Microscopic Perspective of Synergy between Localized Surface Plasmon Resonance and Disruption of Dye Aggregates in Metal Nanoparticle-Enhanced Fluorescence

The interaction of rose bengal (RB) aggregates with silver nanoparticles (AgNPs) is investigated to understand the factors that contribute toward metal nanopaticle enhanced fluorescence (MEF), such as reproducibility, spectral shift, and distortion. Various shapes and sizes of RB aggregates (spherical, rods, and fibrils) are formed upon preparing films from their solution in solvent with different polarities. These molecular aggregates are disrupted in the presence of AgNPs, resulting in different enhancement factors, not only because of MEF but also due to hindrance to aggregation-caused quenching. Microspectroscopic studies provide valuable insights into the microheterogeneity of these mixed aggregates. Interestingly, the excited state decay pathways remain the same at the nanosecond time scale for different emission wavelengths. Additionally, the lifetime distribution is very narrow due to the interaction of RB deaggregates with the plasmonic AgNPs

Spectrally Resolved Photoluminescence Imaging of ZnO Nanocrystals at Single-Particle Levels

The intrinsic spectral line widths of defect-related transitions in quantum-confined semiconductor nanocrystals are often difficult to estimate using ensemble measurements because the extent of inhomogeneous broadening due to particle size distributions is not known precisely. To address this problem, we performed spectrally resolved photoluminescence (PL) microscopy of individual ZnO NC by directly populating the defects states using low-energy laser excitation. The temporal evolution of PL intensities shows discrete blinking behaviors, suggesting that the NCs are detected near single-particle levels. The transition energies of individual NCs are found to fluctuate around their mean position (2.25 eV) by ∼0.130 eV, which is attributed to particle size distribution and defects densities associated with each NC. The spectral line width associated with defect emission envelope of ZnO NCs is found to be inherently broad (200–400 meV), which further establishes the presence of multiple closely spaced defect energy levels within every ZnO NC

Photoluminescence Flickering of Micron-Sized Crystals of Methylammonium Lead Bromide: Effect of Ambience and Light Exposure

Recent reports on temporal photoluminescence (PL) intensity fluctuations (<i>blinking</i>) within localized domains of organo-metal lead halide (hybrid) perovskite microcrystals have invoked considerable interest to understand their origins. Using PL microscopy, we have investigated the effect of atmospheric constituents and photoillumination on spatially extended intensity fluctuations in methylammonium lead bromide (MAPbBr₃) perovskite materials, explicitly for micrometer (ca. 1–2 μm)-sized crystals. Increase in the relative humidity of the ambience results in progressive reduction in the PL intensity, and beyond a threshold value, individual microcrystalline grains exhibit multistate PL intermittency (<i>flickering</i>), which is characteristically different from quasi two-state blinking observed in nanocrystals. Such flickering disappears upon removal of moisture, accompanied by considerable enhancement of the overall PL efficiency. We hypothesize that initiation of moisture-induced degradation marked by the lowering of PL intensity correlates with the appearance of PL flickering, and such processes further accelerate in the presence of oxygen as opposed to an inert (nitrogen) environment. We find that the intrinsic defects not only increase the threshold level of ambient moisture needed to initiate flickering but also modulate the nature of PL intermittency. Our results therefore establish a strong correlation between initiation of material degradation and PL flickering of hybrid perovskite microcrystals, induced by transient defects formed via interaction with the ambience

Bioactive Polymersomes Self-Assembled from Amphiphilic PPO-<i>Glyco</i>Polypeptides: Synthesis, Characterization, and Dual-Dye Encapsulation

Glycopolypeptide-based polymersomes have promising applications as vehicles for targeted drug delivery because they are capable of encapsulating different pharmaceuticals of diverse polarity as well as interacting with specific cell surfaces due to their hollow structural morphology and bioactive surfaces. We have synthesized glycopolypeptide-<i>b</i>-poly­(propylene oxide) by ROP of glyco-<i>N</i>-carboxyanhydride (NCA) using the hydrophobic amine-terminated poly­(propylene oxide) (PPO) as the initiator. This block copolymer is composed of an FDA-approved PPO hydrophobic block in conjugation with hydrophilic glycopolypeptides which are expected to be biocompatible. We demonstrate the formation of glycopolypeptide-based polymersomes from the self-assembly of glycopolypeptide-<i>b</i>-poly­(propylene oxide) in which the presence of an ordered helical glycopolypeptide segment is required for their self-assembly into spherical nanoscale (∼50 nm) polymersomes. The polymersomes were characterized in detail using a variety of techniques such as TEM, AFM, cryo-SEM, and light-scattering measurements. As a model for drugs, both hydrophobic (RBOE) and hydrophilic (calcein) dyes have been incorporated within the polymersomes from solution. To substantiate the simultaneous entrapment of the two dyes, spectrally resolved fluorescence microscopy was performed on the glycopeptide polymersomes cast on a glass substrate. We show that it is possible to visualize individual nanoscale polymersomes and effectively probe the dyes’ colocalization and energy-transfer behaviors therein as well as investigate the variation in dual-dye encapsulation over a large number of single polymersomes. Finally, we show that the galactose moieties present on the surface can specifically recognize lectin RCA₁₂₀, which reveals that the polymersomes’ surface is indeed biologically active

Sensing Hg(II) <i>in Vitro</i> and <i>in Vivo</i> Using a Benzimidazole Substituted BODIPY

A multisignaling Hg­(II) sensor based on a benzimidazole substituted BODIPY framework was designed, which displays excellent selectively toward Hg­(II) <i>in vitro</i> and <i>in vivo</i>. Optical and fluorogenic measurements in solution reveal that the sensor can detect mercury ions at submicromolar concentrations, with high specificity. The detection of Hg­(II) is associated with a blue-shift in optical spectra and a simultaneous increase in the fluorescence quantum yield of the sensor, which is attributed to a decrease in charge delocalization and inhibition of photoinduced electron transfer upon binding to Hg­(II). Using several spectroscopic measurements, it is shown that the binding mechanism involves two sensor molecules, where lone pairs of the benzimidazole nitrogen coordinate to a single mercury ion. The utility of this BODIPY sensor to detect Hg­(II) <i>in vivo</i> was demonstrated by fluorescence imaging and spectroscopy of labeled human breast adenocarcinoma cells. While average emission intensity of the sensor over a large number of cells increases with incubated mercury concentrations, spatially resolved fluorescence spectroscopy performed on <i>individual cells</i> reveals clear spectral blue-shifts from a subensemble of sensors, corroborating the detection of Hg­(II). Interestingly, the emission spectra at various submicrometer locations within cells exhibited considerable inhomogeneity in the extent of blue-shift, which demonstrates the potential of this sensor to monitor the local (effective) concentration of mercury ions within various subcellular environments

Sensing Hg(II) <i>in Vitro</i> and <i>in Vivo</i> Using a Benzimidazole Substituted BODIPY

A multisignaling Hg­(II) sensor based on a benzimidazole substituted BODIPY framework was designed, which displays excellent selectively toward Hg­(II) <i>in vitro</i> and <i>in vivo</i>. Optical and fluorogenic measurements in solution reveal that the sensor can detect mercury ions at submicromolar concentrations, with high specificity. The detection of Hg­(II) is associated with a blue-shift in optical spectra and a simultaneous increase in the fluorescence quantum yield of the sensor, which is attributed to a decrease in charge delocalization and inhibition of photoinduced electron transfer upon binding to Hg­(II). Using several spectroscopic measurements, it is shown that the binding mechanism involves two sensor molecules, where lone pairs of the benzimidazole nitrogen coordinate to a single mercury ion. The utility of this BODIPY sensor to detect Hg­(II) <i>in vivo</i> was demonstrated by fluorescence imaging and spectroscopy of labeled human breast adenocarcinoma cells. While average emission intensity of the sensor over a large number of cells increases with incubated mercury concentrations, spatially resolved fluorescence spectroscopy performed on <i>individual cells</i> reveals clear spectral blue-shifts from a subensemble of sensors, corroborating the detection of Hg­(II). Interestingly, the emission spectra at various submicrometer locations within cells exhibited considerable inhomogeneity in the extent of blue-shift, which demonstrates the potential of this sensor to monitor the local (effective) concentration of mercury ions within various subcellular environments

Plasticization of Poly(vinylpyrrolidone) Thin Films under Ambient Humidity: Insight from Single-Molecule Tracer Diffusion Dynamics

Studies on diffusion dynamics of single molecules (SMs) have been useful in revealing inhomogeneity of polymer thin films near and above the glass-transition temperature (<i>T</i>_g). However, despite several applications of polymer thin films where exposure to solvent (or vapor) is common, the effect of absorbed solvent molecules on local morphology and rigidity of polymer matrices is yet to be explored in detail. High-<i>T</i>_g hydrophilic polymers such as poly­(vinylpyrrolidone) (PVP) are used as pharmaceutical coatings for drug release in aqueous medium, as they readily absorb moisture, which results in effective lowering of the <i>T</i>_g and thereby leads to plasticization. The effect of moisture absorption on swelling and softening of PVP thin films was investigated by visualizing the diffusion dynamics of rhodamine 6G (Rh6G) tracer molecules at various ambient relative humidities (RH). Wide-field epifluorescence microscopy, in conjunction with high-resolution SM tracking, was used to monitor the spatiotemporal evolution of individual tracers under varied moisture contents of the matrix. In the absence of atmospheric moisture, Rh6G molecules in dry PVP films are translationally inactive, suggestive of rigid local environments. Under low moisture contents (RH 30–50%), translational mobility remains arrested but rotational motion is augmented, indicating slight swelling of the polymer network which marks the onset of plasticization. The translational mobility of Rh6G was found to be triggered only at a threshold ambient RH, beyond which a large proportion of tracers exhibit extensive diffusion dynamics. Interestingly, SM tracking data at higher moisture contents of the film (RH ≥ 60%) reveal that the distributions of dynamic parameters (such as diffusivity) are remarkably broad, spanning several orders of magnitude. Furthermore, Rh6G molecules display a wide variety of translational motion even at a fixed ambient RH, clearly pointing out the extremely inhomogeneous environment of plasticized PVP network. Intriguingly, it is observed that a majority of tracers undergo anomalous subdiffusion even under high moisture contents of the matrix. Analyses of SM trajectories using velocity autocorrelation function reveal that subdiffusive behaviors of Rh6G are likely to originate from fractional Brownian motion, a signature of tracer dynamics in viscoelastic medium

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide₂₀-<i>b</i>-poly-l-lysine₁₀₀ and PEG_2k-<i>b</i>-poly-l-glutamate₁₀₀ [PEG = poly­(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of <i>N</i>-carboxyanhydrides and “click” chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes

Plasticization of Poly(vinylpyrrolidone) Thin Films under Ambient Humidity: Insight from Single-Molecule Tracer Diffusion Dynamics

Studies on diffusion dynamics of single molecules (SMs) have been useful in revealing inhomogeneity of polymer thin films near and above the glass-transition temperature (<i>T</i>_g). However, despite several applications of polymer thin films where exposure to solvent (or vapor) is common, the effect of absorbed solvent molecules on local morphology and rigidity of polymer matrices is yet to be explored in detail. High-<i>T</i>_g hydrophilic polymers such as poly­(vinylpyrrolidone) (PVP) are used as pharmaceutical coatings for drug release in aqueous medium, as they readily absorb moisture, which results in effective lowering of the <i>T</i>_g and thereby leads to plasticization. The effect of moisture absorption on swelling and softening of PVP thin films was investigated by visualizing the diffusion dynamics of rhodamine 6G (Rh6G) tracer molecules at various ambient relative humidities (RH). Wide-field epifluorescence microscopy, in conjunction with high-resolution SM tracking, was used to monitor the spatiotemporal evolution of individual tracers under varied moisture contents of the matrix. In the absence of atmospheric moisture, Rh6G molecules in dry PVP films are translationally inactive, suggestive of rigid local environments. Under low moisture contents (RH 30–50%), translational mobility remains arrested but rotational motion is augmented, indicating slight swelling of the polymer network which marks the onset of plasticization. The translational mobility of Rh6G was found to be triggered only at a threshold ambient RH, beyond which a large proportion of tracers exhibit extensive diffusion dynamics. Interestingly, SM tracking data at higher moisture contents of the film (RH ≥ 60%) reveal that the distributions of dynamic parameters (such as diffusivity) are remarkably broad, spanning several orders of magnitude. Furthermore, Rh6G molecules display a wide variety of translational motion even at a fixed ambient RH, clearly pointing out the extremely inhomogeneous environment of plasticized PVP network. Intriguingly, it is observed that a majority of tracers undergo anomalous subdiffusion even under high moisture contents of the matrix. Analyses of SM trajectories using velocity autocorrelation function reveal that subdiffusive behaviors of Rh6G are likely to originate from fractional Brownian motion, a signature of tracer dynamics in viscoelastic medium

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide₂₀-<i>b</i>-poly-l-lysine₁₀₀ and PEG_2k-<i>b</i>-poly-l-glutamate₁₀₀ [PEG = poly­(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of <i>N</i>-carboxyanhydrides and “click” chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes