Protein‐Specific Imaging of O‐GlcNAcylation in Single Cells

Thousands of intracellular proteins are post‐translationally modified with O‐GlcNAc, and O‐GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O‐GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM–FRET‐based strategy, which exploits the spatial proximity of the O‐GlcNAc moiety and the attaching protein, for protein‐specific imaging of O‐GlcNAcylation in single cells. We demonstrated this strategy by imaging the O‐GlcNAcylation state of tau and β‐catenin inside the cells. Furthermore, the changes in tau O‐GlcNAcylation were monitored when the overall cellular O‐GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM–FRET strategy will be broadly applicable to probe the O‐GlcNAcylation state of various proteins in the cells.     

Functionalization of manganite nanoparticles and their interaction with biologically relevant small ligands: picosecond time-resolved FRET studies

We report molecular functionalization of the promising manganite nanoparticles La0.67Sr0.33MnO3 (LSMO) for their solubilization in aqueous environments. The functionalization of individual NPs with the biocompatible citrate ligand, as confirmed by Fourier transform infrared (FTIR) spectroscopy, reveals that citrates are covalently attached to the surface of the NPs. UV-VIS spectroscopic studies on the citrate functionalized NPs reveals an optical band in the visible region. Uniform size selectivity (2.6 nm) of the functionalization process is confirmed from high resolution transmission electron microscope (HRTEM). In the present study we have used the optical band of the functionalized NPs to monitor their interaction with other biologically important ligands. F?rster resonance energy transfer (FRET) of a covalently attached probe 4-nitrophenylanthranilate (NPA) with the capped NPs confirm the attachment of the NPA ligands to the surface functional group (-OH) of the citrate ligand. The FRET of a DNA base mimic, 2-aminopurine (2AP), with the NPs confirms the surface adsorption of 2AP. Our study may find relevance in the study of the interaction of individual manganite NPs with drug/ligand molecules.     

Preparation,Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme

A bicyclo[6,1,0]nonyne‐substituted 9‐mesityl‐10‐methyl‐acridinium cofactor was prepared and covalently linked to a prolyl oligopeptidase scaffold containing a genetically encoded 4‐azido‐L ‐phenylalanine residue in its active site. The resulting artificial enzyme catalyzed sulfoxidation when irradiated with visible light in the presence of air. This reaction proceeds by initial electron abstraction from the sulfide within the enzyme active site, and the protein scaffold extended the fluorescence lifetime of the acridium cofactor. The mode of sulfide activation and placement of the acridinium cofactor ( 5 ) in POP‐ZA₄‐ 5 make this artificial enzyme a promising platform for developing selective photocatalytic transformations.     

Fluorescence properties of fluorescein and rhodamine supported on alumina nanowire films

In this paper, alumina nanopore array films and nanowire films were prepared by anodic oxidation method, and fluorescein and rhodamine molecules were loaded onto their surfaces to fabricate composite fluorescent films. The effects of morphologies and annealing temperatures of alumina films on the fluorescence properties of the composite films were investigated. When loaded on alumina nanowire films, the properties of rhodamine B (RB), fluorescein (FL), rhodamine 110 (R110), rhodamine 6G (R6G) and the FRET (fluorescence resonance energy transfer) systems they constituted were studied in detail. Results show that the fluorescence emission of RB is stronger on alumina nanowire films than on alumina nanopore array films. The molecular structure of donor and acceptor can affect the energy transfer efficiency of FRET system (E), the fluorescence emission enhancement multiple of acceptor (I_A(DA)/I_A) and the transferred energy utilization factor (η). Compared to the systems of FL-R110, FL-R6G, R110-R6G, R110-RB and R6G-RB, FL-RB has the best comprehensive performance with the largest I_A(DA)/I_A (4.90) and η (6.42), despite its medium E (76.3%).     

Study of polymer gelation by fluorescence spectroscopy

A fluorescence technique is used to study the mechanism and dynamics of thermoreversible gel formation in concentrated polymer solutions. Two examples of these ordered structures were studied: isotactic polystyrene (iPS) in decalin and syndiotactic poly(methyl methacrylate) (sPMMA) in toluene. iPS can form two types of gels in decalin solutions. Turbid suspensions are formed between 60°C and 130°C. When the solution is quenched beneath 20°C, transparent gels are formed. In the former solution the polymer has a 3₁ helix structure; in the latter a 12₁ helix is formed. These structures formed by iPS in decalin solution have different fluorescence spectra. The ratio (I_m/I_e) of the intensity of the monomer fluorescence band in the spectrum over the intensity of the excimer emission band is higher in the spectra of transparent gels. The formation of the transparent gels from a solution can be followed by plotting I_m/I_e as a function of temperature. The kinetics of the gel formation are studied by following I_m/I_e as a function of time at a fixed temperature. sPMMA forms gels in toluene by a mechanism called “conformational gelation.” This mechanism consists of two steps: a fast conformational change followed by a slower aggregation of polymer chains. Both steps were followed by measuring fluorescence of a pyrene label attached to the polymer. The fluorescence lifetime of this probe is sensitive to the polarity of the environment. When the polymer conformation goes from a random coil to an all trans conformation, the environment of the fluorescent label becomes increasingly richer in solvent. This change in polarity is reflected in a change in fluorescence lifetime. The change in fluorescence anisotropy of the label reflects the second step of the mechanism and correlates with the occurrence of chain aggregation.     

Synthesis of magnetic citric‐acid‐functionalized graphene oxide and its application in the removal of methylene blue from contaminated water

A magnetic nanocomposite of citric‐acid‐functionalized graphene oxide was prepared by an easy method. First, citric acid (CA) was covalently attached to acyl‐chloride‐functionalized graphene oxide (GO). Then, Fe₃O₄ magnetic nanoparticles (MNPs) were chemically deposited onto the resulting adsorbent. CA, as a good stabilizer for MNPs, was covalently attached to the GO; thus MNPs were adsorbed much more strongly to this framework and subsequent leaching decreased and less agglomeration occurred. The attachment of CA onto GO and the formation of the hybrid were confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, X‐ray diffraction spectrometry and transmission electron microscopy. The specific saturation magnetization of the magnetic CA‐grafted GO (GO‐CA‐Fe₃O₄) was 57.8 emu g^?1 and the average size of the nanoparticles was found to be 25 nm by transmission electron microscopy. The magnetic nanocomposite was employed as an adsorbent of methylene blue from contaminated water. The adsorption tests demonstrated that it took only 30 min to attain equilibrium. The adsorption capacity in the concentration range studied was 112 mg g^?1. The GO‐CA‐Fe₃O₄ nanocomposite was easily manipulated in an external magnetic field which eases the separation and leads to the removal of dyes. Thus the prepared nanocomposite has great potential in removing organic dyes. © 2014 Society of Chemical Industry     

Temperature effect on the swelling of PAAm‐κ‐carrageenan composites

The steady‐state fluorescence (SSF) technique was used for studying swelling of disc‐shaped polyacrylamide (PAAm)‐κ‐carrageenan (κC) composites which were prepared by free‐radical crosslinking copolymerization at 80°C. Pyranine was introduced as a fluorescence probe during polymerization. Swelling experiments were performed in water at various temperatures by real‐time monitoring of the pyranine (Py) fluorescence intensity, I which decreased as swelling proceeded. Stern–Volmer equation is modified for low quenching efficiencies to interpret the behavior of Py intensity during the swelling of PAAm‐κC composites. The Li‐Tanaka equation was used to determine the swelling time constants, τ₁, and cooperative diffusion coefficients, D₀, from fluorescence intensity, weight, and volume variations of the composites at various temperatures. It was observed that τ₁ first decreased up to 40°C and then increased; naturally, D₀ increased up to 40°C and then decrease for all κC content gels. Swelling activation energies, ΔE, were measured for the swelling composites, which are found to be exothermic and endothermic in between 30–40 and 40–60°C, respectively. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

A fluorescence resonance energy transfer probe for sensing pH in aqueous solution

A fluorescence resonance energy transfer (FRET) sensor, which has a FRET donor and an acceptor attached to each chain end of pH-sensitive polysulfonamide, is synthesized and its pH sensitivity is examined in terms of the FRET efficiency. This polymeric sensor exhibits an instantaneous conformation change from coil to globule at a specific pH, which results in the drastic on-and-off FRET efficiency. To detect a specific pH region, sulfadimethoxine and sulfamethizole are selected among various sulfonamides since their pK_a values are in the physiological pH. For tuning the emission color arising from FRET, 7-hydroxy-4-bromomethyl coumarin and coumarin 343 are used as a FRET donor and an acceptor, respectively, for a blue-to-green FRET sensor, and fluorescence amine isomer I and rhodamine B are used for a green-to-red FRET sensor. Each sensor shows a distinct color change from the emission wavelength of FRET donor to the emission wavelength of FRET acceptor, which well explains their feasibility as a useful optical sensor.     

Fluorescence studies on the interaction between pyrene‐labelled poly(acrylic acid) and cyclodextrins

The interactions, in aqueous media, between a pyrene‐labelled polyelectrolyte poly(acrylic acid) (PAAMePy) with two different degrees of labelling and β‐ and γ‐cyclodextrins (β‐ and γ‐CD) were studied using absorption and fluorescence (steady‐state and time‐resolved) techniques. In addition to qualitative and quantitative parameters obtained from absorption and steady‐state fluorescence spectra, time‐resolved fluorescence data are presented, allowing additional important observations regarding the nature of the interactions. From the overall data it was possible to conclude that in the case of interaction with γ‐CD the efficient encapsulation of two pyrene units into the cavity of the cyclodextrin molecule leads to a decrease in the number of available free monomers and an increase in the number of preformed ground‐state dimers (GSDs) of pyrene. It was also shown that contrary to the situation in water, where only intramolecular interactions are present, the addition of γ‐CD leads to new interpolymeric interactions. The absence of significant changes is noted when the interactions of PAAMePy polymers take place with β‐CD. The excimer‐to‐monomer fluorescence intensity ratio (I_E/I_M) was found to increase with the added amount of γ‐CD but not with β‐CD. This increase is justified on the basis of the increase of the GSD contribution. The photophysical behaviour was found to be dependent on the pH of the media, but with the absence of relevant interactions between CD and PAAMePy polymer at alkaline values. Copyright © 2007 Society of Chemical Industry     

Film formation of poly (methyl methacrylate) latex with pyrene functional poly (divinylbenzene) microspheres prepared by click chemistry

This work reports on the application of steady state fluorescence (SSF) technique for studying film formation from poly(methyl methacrylate) (PMMA) latex and poly(divinylbenzene) (PDVB) microsphere composites. Pyrene (P) functionalized PDVB cross‐linked spherical microspheres with diameters of 2.5 μm were synthesized by using precipitation polymerization technique followed by click coupling reaction. The diameter of the PMMA particles prepared by emulsion polymerization were in the range of 0.5–0.7 μm. PMMA/PDVB composite films were then prepared by physically blending of PMMA latex with PDVB microspheres at various composition (0, 1, 3, 5, 10, 20, 40, and 60 wt%). After drying, films were annealed at elevated temperatures above T_g of PMMA ranging from 100 to 270°C for 10 min time intervals. Evolution of transparency of the composite films was monitored by using photon transmission intensity, I_tr. Monomer (I_P) and excimer (I_E) fluorescence intensities from P were measured after each annealing step. The possibility of using the excimer‐to‐monomer intensity ratio (I_E/I_P) from PDVB microparticles as a measure of PMMA latex coalescence was demonstrated. Diffusion of the PMMA chains across the particle–particle interfaces dilutes the dyes, increasing their separation. The film formation stages of PMMA latexes were modeled by monitoring the I_E/I_P ratios and related activation energies were determined. There was no observable change in activation energies confirming that film formation behavior is not affected by varying the PDVB composition in the studied range. SEM images of PMMA/PDVB composites confirmed that the PMMA particles undergo complete coalescence forming a continuous phase in where PDVB microspheres are dispersed. POLYM. COMPOS., 2011. © 2011 Society of Plastics Engineers     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

High‐performance varistors prepared by hot‐dipping tin oxide thin films in Sb2O3 powder: Influence of temperature

A series of SnO_x–Sb₂O₃ thin film varistors were fabricated through hot‐dipping tin oxide films deposited by radio‐frequency magnetron sputtering in Sb₂O₃ powder at varied temperatures in air. With the increase in hot‐dipping temperature (HDT) from 200°C to 600°C, the nonlinear coefficient (α) of the samples increased first and then decreased, reaching the maximum at 500°C, which was mainly determined by the completeness of high‐resistant Sb₂O₃ layer at tin oxide grain boundary and the chemical composition of tin oxide films. Correspondingly, the leakage current (I_L) decreased first and increased later. The breakdown electric field (E_100 mA) decreased constantly with increasing HDT. The SnO_x–Sb₂O₃ film varistors prepared at 500°C exhibited the optimum nonlinear properties with the maximum α of 10.88, the minimum I_L of 36.3 mA/cm², and an E_100mA of 0.0188 V/nm. The obtained nanoscaled film varistors would be promising in electrical/electronic devices working in low voltage.     

Film formation from PS/AL2O3 nanocomposites prepared by dip‐drawing method

In this study, steady state fluorescence (SSF) and UV–vis (UVV) techniques were used to examine film formation from pyrene (P) labeled polystyrene (PS) latex/Al₂O₃ (PS/Al₂O₃) composites prepared by the dip‐drawing method. The effects of dip‐drawing rates and dipping time in Al₂O₃ sol on film formation behavior and the morphology of PS/Al₂O₃ films were investigated. Films were prepared first by casting PS dispersion on clean glass substrates which creates a close‐packed array of PS sphere (203 nm) templates. These templates were then covered with Al₂O₃ utilizing the dip‐drawing method for various dip‐drawing rates and dipping times in Al₂O₃ sol. The film formation of these composites was studied by annealing them at a temperature range of 100°C to 270°C and monitoring the scattered light (I_sc), fluorescence (I_P), and transmitted light (I_tr) intensities after each annealing step. The structural properties of the composite films were analyzed with a scanning electron microscope (SEM). The results demonstrated that the film formation behavior and morphology of composites depended mainly on dipping time, and no dependence on the dip‐drawing rate was observed. The optical results indicated that PS/Al₂O₃ films undergo complete film formation independent of the dip‐drawing rate and dipping time. Additionally, the film formation stages were modeled and the corresponding activation energies were determined. After completion of film formation, PS polymers were extracted to obtain porous Al₂O₃ thin films. Highly ordered porous structures were observed for long dipping time in Al₂O₃ sol but no change was observed for different dip‐drawing rates, confirming the optical data. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers     

Excimer fluorescence as a probe of miscibility in styrene copolymer blends

The miscibility of styrene-alt-maleic anhydride (SMA) and a series of styrene-acrylonitrile (SAN) copolymers in blends with either PMMA or PVC were examined by fluorescence and FTIR spectroscopic techniques. In the case of the alternating copolymer intra-molecular excimer formation is precluded and changes in the excimer/monomer fluorescence intensity (I_E/I_M) reflect changes in the amount of inter-chain excimer formation. The value of I_E/I_M in blends, therefore, provides a sensitive measure of phase separation (miscibility). In the case of the acrylonitrile copolymers the I_E/I_M of the blend had to be corrected to account for intra-molecular excimers formed between adjacent chromophores in the SAN. These results compared favourably with observed frequency shifts in the phenyl C—H out-of-plane bend (～700 cm^?1).     

Studies on drying and swelling of PAAm‐NIPA composites in various compositions

The steady‐state fluorescence technique was introduced for studying the drying and swelling of disc‐shaped PAAm‐NIPA composites. Disc‐shaped gels were formed with various acrylamides (AAm) and N‐isopropylacrylamides (NIPA) by free radical crosslinking copolymerization in water. Composites were prepared with pyranine (Py) doped as a fluorescence probe. Scattered light, I_sc, and fluorescence intensities, I, were monitored during drying of these gels. The fluorescence intensity of pyranine increased and decreased as drying and swelling time increased respectively for all samples. The Stern‐Volmer equation combined with moving boundary and Li‐Tanaka models were used to explain the behavior of I during drying and swelling, respectively. It was found that the desorption coefficient, D, increased as NIPA contents were increased for a given temperature during drying. However, the cooperative diffusion coefficient, D₀, increased as NIPA contents were decreased during swelling at a given temperature. Supporting gravimetrical and volumetric experiments were also carried out during drying and swelling of PAAm‐NIPA composites. It was observed that NIPA contents affect the drying and swelling process. POLYM. COMPOS., 2011. © 2011 Society of Plastics Engineers     

Self-association of hyperbranched poly(sulfone-amine) in water: studies with pyrene-fluorescence probe and fluorescence label

Both pyrene-fluorescence probe and fluorescence label techniques are used to investigate the association behaviors of hyperbranched poly(sulfone-amine) (HPSA) in aqueous solution. In the presence of HPSA, excimer emission peak evidently appeared, while no excimer peak was observed in the emission spectra in the absence of HPSA. The excitation spectrum monitored at excimer emission red shifts by about 38-40 nm compared to that monitored at monomer emission, which shows that the excimer is formed by preassociated pyrene chromophores. In the same concentration of pyrene, monomer emission of pyrene decreases but excimer emission increases with increasing the concentration of HPSA; the ratio of excimer-to-monomer emission intensity (I_E/I_M) gradually increases, reaches a critical point at 5-7 g/l, and sharply increases with the concentration. Pyrene-labeled hyperbranched poly(sulfone-amine) (Py-HPSA) was synthesized from 4-(1-Pyrene)butyroyl chloride and HPSA. The monomer emission and excimer emission of Py-HPSA show the concentration-quenching effect, while I_E/I_M increases monotonously, approaches a critical point, and then suddenly increases with increasing the concentration of Py-HPSA. Influences of acidity and solvents on the fluorescence emission were studied. In high concentrations of hyperbranched polymer, pH and DMSO significantly influence the emission of pyrene, and excimer peak disappears at 72% of DMSO fraction.     

Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein

Each of the two domains of mammalian metallothioneins contains a zinc-thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the alpha-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ([[(amino)ethyl]amino]naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the alpha-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.     

Film formation of nano‐sized hard latex (PS) in soft polymer matrix (PBA): An excimer study

This work reports steady state fluorescence (SSF) technique for studying film formation from pyrene (P)‐labeled nano‐sized polystyrene (PS) and poly(n‐butyl acrylate) (PBA) hard/soft latex blends. Blend films were prepared from mixtures of PS and PBA in dispersion. Eight different blend films were prepared in various hard/soft latex compositions at room temperature and annealed at elevated temperatures above glass transition temperature (T_g) of polystyerene. Monomer (I_P) and excimer (I_E) intensities from P was measured after each annealing step to monitor the stages of film formation. The evolution of transparency of latex films was monitored using photon transmission intensity, I_tr. Film morphologies were examined by atomic force microscopy (AFM). The results showed that as the amount of hard component (PS) in the blend is decreased, a significant change occurred in both I_E/I_P and I_tr curves at a certain critical weight fraction (50 wt%) of PS hard latex. Two distinct film formation stages, which are named as void closure and interdiffusion were seen in (I_E/I_P) data above this fraction. However, below 50 wt% PS no film formation was observed. AFM pictures also confirmed these findings. Void closure and interdiffusion stages for (50–100) wt% range of PS were modeled and related activation energies were determined. There was no observable change in activation energies confirming that film formation behavior is not affected by varying the blend composition in this range. POLYM. COMPOS., 31:1611–1619, 2010. © 2009 Society of Plastics Engineers