Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence.

The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach fluorochromes, and more worrisomely, lead to phototoxicity. Upon light excitation, noble metal nanoparticles such as silver nanoparticles (AgNPs) generate plasmons, which can then amplify excitation in direct proximity of the nanoparticle's surface and couple to the oscillating dipole of nearby radiating fluorophores, modifying their rate of emission and thus, enhancing their fluorescence. Here, we show that AgNPs fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted Alexa488-conjugated dextran, BODIPY-cholesterol, and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence can occur across the lysosomal membrane. The inclusion of AgNPs in lysosomes did not disturb lysosomal properties such as lysosomal pH, degradative capacity, autophagy and autophagic flux, and membrane integrity, though AgNP seemed to increase basal lysosome tubulation. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing phototoxicity.

Direct photochemical route to azoxybenzenes via nitroarene homocoupling.

We report on a direct photochemical method for the one-pot, catalyst- and additive-free synthesis of azoxybenzene and substituted azoxy derivatives from nitrobenzene building blocks. This reaction is conducted at room temperature and under air, and can be applied to substrates with a wide range of substituents. Yields of products derived from para- and meta-substituted nitrobenzenes are typically good, while sterically encumbered ortho-substituted substrates are not as fruitful. Photochemical Wallach rearrangement of generated azoxybenzenes to ortho-hydroxyazoxybenzenes was observed in some cases, most markedly in selected ortho-halogenated nitrobenzenes. Overall, this method provides an efficient, green pathway to highly value-added azoxybenzene products.

Design and engineering of a dual-mode absorption/emission molecular switch for all-optical encryption.

Photochemical reactions that produce a detectable change in the spectroscopic properties of organic chromophores can be exploited to harness the principles of Boolean algebra and design molecule-based logic circuits. Moreover, the logic processing capabilities of these photoactive molecules can be directed to protect, encode, and conceal information at the molecular level. We have designed a photochemical strategy to read, write and encrypt data in the form of optical signals. We have synthesized a supramolecular system based on the known dye resazurin, and investigated a series of photochemical transformations that can be used to regulate its absorption and emission properties upon illumination with ultraviolet or visible light. We have then examined the logic behaviour of the photochemistry involved, and illustrated its potential application in data encryption.

A Reconfigurable, Dual-Output INHIBIT and IMPLICATION Molecular Logic Gate.

Molecules that respond to input stimulations to produce detectable outputs can be exploited to mimic Boolean logic operators and reproduce basic arithmetic functions. We have designed a two-state fluorescent probe with tunable emission wavelength for the construction of a molecular logic gate with reconfigurable single- or dual-output capability. The system is based on a BODIPY skeleton coupled with 4-(dimethylamino)benzaldehyde. The behavior of the molecular logic gate can be easily investigated in solution with fluorescence spectroscopy, and the optical readout (fluorescence) can be monitored in one (green) or two (green and red) channels. Depending on the solvent of choice, single INHIBIT or dual INHIBIT/IMPLY logic functions can be achieved using chemical inputs (acid and base). Reconfiguration from single- to dual-output is thus made possible by operating the system in acetonitrile (single output) or toluene (dual output), respectively. The logic gate can be switched by manipulating the fluorescence emission via protonation or deprotonation, even when immobilized onto a glass substrate. At the solid state, the resulting output can be stored for extended periods of time. This feature provides two added benefits: (i) memory function and (ii) "set/reset" capability of the logic gate. Our design thus provides a proof-of-concept interface between the molecular and electronic domains.

Optical writing and single molecule reading of photoactivatable and silver nanoparticle-enhanced fluorescence.

We designed a hybrid nanoparticle-molecular system composed of silver nanostructures (AgNP) and a fluorogenic boron dipyrromethene (BODIPY) that can be selectively activated by UVA or UVC light in the presence of an appropriate photoacid generator (PAG). Light irradiation of the PAG encourages the release of p-toluenesulfonic, triflic or hydrobromic acid, any of which facilitate optical 'writing' by promoting the formation of a fluorescent species. Metal-enhanced fluorescence (MEF) by AgNP was achieved through rational design of the nano-molecular system in accordance with the principles of radiative decay engineering. In addition to increasing signal to noise, AgNP permitted shorter reaction times and low irradiance - all of which have important implications for applications of fluorescence activation in portable fluorescence patterning, bioimaging and super-resolution microscopy. Single molecule fluorescence microscopy provided unique insights into the MEF mechanism which were hidden by ensemble-averaged measurements, demonstrating that single molecule 'reading' is a valuable tool for characterizing particle-molecule interactions such as those responsible for the relative contributions of increased excitation and plasmophoric emission toward overall MEF. This work represents a step forward in the contemporary design of synergistic nano-molecular systems, and showcases the advantage of fusion between classic spectroscopic techniques and single molecule methods in terms of improved quantitative understanding of fluorophore-nanoparticle interactions, and how these interactions can be exploited to the fullest extent possible.

Two-Photon Excitation of a Plasmonic Nanoswitch Monitored by Single-Molecule Fluorescence Microscopy.

Visible-light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single-molecule study of the plasmon-assisted isomerization of a photochrome-fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser. The metallic nanostructures significantly enhance the visible light-induced performance of the photoconversion, which normally requires ultraviolet excitation. The resulting ring-open isomer is strongly fluorescent and can also be excited at 633 nm. These stochastic emission events are used to monitor photochromic activation and show quadratic dependence on incident power. The utilization of a single laser wavelength for both photochromic activation and excitation effectively mimics a pseudo two-colours system.

Dye synthesis in the Pechmann reaction: catalytic behaviour of samarium oxide nanoparticles studied using single molecule fluorescence microscopy.

Photochemically prepared samarium oxide nanoparticles (Sm2O3NP) efficiently catalyze the formation of coumarin 153 via the Pechmann trans-esterification and condensation process. The formation of the fluorescent coumarin allowed the catalytic process to be monitored in real time at the single molecule level using Total Internal Reflection Fluorescence Microscopy (TIRFM). Benchtop experiments conducted in parallel demonstrated that the observed catalysis occurred in solution rather than by pure heterogeneous catalysis and is due to a mobile population of small Sm2O3NP released from a polydisperse original sample containing larger particles. TIRFM provided unique insights by demonstrating that catalysis by these smaller colloidal particles is in fact a surface process, while the larger particles are merely suppliers of the small catalytic nanostructures. We refer to this behaviour as a semi-heterogeneous catalytic system. This work showcases the opportunity that single molecule fluorescence techniques can offer in terms of understanding and ultimately improving benchtop and scaled-up synthesis. This specific example highlights the general applicability of this approach to the study of widely-utilized chemical reactions and lays the groundwork for researchers to adopt similar strategies in other systems.

Copper nanoparticle heterogeneous catalytic 'click' cycloaddition confirmed by single-molecule spectroscopy.

Colloidal or heterogeneous nanocatalysts can improve the range and diversity of Cu(I)-catalysed click reactions and facilitate catalyst separation and reuse. Catalysis by metal nanoparticles raises the question as to whether heterogeneous catalysts may cause homogeneous catalysis through metal ion leaching, since the catalytic process could be mediated by the particle, or by metal ions released from it. The question is critical as unwanted homogeneous processes could offset the benefits of heterogeneous catalysis. Here, we combine standard bench scale techniques with single-molecule spectroscopy to monitor single catalytic events in real time and demonstrate that click catalysis occurs directly at the surface of copper nanoparticles; this general approach could be implemented in other systems. We use 'from the mole to the molecule' to describe this emerging idea in which mole scale reactions can be optimized through an intimate understanding of the catalytic process at the single-molecule-single catalytic nanoparticle level.

Sensitized excited free-radical processes as read-write tools: impact on non-linear lithographic processes.

A prefluorescent radical probe in which a 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) unit is sensitized with visible light via energy transfer from a tethered coumarin dye has been synthesized. The excited TEMPO moiety undergoes hydrogen abstraction from a polymer with concomitant functionalization of the polymeric matrix; this writing process can be reversed thermally. The fluorescent probes can be bleached under high intensity illumination, making these new materials suitable for dual-tone lithography with potential applications in sub-diffraction lithographic imaging.

Photoinduced enhancement in the luminescence of hydrophilic quantum dots coated with photocleavable ligands.

In search of strategies to photoactivate the luminescence of semiconductor quantum dots, we devised a synthetic approach to attach photocleavable 2-nitrobenzyl groups to CdSe-ZnS core-shell quantum dots coated with hydrophilic polymeric ligands. The emission intensity of the resulting nanostructured constructs increases by more than 60% with the photolysis of the 2-nitrobenzyl appendages. Indeed, the photoinduced separation of the organic chromophores from the inorganic nanoparticles suppresses an electron-transfer pathway from the latter to the former and is mostly responsible for the luminescence enhancement. However, the thiol groups anchoring the polymeric envelope to the ZnS shell also contribute to the photoinduced emission increase. Presumably, their photooxidation eliminates defects on the nanoparticle surface and promotes the radiative deactivation of the excited quantum dots. This effect is fully reversible but its magnitude is only a fraction of the change caused by the photocleavage of the 2-nitrobenzyl groups. In addition, these particular quantum dots can cross the membrane of model cells and their luminescence increases by ~80% after the intracellular photocleavage of the 2-nitrobenzyl quenchers. Thus, photoswitchable luminescent constructs with biocompatible character can be assembled combining the established photochemistry of the 2-nitrobenzyl photocage with the outstanding photophysical properties of semiconductor quantum dots and the hydrophilic character of appropriate polymeric ligands.

Structural and size effects on the spectroscopic and redox properties of CdSe nanocrystals in solution: the role of defect states.

Two series of CdSe quantum dots (QDs) with different diameters are prepared, according to frequently used protocols of the same synthetic procedure. For each sample the photophysical properties and the potentials for the first reduction and oxidation processes in organic solution are determined. The band gap obtained from electrochemical experiments is compared with that determined from the absorption and luminescence spectra. While the optical band gap decreases upon increasing the nanocrystal diameter, as expected on the basis of quantum confinement, the redox potentials and the electrochemical band gap are not monotonously related to the QD size. For both series, the smallest and largest QDs are both easier to oxidize and reduce than mid-sized QDs. In fact, the latter samples exhibit very broad voltammetric profiles, which suggests that the heterogeneous electron-transfer processes from/to the electrode are kinetically hindered. Conversely, the electrochemical band gap for the smallest and largest particles of each series is somewhat smaller than the optical band gap. These results indicate that, while the optical band gap depends on the actual electron-hole recombination within the nanocrystal, and therefore follows the size dependence expected from the particle-in-a-box model, the electrochemical processes of these QDs are strongly affected by other factors, such as the presence of surface defects. The investigations suggest that the influence of these defects on the potential values is more important for the smallest and largest QDs of each series, as confirmed by the respective luminescence bands and quantum yields. An interpretation for the size-dependent evolution of the surface defects in these nanocrystals is proposed based on the mechanism of their formation and growth.

Molecular strategies to read and write at the nanoscale with far-field optics.

Diffraction prevents the focusing of ultraviolet and visible radiations within nanoscaled volumes and, as a result, the imaging and patterning of nanostructures with conventional far-field illumination. Specifically, the irradiation of a fluorescent or photosensitive material with focused light results in the simultaneous excitation of multiple chromophores distributed over a large area, relative to the dimensions of single molecules. It follows that the spatial control of fluorescence and photochemical reactions with molecular precision is impossible with conventional illumination configurations. However, the photochemical and photophysical properties of organic chromophores can be engineered to overcome diffraction in combination with patterned or reiterative illumination. These ingenious strategies offer the opportunity to confine excited chromophores within nanoscaled volumes and, therefore, restrict fluorescence or photochemical reactions within subdiffraction areas. Indeed, information can be "read" in the form of fluorescence and "written" in the form of photochemical products with resolution down to the nanometre level on the basis of these innovative approaches. In fact, these promising far-field optical methods permit the convenient imaging of biological samples and fabrication of miniaturized objects with unprecedented resolution and can have long-term and profound implications in biomedical research and information technology.

Supramolecular strategies to construct biocompatible and photoswitchable fluorescent assemblies.

We designed and synthesized an amphiphilic copolymer with pendant hydrophobic decyl and hydrophilic poly(ethylene glycol) chains along a common poly(methacrylate) backbone. This macromolecular construct captures hydrophobic boron dipyrromethene fluorophores and hydrophobic spiropyran photochromes and transfers mixtures of both components in aqueous environments. Within the resulting hydrophilic supramolecular assemblies, the spiropyran components retain their photochemical properties and switch reversibly to the corresponding merocyanine isomers upon ultraviolet illumination. Their photoinduced transformations activate intermolecular electron and energy transfer pathways, which culminate in the quenching of the boron dipyrromethene fluorescence. As a result, the emission intensity of these supramolecular constructs can be modulated in aqueous environments under optical control. Furthermore, the macromolecular envelope around the fluorescent and photochromic components can cross the membrane of Chinese hamster ovarian cells and transport its cargo unaffected into the cytosol. Indeed, the fluorescence of these supramolecular constructs can be modulated also intracellularly by operating the photochromic component with optical inputs. In addition, cytotoxicity tests demonstrate that these supramolecular assemblies and the illumination conditions required for their operation have essentially no influence on cell viability. Thus, supramolecular events can be invoked to construct fluorescent and photoswitchable systems from separate components, while imposing aqueous solubility and biocompatibility on the resulting assemblies. In principle, this simple protocol can evolve into a general strategy to deliver and operate intracellularly functional molecular components under optical control.

Photoswitchable fluorescent dyads incorporating BODIPY and [1,3]oxazine components.

We designed and synthesized three compounds incorporating a BODIPY fluorophore and an oxazine photochrome within the same molecular skeleton and differing in the nature of the linker bridging the two functional components. The [1,3]oxazine ring of the photochrome opens in less than 6 ns upon laser excitation in two of the three fluorophore-photochrome dyads. This process generates a 3H-indolium cation with a quantum yield of 0.02-0.05. The photogenerated isomer has a lifetime of 1-3 µs and reverts to the original species with first-order kinetics. Both photochromic systems tolerate hundreds of switching cycles with no sign of degradation. The visible excitation of the dyads is accompanied by the characteristic fluorescence of the BODIPY component. However, the cationic fragment of their photogenerated isomers can accept an electron or energy from the excited fluorophore. As a result, the photoinduced transformation of the photochromic component within each dyad results in the effective quenching of the BODIPY emission. Indeed, the fluorescence of these photoswitchable compounds can be modulated on a microsecond time scale with excellent fatigue resistance under optical control. Thus, our operating principles and choice of functional components can ultimately lead to the development of valuable photoswitchable fluorescent probes for the super-resolution imaging of biological samples.

Anti-proliferative and anti-cancer properties of Achyranthes aspera: specific inhibitory activity against pancreatic cancer cells.

AIMS OF THE STUDY: Achyranthes aspera (Family: Amaranthacea) is a medicinal plant used as an anti-cancer agent in ayurveda, a traditional system of medicine practiced in subcontinental India. The aim of the study was to systematically investigate the anti-proliferative properties of Achyranthes aspera leaves extracted in methanol (LE) on human cancer cells in vitro. MATERIALS AND METHODS: We tested time, dose dependent and specific anti-proliferative activity of LE by clonogenic cell survival assay on human cancer and normal epithelial cell lines in vitro. We further investigated its effect on the expression of metastatic and angiogenic genes by real time polymerase chain reaction. On silica gel column, we carried out initial fractionation analysis. RESULTS: LE exhibited time and dose dependent cytotoxicity on several tumor cells. Compared to cancer cells of colon, breast, lung and prostate origin, pancreatic cancer cells were significantly more sensitive to LE. Preliminary mechanistic studies suggested that LE selectively suppressed the transcription of metalloproteases (MMP-1 and -2), inhibitors of MMPs (TIMP-2) and angiogenic factors (VEGF-A and VEGF-B). Fractionation of LE on methanol equilibrated silica gel column resolved into three fractions of which fraction (F 3) was found to be enriched with anti-proliferative activity. CONCLUSION: Methanolic extract of Achyranthes aspera contains potent anti-proliferative compound with specific activity against pancreatic cancer. Further studies are needed to confirm the in vivo anti-tumorigenicity and subsequent chemical characterization of the active molecule(s).

A redox-driven multicomponent molecular shuttle.

A multicomponent [2]rotaxane designed to operate as a molecular shuttle driven by light energy has been constructed, and its properties have been investigated. The system is composed of (1) a light-fueled power station, capable of using the photon energy to create a charge-separated state, and (2) a mechanical switch, capable of utilizing such a photochemically generated driving force to bring about controllable molecular shuttling motions. The light-fueled power station is, in turn, a dyad comprising (i) a pi-electron-accepting fullerene (C60) component and (ii) a light-harvesting porphyrin (P) unit which acts as an electron donor in the excited state. The mechanical switch is a redox-active bistable [2]rotaxane moiety that consists of (i) a tetrathiafulvalene (TTF) unit as an efficient pi-electron-donor station, (ii) a dioxynaphthalene (DNP) unit as a second pi-electron-rich station, and (iii) a tetracationic cyclobis(paraquat-p-phenylene) (CBPQT4+) pi-electron-acceptor cyclophane, which encapsulates the better pi-electron-donating TTF station. Diethylene glycol spacers were conveniently introduced between the electroactive components in the dumbbell-shaped thread to facilitate the template-directed synthesis of the [2]rotaxane. A modular synthetic approach was undertaken for the overall synthesis of this multicomponent bistable [2]rotaxane, beginning with the syntheses of the P-C60 dyad unit and the two-station TTF-DNP-based [2]rotaxane separately, using conventional synthetic methodologies. These two components were finally stitched together by an esterification to afford the target rotaxane. Its structure was characterized by 1H NMR spectroscopy and mass spectrometry as well as by UV-vis-NIR absorption spectroscopy and voltammetry. The observations reflect remarkable electronic interactions between the various units, pointing to the existence of folded conformations in solution. The redox-driven shuttling process of the CBPQT4+ ring between the two competitive electron-rich recognition units, namely, TTF and DNP, was investigated by electrochemistry and spectroelectrochemistry as a means to verify its operational behavior prior to the photophysical studies related to light-driven operation. The oxidation process of the TTF unit is dramatically hampered in the rotaxane, thereby reducing the efficiency of the shuttling motion. These results confirm that, as the structural complexity increases, the overall function of the system no longer depends simply on its "primary" structure but also on higher-level effects which are reminiscent of the secondary and tertiary structures of biomolecules.