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.

Core-Assisted Formation of Porphyrin J-Aggregates in pH-Sensitive Polyelectrolyte Microcapsules Followed by Fluorescence Lifetime Imaging Microscopy.

A strategy assisted by an inorganic template was developed to promote the organized self-assembly of meso-(tetrakis)-(p-sulfonatophenyl)porphyrin (TPPS) on pH-sensitive core-shell polyelectrolyte microcapsules (PECs) of poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). A key feature of this strategy is the use of template CaCO3 microparticles as a nucleation site endorsing inside-outside directional growth of porphyrin aggregates. Using this approach, TPPS self-assembly in positively charged PECs with CaCO3 (PAH/PSS)2PAH as a sequence of layers was successfully achieved using mild pH conditions (pH 3). Evidence for porphyrin aggregation was obtained by UV-vis with the characteristic absorption bands in PECs functionalized with porphyrins. Fluorescence lifetime imaging microscopy (FLIM) of the polyelectrolyte core-shell confirmed the presence of radially distributed needlelike structures sticking out from polyelectrolyte shells. Microscopic images also revealed a sequential process (adsorption, redistribution, and aggregation) for the directional growth (inside/outside) of TPPS aggregates, which highlights the importance of the core in the aggregation induction. Removing the CaCO3 core alters the porphyrin interaction in the PEC environment, and aggregate growth is no longer favored.

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.

Polyelectrolyte-assisted noncovalent functionalization of carbon nanotubes with ordered self-assemblies of a water-soluble porphyrin.

The self-assembly and induced supramolecular chirality of meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) on both single-wall (SWCNT) and multiwall carbon nanotubes (MWCNT) are investigated. Under mild pH conditions (pH 3), TSPP forms aggregates when CNTs are dispersed in an aqueous solution containing positively charged polyelectrolytes such as poly-L-lysine (PLL) or poly(allylamine hydrochloride) (PAH). Evidence for the geometry of the porphyrin aggregates is obtained from absorption spectra, whereby the fingerprints of J- and H-aggregates are clearly seen only in the presence of smaller-diameter nanotubes. J-aggregates are better stabilized with PLL, whereas in the presence of PAH mainly H-aggregates prevail. Excited-state interactions within these nanohybrids are studied by steady-state and time-resolved fluorescence. The porphyrin emission intensity in the nanohybrid solution is significantly quenched compared to that of TSPP alone, and this implies strong electronic interaction between CNTs and porphyrin molecules. Fluorescence lifetime imaging microscopy (FLIM) further supports that porphyrin arrays are associated with the MWCNT sidewalls wrapped in PLL. In the case of the SWCNT hybrid, spherical structures associated with longer fluorescence lifetime appeared after one week, indicative of H-aggregates of TSPP. The latter are the result of π-π stacking of porphyrin units on neighboring nanotubes facilitated by the strong tendency of these nanotubes to interact with each other. These results highlight the importance of optimum dimensions and surface-area architectures of CNTs in the control/stability of the porphyrin aggregates with promising properties for light harvesting.

Electrophilic reactivity of tetrabromorhodamine 123 is bromine induced: convergent interpretation through complementary molecular descriptors.

Nucleophilic addition of water and of methanol to 3,6-diamino-2,4,5,7-tetrabromo-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, 4BrR123, yields respectively 2-(3,6-diamino-2,4,5,7-tetrabromo-9-hydroxy-9H-xanthen-9-yl)xanthyl benzoate, HO4BrR123 and 2-(3,6-diamino-2,4,5,7-tetrabromo-9-methoxy-9H-xanthen-9-yl)xanthyl benzoate, MeO4BrR123. The novel experimental results are addressed theoretically. The linear free energy relationship, LFER, second-order perturbation theory analysis of the natural bond orbital, NBO, and quantum theory of atoms in molecules, QTAIM, lead to the same conclusion: the electron-withdrawing effect of bonded Br atoms in 4BrR123 extremely enhances the molecular electrophilicity, as compared to 3,6-diamino-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, R123. The reactivity of these diaminoxanthylium cations is discussed in the context of local and global softness in extended conjugated systems.

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.

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.

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.

Ordered self-assembly of protonated porphyrin induced by the aqueous environment of biomimetic systems.

Water-soluble ionic porphyrins show an interesting self-association pattern in acidic conditions under which highly ordered aggregates are stabilized. Herein we give an overview of the template effect of biological interfaces in tetrakis(4-sulfonatophenyl)porphyrin TSPP self-aggregation, in which we point out the possibility of tuning this process by screening the charge repulsion through ionic strength and pH. We give special emphasis to the use of fluorescence lifetime imaging microscopy and of fluorescence correlation spectroscopy to visualize and characterize these molecular aggregates.

Interaction of zinc tetrasulfonated phthalocyanine with cytochrome C in water and Triton-X 100 micelles.

The interaction of a zinc tetrasulfonated phthalocyanine with cytochrome c was studied using steady-state spectroscopic techniques and time-correlated single photon counting in water and Triton-X 100 micelles. The dye forms dimers in water with a high equilibrium constant (70 x 10(6) M(-1)). Because of a specific electrostatic interaction, the presence of cytochrome c does not lead to a dissociation of this dimer, but increases its formation, with an equilibrium constant of about 7.9 x 10(9) M(-1). Triton-X 100 micelles dissociate the dimer, creating two populations of dye molecules: one in a hydrophilic media, probably on the surface of the micelles, another on a hydrophobic environment, probably inside the micelles. However, when cytochrome c is added the dye aggregation is again induced leading to a strong fluorescence quenching. This fluorescence quenching may also be caused by a photoinduced electron-transfer due to the formation of a 1:1 complex between the dye and the protein, but the present work does not give direct evidence of such an effect because the fluorescence decays did not show the presence of an extra component. The results presented here are quite different from those reported for aluminum sulfonated phthalocyanines, where aggregation does not occur and the fluorescence quenching is solely due to photoinduced electron-transfer reactions.

Molecular dynamics simulations of porphyrin-dendrimer systems: toward modeling electron transfer in solution.

We have performed computational simulations of porphyrin-dendrimer systems--a cationic porphyrin electrostatically associated to a negatively charged dendrimer--using the method of classical molecular dynamics (MD) with an atomistic force field. Previous experimental studies have shown a strong quenching effect of the porphyrin fluorescence that was assigned to electron transfer (ET) from the dendrimer's tertiary amines (Paulo, P. M. R.; Costa, S. M. B. J. Phys. Chem. B 2005, 109, 13928). In the present contribution, we evaluate computationally the role of the porphyrin-dendrimer conformation in the development of a statistical distribution of ET rates through its dependence on the donor-acceptor distance. We started from simulations without explicit solvent to obtain trajectories of the donor-acceptor distance and the respective time-averaged distributions for two dendrimer sizes and different initial configurations of the porphyrin-dendrimer pair. By introducing explicit solvent (water) in our simulations, we were able to estimate the reorganization energy of the medium for the systems with the dendrimer of smaller size. The values obtained are in the range 0.6-1.5 eV and show a linear dependence with the inverse of the donor-acceptor distance, which can be explained by a two-phase dielectric continuum model taking into account the medium heterogeneity provided by the dendrimer organic core. Dielectric relaxation accompanying ET was evaluated from the simulations with explicit solvent showing fast decay times of some tens of femtoseconds and slow decay times in the range of hundreds of femtoseconds to a few picoseconds. The variations of the slow relaxation times reflect the heterogeneity of the dendrimer donor sites which add to the complexity of ET kinetics as inferred from the experimental fluorescence decays.

Self-aggregation of free base porphyrins in aqueous solution and in DMPC vesicles.

Free base porphyrin (PPhe), derivatized with aminosulfonyl groups linked to the aromatic amino acid phenylalanine at the meso-positions, was mixed with DMPC vesicles. The resulting interaction was studied by absorption, steady-state and transient state fluorescence, at different pHs. At pH=2 to pH=9, the aforementioned porphyrin predominates as an aggregated species, with a co-facial arrangement of the molecules taking into account the blue shift of the Soret band (414 nm for the monomer and 401 nm for the aggregate). Upon interaction with DMPC vesicles, the competing hydrophobic interactions with the bilayer destabilize the aggregated species in favor of monomer incorporation. Fluorescence lifetimes also show that the long component assigned to the monomer contributes only 30% to the overall decay in solution (e.g. pH=7.0) whereas in DMPC vesicles this contribution increases up to 85% independent of the solution pH, which confirms a location of the probe in an environment "protected" from free water. The picture changes completely in the case of TSPP, an anionic porphyrin which does not incorporate in DMPC vesicles. Remarkably, at pH=2.5 all the experimental findings point to the self-assembling of the porphyrin units in J-aggregates induced at the surface of the DMPC vesicle. In fact, upon removal of the aqueous solvent, we could define by fluorescence lifetime imaging microscopy (FLIM) regions where the fluorescence lifetime is that characteristic of the J-aggregate ( 0.11 ns).

Molecular dynamics simulations of charged dendrimers: low-to-intermediate half-generation PAMAMs.

We have performed atomistic molecular dynamics simulations of PAMAM dendrimers of generations 0.5, 1.5, 2.5, 3.5, and 4.5. The simulated systems comprise the charged dendrimer and its counterions embedded in a dielectric continuum (i.e., without explicit solvent). Structural properties of these dendrimers, like the radius of gyration, the principal moments of inertia, and the segment density profiles, were evaluated from the simulations. The average radius of gyration obtained for the intermediate half-generations 2.5, 3.5, and 4.5 follows the same scaling law that was previously inferred from simulations of full-generation PAMAMs, Rg approximately M1/3, and is characteristic of space-filling objects. The low half-generations 0.5 and 1.5 deviate, however, to greater Rg values. The shape of the smaller dendrimers is approximately that of a prolate ellipsoid, which becomes more spherical for higher generations. The segment density profiles show features identical to those obtained in other simulations of flexible-chain dendrimers, like dendron-backfolding. Two slightly different configurations, in terms of size and shape, were identified for generation 2.5. The radial distributions of counterions extracted from the simulations compare well with the solutions of Poisson-Boltzmann cell model, and the dendrimer's effective charge was estimated using the Bjerrum criterion. The influence of electrostatic interactions in the dendrimer's conformation due to repulsion between the charged end-groups and its relation to counterion effects is discussed for the several generations simulated. The form factors calculated from the simulations are compared with the model of a homogeneous ellipsoid of revolution. The overall results are in agreement with the previously established morphological transition of PAMAM dendrimers toward a more spherical and compact conformation above generations 3 or 4.

Lipophilic porphyrin microparticles induced by AOT reverse micelles: a fluorescence lifetime imaging study.

Fluorescence Lifetime Imaging Microscopy (FLIM) technique was applied to investigate the fluorescence dynamics and structural features of large colloidal aggregates of meso-tetra(N-dodecyl-4-amino sulfonyl-phenyl)porphyrin (PC12) induced by Sodium 1,4-bis(2-ethyl hexyl)sulfosuccinate (AOT) reverse micelles. The aggregate's particle sizes (down to 1 microm) obtained from the confocal fluorescence images matched with the particle sizes measured in the images obtained from Scanning Electron Microscopy (SEM). The fluorescence decays for those aggregates in the micro spatial domain show triexponential fluorescence lifetimes (tau1 approximately 12 ns, tau2 approximately 3 ns and tau3 approximately 1 ns) which are independent of the aggregate's size.

Electron-transfer mechanism of the triplet state quenching of aluminium tetrasulfonated phthalocyanine by cytochrome c.

The mechanism of electron-transfer from aluminium tetrasulfonated phthalocyanine triplet state to cytochrome c was investigated in this work. This reaction successfully quenches the dye triplet state due to the formation of complexes between the solute and the protein at the active site. The electron-transfer rate constant is around 3x10(7) s(-1), and is in accordance with previous results for the singlet excited state quenching [C.A.T. Laia, S.M.B. Costa, D. Phillips, A. Beeby. Electron-transfer kinetics in sulfonated aluminum phthalocyanines/cytochrome c complexes, J. Phys. Chem. B 108 (2004) 7506-7514.] in the framework of the Marcus theory, with a reorganization energy equal to 0.94 eV. The complex formation is diffusion controlled, but heterogeneities of the protein surface charge distribution lead to quenching rate constants smaller than predicted on a hard-spheres model with electrostatic interactions. Also the binding equilibrium constant is strongly affected by this phenomenon. Ionic strength plays an important role on the complex formation, but its effect on the unimolecular electron-transfer rate constant is negligible within experimental error.

Design of polyelectrolyte core-shells with DNA to control TMPyP binding.

The interaction of DNA with 5,10,15,20-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) in polyelectrolyte core-shells obtained via layer by layer adsorption of poly(sodium 4-styrenesulfonate), PSS, and poly(allylamine hydrochloride), PAH, polyelectrolytes was followed by steady state, time resolved fluorescence and by Fluorescence Lifetime Imaging Microscopy (FLIM). Our results show that DNA adsorption onto polyelectrolyte core-shell changes the TMPyP interaction within PSS/PAH core-shells structure and increase significantly the TMPyP uptake. Specific DNA/TMPyP interactions are also altered by DNA adsorption favouring porphyrin intercalation onto GC pair rich regions. Circular dichroism (CD) spectra reveal that DNA undergoes important conformational changes upon adsorption onto the core-shell surface, which are reverted upon TMPyP encapsulation.

Interactions in noncovalent PAMAM/TMPyP systems studied by fluorescence spectroscopy.

Steady-state absorption and emission spectroscopy and time-resolved fluorescence measurements were employed in the study of meso-tetrakis(4-N-methylpyridinium)porphine (TMPyP) interactions with half-generation carboxyl-terminated poly(amidoamine) (PAMAM) dendrimers in water. TMPyP experiences a less polar environment and a strong fluorescence quenching effect upon dendrimer association. The tertiary amine functional groups in PAMAM dendrimers are likely to be responsible for the fluorescence quenching of TMPyP through an electron-transfer mechanism. The Stern-Volmer plots achieve a plateau at high dendrimer concentrations that was attributed to full porphyrin-dendrimer association, and an average fluorescence quantum yield of 15-20% relative to aqueous TMPyP was estimated. The association constant for the 1:1 complex with generation 2.5 at dendrimer-porphyrin ratio D/P = 1 is 5.75 x 10(7) M(-1), indicating a strong binding affinity. The dissociation of the complex with increasing ionic strength reinforces the role of electrostatic forces in porphyrin-dendrimer association. Comparison of Stern-Volmer plots obtained from quantum yields or lifetimes showed the importance of a static effect in these systems. The fluorescence decays of the porphyrin-dendrimer complex were fitted with a dispersed kinetics model. At intermediate dendrimer-porphyrin ratios (D/P approximately 1), diffusional quenching processes between free porphyrin and dendrimer were modeled with the Sano-Tachiya pair survival probability equation. Transient diffusional effects were dismissed as a possible explanation for the static effect detected.

Complexation of polymethine dyes with human serum albumin: a spectroscopic study.

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.

Energy transfer and fluorescence quenching in complexes of polymethine dyes with human serum albumin.

Electronic excitation energy transfer (EET) between molecules of polymethine dyes bound to human serum albumin (HSA) has been established and studied by absorption and fluorescence spectroscopy as well as by fluorescence decay measurements. In this system, excitation of the donor dye molecule leads to fluorescence of the acceptor dye molecule, both bound to HSA, with donor fluorescence quenching by the acceptor. The short distance between the donor and the acceptor (25-28 A) revealed from the Forster model of EET as well as some spectroscopic data show that both molecules are probably located in the same binding domain of HSA. The role of HSA is to bring donor and acceptor molecules together to a distance adequate to achieve EET as well as to increase the donor and acceptor fluorescence quantum yields. Efficient quenching of the intrinsic HSA fluorescence by some polymethine dyes (oxonols) is observed. The experimental results fit well a model for the formation of a weakly fluorescent dye-HSA complex; the quencher in this complex should be located in the immediate vicinity of the HSA fluorophore group (Trp(214)).