellent biocompatibility, free of random blinking, and strong photobleaching resistance. These features enable cancer cell detection, long term cell tracing, and tumor imaging in a noninvasive and high contrast manner. Recent research has significantly expanded the scope of biological applications of AIE fluorogens and offers new strategies to fluorescent bioprobe design. We anticipate that future development on AIE bioprobes will combine one- or multiphoton fluorescence with other modalities (e.g., magnetic resonance imaging) or functionalities (e.g. therapy) to fully demonstrate their potential as a new generation of theranostic reagent. In parallel, the advances in molecular biology will provide more specific bioreceptors, which will enable the development of next generation AIE bioprobes with high selectivity and sensitivity for molecular sensing and imaging.

Small molecule dye rubrene doped organic bulk heterojunction solar cells

Poly(3-hexylthiphene) (P3HT):[6,6]-phenyl C60-butyric acid methyl ester (PCBM) bulk heterojunction solar cells doped with a small molecule dye of rubrene were fabricated. The effect of different doping concentration on the performance of solar cells was investigated. The results showed that by doping 3.5wt% rubrene, the short circuit current and the power conversion efficiency (PCE) of the cell were improved by 23.1% and 23.4%, respectively. The increased light absorption and effective charge separation of blend films contributed to the enhancement of PCE in doping devices. Moreover, by simulating the photocurrent of solar cells using Onsager–Braun theory, it was found that doping rubrene was responsible for the enhancement of charge transfer generation and dissociation, thereby improving the performance of P3HT:PCBM solar cells.

Passive tumor targeting of renal-clearable luminescent gold nanoparticles: long tumor retention and fast normal tissue clearance.

Glutathione-coated luminescent gold nanoparticles (GS-AuNPs) with diameters of ∼2.5 nm behave like small dye molecules (IRDye 800CW) in physiological stability and renal clearance but exhibit a much longer tumor retention time and faster normal tissue clearance, indicating that the well-known enhanced permeability and retention effect, a unique strength of conventional NPs in tumor targeting, still exists in such small NPs. These merits enable the AuNPs to detect tumor more rapidly than the dye molecules without severe accumulation in reticuloendothelial system organs, making them very promising for cancer diagnosis and therapy.

Research Highlights: Highlights from the latest articles on molecular imaging with Raman spectroscopy

Raman spectroscopy using surfaceenhanced Raman scattering (SERS) nanoparticles is an exciting new molecular imaging modality that has emerged over the past decade [1,2]. The approach is based on the inelastic scatter of photons by molecular bonds. Small-molecule dyes adsorbed on a rough metal (usually gold) surface experience a dramatic increase in their Raman scatter intensity due to surface plasmons, which induce strong localized electric fields near the surface [3]. As each bond within a Raman dye has a characteristic vibrational energy, the SERS spectrum is unique for that dye. The nanoparticle for Raman molecular imaging is composed of: a gold core to provide the signal enhancement, an adsorbed dye to provide the SERS spectrum, a biocompatible coating (e.g., polyethylene glycol or silica) and a targeting moiety (e.g., an antibody). By combining unique dye spectra and targeting moieties for different biological targets, highsensitivity, multiplexed molecular imaging is possible in vivo with an environmentally and optically stable contrast agent [4–8]. Summarized below are four exciting recent reports that significantly advance the field of SERS-based molecular imaging.

A novel molecular logic system based on lead-induced substitution of potassium from a G-quadruplex as a fluorescent lead sensor

A label-free molecular logic system is developed based on a G-quadruplex conformation change, which can be reported using a small dye molecule N-methyl mesoporphyrin IX (NMM) as the fluorescent signaling indicator. In the absence of Pb2+, the K+-stabilized G-quadruplex could bind with NMM, resulting in strong fluorescent intensity. In contrast, the fluorescence decreases greatly when Pb2+ is present. This molecular logic system provides a simple biosensor for Pb2+. The biosensor has shown high accuracy, selectivity, and sensitivity, as well as linearity within a wide concentration range with a detection limit of 5 nM, which meets the EPA standard for Pb2+ (50 nM).

Photophysics of the Geminate Polaron‐Pair State in Copper Phthalocyanine Organic Photovoltaic Blends: Evidence for Enhanced Intersystem Crossing

Geminate polaron-pair recombination directly to the triplet state of the small dye molecule copper(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H- phthalocyanine (CuPC) and exciton trapping in CuPC domains, combine to reduce the internal quantum efficiency of free polaron formation in the bulk-heterojunction blends of CuPC doped with [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) as the electron acceptor.

Using electrophoretic exclusion to manipulate small molecules and particles on a microdevice

Electrophoretic exclusion, a novel separations technique that differentiates species in bulk solution using the opposing forces of electrophoretic velocity and hydrodynamic flow, has been adapted to a microscale device. Proof-of-principle experiments indicate that the device was able to exclude small particles (1 μm polystyrene microspheres) and fluorescent dye molecules (rhodamine 123) from the entrance of a channel. Additionally, differentiation of the rhodamine 123 and polystyrene spheres was demonstrated. The current studies focus on the direct observation of the electrophoretic exclusion behavior on a microchip.

A New Approach for the Identification of Allosteric Binding Sites in Proteins

Recent results indicate that small‐molecule‐protein interactions modulate the function of a large fraction of the enzyme complement in eukaryotic cells. In particular, modulation of enzyme functions by small metabolites that are not their substrates or products is increasingly recognized as a mechanism by which cells can adapt to changes in environmental conditions. While the effect of metabolite‐binding on conformational transitions and thus protein folding, stability, and catalytic activity is clearly of considerable importance, studying metabolite‐protein interactions is in most instances impeded by the low‐affinity nature of the interactions, their moderate effect on enzyme activity, and the complex nature of their interference with substrate turnover. Here, we describe an approach for the efficient mapping of the binding sites of nutrient polyphenols and small dye molecules to the 80 kDa myosin motor domain. We speculate that their binding occurs preferentially with sites that are important for protein‐metabolite interactions and can also be exploited as binding sites for therapeutic drugs. To determine the affinity of the prolific binders for the myosin‐2 motor domain and to select the most suitable compounds, we performed microscale thermophoresis experiments. Subsequently, we determined the structure of the binding pockets for the most promising compounds by X‐ray crystallography.

Biological Interactions of Graphene-Family Nanomaterials: An Interdisciplinary Review

Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms isolated from its three-dimensional parent material, graphite. Related materials include few-layer-graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (rGO), and graphene nanosheets (GNS). This review proposes a systematic nomenclature for this set of Graphene-Family Nanomaterials (GFNs) and discusses specific materials properties relevant for biomolecular and cellular interactions. We discuss several unique modes of interaction between GFNs and nucleic acids, lipid bilayers, and conjugated small molecule drugs and dyes. Some GFNs are produced as dry powders using thermal exfoliation, and in these cases, inhalation is a likely route of human exposure. Some GFNs have aerodynamic sizes that can lead to inhalation and substantial deposition in the human respiratory tract, which may impair lung defense and clearance leading to the formation of granulomas and lung fibrosis. The limited literature on in vitro toxicity suggests that GFNs can be either benign or toxic to cells, and it is hypothesized that the biological response will vary across the material family depending on layer number, lateral size, stiffness, hydrophobicity, surface functionalization, and dose. Generation of reactive oxygen species (ROS) in target cells is a potential mechanism for toxicity, although the extremely high hydrophobic surface area of some GFNs may also lead to significant interactions with membrane lipids leading to direct physical toxicity or adsorption of biological molecules leading to indirect toxicity. Limited in vivo studies demonstrate systemic biodistribution and biopersistence of GFNs following intravenous delivery. Similar to other smooth, continuous, biopersistent implants or foreign bodies, GFNs have the potential to induce foreign body tumors. Long-term adverse health impacts must be considered in the design of GFNs for drug delivery, tissue engineering, and fluorescence-based biomolecular sensing. Future research is needed to explore fundamental biological responses to GFNs including systematic assessment of the physical and chemical material properties related to toxicity. Complete materials characterization and mechanistic toxicity studies are essential for safer design and manufacturing of GFNs in order to optimize biological applications with minimal risks for environmental health and safety.

Surfactant Effects on Amyloid Aggregation Kinetics

There is strong experimental evidence of the influence of surfactants (e.g., fatty acids) on the kinetics of amyloid fibril formation. However, the structures of mixed assemblies and interactions between surfactants and fibril-forming peptides are still not clear. Here, coarse-grained simulations are employed to study the aggregation kinetics of amyloidogenic peptides in the presence of amphiphilic lipids. The simulations show that the lower the fibril formation propensity of the peptides, the higher the influence of the surfactants on the peptide self-assembly kinetics. In particular, the lag phase of weakly aggregating peptides increases because of the formation of mixed oligomers, which are promoted by hydrophobic interactions and favorable entropy of mixing. A transient peak in the number of surfactants attached to the growing fibril is observed before reaching the mature fibril in some of the simulations. This peak originates from transient fibrillar defects consisting of exposed hydrophobic patches on the fibril surface, which provide a possible explanation for the temporary maximum of fluorescence observed sometimes in kinetic traces of the binding of small-molecule dyes to amyloid fibrils.

Structure of the Escherichia coli Phosphonate Binding Protein PhnD and Rationally Optimized Phosphonate Biosensors

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ∼ 70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.