Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time.

We have established a label-free plasmonic platform that monitors proteolytic activity in real time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin, resulting in a blueshift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods, we determined thrombin's proteolytic activity in real time and inferred relevant kinetic parameters. Furthermore, a comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors to open the window to a range of applications stretching from the diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Optical Binding-Driven Micropatterning and Photosculpting with Silver Nanorods.

Controlling the nano- and micropatterning of metal structures is an important requirement for various technological applications in photonics and biosensing. This work presents a method for controllably creating silver micropatterns by laser-induced photosculpting. Photosculpting is driven by plasmonic interactions between pulsed laser radiation and silver nanorods (AgNRs) in aqueous suspension; this process leads to optical binding forces transporting the AgNRs in the surroundings, while electronic thermalization results in photooxidation, melting, and ripening of the AgNRs into well-defined 3D structures. This work call these structures Airy castles due to their structural similarity with a diffraction-limited Airy disk. The photosculpted Airy castles contain emissive Ag nanoclusters, allowing for the visualization and examination of the aggregation process using luminescence microscopy. This work comprehensively examines the factors that define the photosculpting process, namely, the concentration and shape of the AgNRs, as well as the energy, power, and repetition rate of the laser. Finally, this work investigates the potential applications by measuring the metal-enhanced luminescence of a europium-based luminophore using Airy castles.

Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP.

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.

Merging Porphyrins with Gold Nanorods: Self Assembly Construct to High Fluorescent Polyelectrolyte Microcapsules.

Dual probe porphyrin-gold nanorod polyelectrolyte microcapsules were developed to explore the enhancing effects of a plasmonic interface of self-assembled gold nanoparticles in the fluorescence emission from porphyrins loaded into the capsules' core. An analysis of fluorescence lifetime imaging microscopy (FLIM) data reports a notable 105-106-fold increase in the maximum detected photon rates from diffraction-limited spots and an overall six-fold increase in fluorescence as averaged over the whole microcapsule area. Large emission enhancements were correlated with decreases in fluorescence lifetimes. The microcapsule's design proved effective in achieving high fluorescent hybrids and may shed light on new possibilities for advanced materials imaging applications.

Fluorescence Spectroscopy of Porphyrins and Phthalocyanines: Some Insights into Supramolecular Self-Assembly, Microencapsulation, and Imaging Microscopy.

The molecular interactions of anionic tetrasulfonate phenyl porphyrin (TPPS) with poly(amido amine) (PAMAM) dendrimers of generation 2.0 and 4.0 (G2 and G4, respectively) forming H- or J-aggregates, as well as with human and bovine serum albumin proteins (HSA and BSA), were reviewed in the context of self-assembly molecular complementarity. The spectroscopic studies were extended to the association of aluminum phthtalocyanine (AlPCS4) detected with a PAMAM G4 dendrimer with fluorescence studies in both steady state and dynamic state, as well as due to the fluorescence quenching associated to electron-transfer with a distribution of lifetimes. The functionalization of TPPS with peripheral substituents enables the assignment of spontaneous pH-induced aggregates with different and well-defined morphologies. Other work reported in the literature, in particular with soft self-assembly materials, fall in the same area with particular interest for the environment. The microencapsulation of TPPS studies into polyelectrolyte capsules was developed quite recently and aroused much interest, which is well supported and complemented by the extensive data reported on the Imaging Microscopy section of the Luminescence of Porphyrins and Phthalocyanines included in the present review.

Co-localization and crosstalk between CD44 and RHAMM depend on hyaluronan presentation.

CD44 and the receptor for hyaluronic acid-mediated motility (RHAMM) are the main hyaluronan (HA) receptors. They are commonly overexpressed in different cancers activating signaling pathways related to tumor progression, metastasis and chemoresistance. Besides their involvement in signal transduction via interaction with HA, currently, there is a little information about the possible crosstalk between CD44 and RHAMM and the role of HA in this process. In the present work, we used immunocytochemistry combined with Förster resonance energy transfer (FRET) microscopy and co-immunoprecipitation to elucidate the involvement of HA in CD44 and RHAMM expression, co-localization and crosstalk. We studied breast cancer cells lines with different degrees of invasiveness and expression of these receptors in the absence of exogenous HA and compared the data with the results obtained for cultures supplemented with either soluble HA or seeded on substrates with end-on immobilized HA. Our results demonstrated that cells response depends on the HA presentation: CD44/RHAMM complexation was upregulated in all cell lines upon interaction with immobilized HA, but not with its soluble form. Moreover, the results showed that the expression of both CD44 and RHAMM is regulated via interactions with HA indicating cell-specific feedback loop(s) in the signaling cascade.

Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

Monitoring Proteolytic Activity in Real Time: A New World of Opportunities for Biosensors.

Proteases play a pivotal role in several biological processes, from digestion, cell proliferation, and differentiation to fertility. Deregulation of protease metabolism can result in several pathological conditions (i.e., cancer, neurodegenerative disorders, and others). Therefore, monitoring proteolytic activity in real time could have a fundamental role in the early diagnosis of these diseases. Herein, the main approaches used to develop biosensors for monitoring proteolytic activity are reviewed. A comparison of the advantages and disadvantages of each approach is provided along with a discussion of their importance and promising opportunities for the early diagnosis of severe diseases. This new era of biosensors can be characterized by the ability to control and monitor biological processes, ultimately improving the potential of personalized medicine.

Fluorescent dye nano-assemblies by thiol attachment directed to the tips of gold nanorods for effective emission enhancement.

The conjugation of dye-labelled DNA oligonucleotides with gold nanorods has been widely explored for the development of multifunctional fluorescent nanoprobes. Here, we show that the functionalization route is crucial to achieve enhanced emission in dye nano-assemblies based on gold nanorods. By using a tip-selective approach for thiol attachment of dye molecules onto gold nanorods, it was possible to effectively increase the emission by more than 10-fold relatively to that of a free dye. On the other hand, a non-selective approach revealed that indiscriminate surface functionalization has a detrimental effect on the enhancement. Simulations of discrete dipole approximation gave further insight into the surface distribution of plasmon-enhanced emission by confirming that tip regions afford an effective enhancement, while side regions exhibit a negligible effect or even emission quenching. The contrast between dye nano-assemblies obtained from tip- and non-selective functionalization was further characterized by single-particle fluorescence emission. These studies showed that tip-functionalized gold nanorods with an average of only 30 dye molecules have a comparable to or even stronger emission than non-selectively functionalized particles with approximately 10 times more dye molecules. The results herein reported could significantly improve the performance of dye nano-assemblies for imaging or sensing applications.

Density Gradient Selection of Colloidal Silver Nanotriangles for Assembling Dye-Particle Plasmophores.

A simple method based on sucrose density gradient centrifugation is proposed here for the fractionation of colloidal silver nanotriangles. This method afforded particle fractions with surface plasmon resonances, spanning from red to infrared spectral ranges that could be used to tune optical properties for plasmonic applications. This feature was exemplified by selecting silver nanotriangle samples with spectral overlap with Atto-655 dye's absorption and emission in order to assemble dye-particle plasmophores. The emission brightness of an individual plasmophore, as characterized by fluorescence correlation spectroscopy, is at least 1000-fold more intense than that of a single Atto-655 dye label, which renders them as promising platforms for the development of fluorescence-based nanosensors.

Lanthanide complexes with phenanthroline-based ligands: insights into cell death mechanisms obtained by microscopy techniques.

Herein we report the synthesis, characterization, and photophysical and biological evaluation of the complexes Ln(DBM)3(RPhen) (Ln = Sm, R = H; Ln = Sm, Eu, Tb, R = 5-NH2) stabilized by three ß-diketonate units (DBM) and a phenanthroline (RPhen) derivative, with the aim of contributing to the development of lanthanide-based compounds with potential application as anticancer agents. The UV-vis spectra of [Sm(DBM)3(Phen)], [Sm(DBM)3(NH2Phen)], [Eu(DBM)3(NH2Phen)] and [Tb(DBM)3(NH2Phen)] measured in DMSO and PBS showed a strong absorption band centered at ca. 350 nm in both solvents. In DMSO, all lanthanide compounds except [Sm(DBM)3(Phen)] show a ligand centered emission band at ca. 520 nm. In PBS only sharp emission peaks are detected. The complexes show similar cytotoxic effects in A2780 ovarian cancer cells, presenting IC50 values at 24 h in the range 16-27 µM. The measurement of the cellular uptake of the complexes in the A2780 cells by inductively coupled plasma mass spectrometry (ICP-MS) revealed preferential accumulation at the membrane and cytoskeleton, with the exception of [Sm(DBM)3(Phen)] that presented higher accumulation in the cytosol than in the cell membranes. All the evaluated lanthanide complexes showed low nuclear uptake, although not negligible. Spectroscopic studies on the interaction of the complexes with calf thymus DNA (ctDNA) revealed a moderate affinity with apparent binding constants in the 104 M-1 range. Complexes bind DNA not by intercalation but probably by electrostatic interactions. A morphological evaluation of the cells treated with the different complexes by electron microscopy (TEM/SEM) proved that all of them induce mitochondrial alterations, which seemed more pronounced for the NH2Phen complexes. In addition, the complex [Eu(DBM)3(NH2Phen)] presented lysosomal uptake that might explain its augmented cytotoxicity.

Extreme Enhancement of Single-Molecule Fluorescence from Porphyrins Induced by Gold Nanodimer Antennas.

Porphyrins are typically weak emitters, which presents challenges to their optical detection by single-molecule fluorescence microscopy. In this contribution, we explore the enhancement effect of gold nanodimer antennas on the fluorescence of porphyrins in order to enable their single-molecule optical detection. Four meso-substituted free-base porphyrins were evaluated: two cationic, one neutral, and one anionic porphyrin. The gold nanodimer antennas are able to enhance the emission from these porphyrins by a factor of 105-106 increase in the maximum detected photon rates. This extreme enhancement is due to the combination of an antenna effect on the excitation rate that is estimated to be above 104-fold and an emission efficiency that corresponds to an increase of 2-10 times in the porphyrin's fluorescence quantum yield.

Tip-Specific Functionalization of Gold Nanorods for Plasmonic Biosensing: Effect of Linker Chain Length.

Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod's surface. The model sensors were further characterized by measuring their dose-response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response.

Evaluation of electrostatic binding of PAMAM dendrimers and charged phthalocyanines by fluorescence correlation spectroscopy.

We have assessed host-guest interactions between PAMAM dendrimers and charged phthalocyanine probes by Fluorescence Correlation Spectroscopy (FCS). Our results show strong binding in water at low ionic strength with an affinity that decreases from KB ∼ 10(9) to 10(8) M(-1) upon decreasing the phthalocyanine charge of z = -4, -2 and -1. The binding affinity also decreases significantly upon salt addition leading to KB values of ca. 10(5)-10(6) M(-1). The changes of binding affinity probed by varying the phthalocyanine charge, and by changing the ionic strength or pH conditions, allowed us to evaluate the electrostatic contribution (Kel) in dendrimer-phthalocyanine interactions. In particular, this approach afforded values of electrostatic potential for PAMAM dendrimers in water at low ionic strength and at dendrimer concentrations in the nanomolar range. The electrostatic potential of PAMAM generations 4 and 7 are around 50 mV in close agreement with theoretical estimates using the Poisson-Boltzmann cell model. Interestingly, the nonelectrostatic binding is significant and contributes even more than electrostatic binding to dendrimer-phthalocyanine interactions. The nonelectrostatic binding contributes to an affinity of KB above 10(5) M(-1), as measured under conditions of low dendrimer charge and high ionic strength, which makes these dendrimers promising hosts as drug carriers.

Resonant plasmonic enhancement of single-molecule fluorescence by individual gold nanorods.

Enhancing the fluorescence of a weak emitter is important to further extend the reach of single-molecule fluorescence imaging to many unexplored systems. Here we study fluorescence enhancement by isolated gold nanorods and explore the role of the surface plasmon resonance (SPR) on the observed enhancements. Gold nanorods can be cheaply synthesized in large volumes, yet we find similar fluorescence enhancements as literature reports on lithographically fabricated nanoparticle assemblies. The fluorescence of a weak emitter, crystal violet, can be enhanced more than 1000-fold by a single nanorod with its SPR at 629 nm excited at 633 nm. This strong enhancement results from both an excitation rate enhancement of ∼130 and an effective emission enhancement of ∼9. The fluorescence enhancement, however, decreases sharply when the SPR wavelength moves away from the excitation laser wavelength or when the SPR has only a partial overlap with the emission spectrum of the fluorophore. The reported measurements of fluorescence enhancement by 11 nanorods with varying SPR wavelengths are consistent with numerical simulations.

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

Deswelling and Electrolyte Dissipation in Free Diffusion of Charged PAMAM Dendrimers.

The diffusion coefficient of charged PAMAM dendrimers was measured by fluorescence correlation spectroscopy in aqueous solution at submicromolar concentrations. The solution pH was varied for conditions ranging from a fully charged to neutral charge dendrimer to infer about electrostatic swelling in the dilute regime. The diffusion coefficient of generation G4 increases by as much as 20% between high and low charge conditions due to the combined effects of polyelectrolyte deswelling and loss of electrolyte dissipation. By taking into account the electrolyte dissipation in the friction factor, we have found that the observed deswelling corresponds to a change of hydrodynamic radius between 7-13% for generation G4 and about 12% for generation G7. Simulations of molecular dynamics of dendrimer G4 show that counterion uptake by the dendrimer structure upon full protonation induces a 16% increase of its radius of gyration. The change in dendrimer size is slightly larger than that previously reported from neutron scattering techniques, thereby suggesting that electrostatic swelling is more pronounced at dilute dendrimer concentration and low ionic strength. It is confirmed that even higher generations, which have more congested molecular structures, can experience some degree of conformational change in response to a change of the dendrimer charge density.

Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod.

Existing methods for the optical detection of single molecules require the molecules to absorb light to produce fluorescence or direct absorption signals. This limits the range of species that can be detected, because most molecules are purely refractive. Metal nanoparticles or dielectric resonators can be used to detect non-absorbing molecules because local changes in the refractive index produce a resonance shift. However, current approaches only detect single molecules when the resonance shift is amplified by a highly polarizable label or by a localized precipitation reaction on the surface of a nanoparticle. Without such amplification, single-molecule events can only be identified in a statistical way. Here, we report the plasmonic detection of single molecules in real time without the need for labelling or amplification. Our sensor consists of a single gold nanorod coated with biotin receptors, and the binding of single proteins is detected by monitoring the plasmon resonance of the nanorod with a sensitive photothermal assay. The sensitivity of our device is ∼700 times higher than state-of-the-art plasmon sensors and is intrinsically limited by spectral diffusion of the surface plasmon resonance.

Reorganization of self-assembled dipeptide porphyrin J-aggregates in water-ethanol mixtures.

The self-assembly of a neutral meso-methoxyphenylporphyrin functionalized with a dipeptide glycilglycine substituent (MGG) in water and in water-ethanol mixtures was studied by absorption and fluorescence spectroscopy. In water, hydrophobic interactions and the noncovalent intermolecular hydrogen bonding between the terminal carboxylate group of one porphyrin and the hydrogen atoms of the pyrrolic nitrogens of another porphyrin originate nonspecific disorganized H- and J-aggregates. The addition of ethanol (0.1-25% v/v) to the water creates small clusters within which porphyrin J-aggregates reorganize as revealed by a narrow intense band detected by the Rayleigh light scattering (RLS) at 443 nm. Similar phenomenology is detected in SDS premicellar aggregates. Computational DFT calculations of a model dimer formation stabilized via intermolecular hydrogen bonding estimate an energy gain of -22 kJ mol(-1) and a center-to-center and interplane distances between porphyrin moieties of 16.8 and 3.7 Å, respectively. The kinetics of the J-aggregate formation could be fitted with a time-dependent model, and an activation energy of 96 kJ mol(-1) was estimated. The aggregate's morphology of MGG was followed by transmission electron microscopy (TEM) which showed rod-type structures of 5-8 µm evolving to spherical particles with increased ethanol content. Similar images and sizes were obtained in analogous samples using fluorescence lifetime imaging microscopy (FLIM) and dynamic light scattering (DLS). The formation of excitonically coupled supramolecular MGG structures of brickwork or staircase types is proposed in these water-ethanol mixtures.