Single-Molecule Orientation Imaging Reveals Two Distinct Binding Configurations on Amyloid Fibrils.

Fluorescence readouts for an amyloid fibril sensor critically depend on its molecular interaction and local environment offered by the available structural motifs. Here we employ polarized points accumulation for imaging in nanoscale topography with intramolecular charge transfer probes transiently bound to amyloid fibrils to investigate the organization of fibril nanostructures and probe binding configurations. Besides the in-plane (θ ≈ 90°) mode for binding on the fibril surface parallel to the long fibril axis, we also observed a sizable population of over 60% out-of-plane (θ < 60°) dipoles for rotor probes experiencing a varying degree of orientational mobility. Highly confined dipoles exhibiting an out-of-plane configuration probably reflect tightly bound dipoles in the inner channel grooves, while the weakly bound ones on amyloid enjoy rotational flexibility. Our observation of an out-of-plane binding mode emphasizes the pivotal role played by the electron donor amino group toward fluorescence detection and hence the emergence of anchored probes alongside conventional groove binders.

Inter-molecular interaction kinetics: tale of photon anti-bunching and bunching in fluorescence correlation spectroscopy (FCS).

Molecular interactions are fundamental to any chemical or biological processes, and their rates define the operational sequence and control for any desirable product. Here, we deliberate on a recently developed novel fluorescence spectroscopic method, which combines fluorescence photon anti-bunching, photon bunching, time-correlated single-photon counting (TCSPC), and steady-state fluorescence spectroscopy, to study composite chemical reactions with single molecule sensitivity. The proposed method captures the full picture of the multifaceted quenching kinetics, which involves static quenching by ground state complexation and collisional quenching in the excited state under dynamic exchange of fluorophore in a heterogeneous media, and which cannot be seen by steady-state or lifetime measurements alone. Photon correlation in fluorescence correlation spectroscopy (FCS) provides access to interrogate interaction dynamics from picosecond to seconds, stitching all possible stages of dye-quencher interaction in a micellar media. This is not possible with the limited time window available to conventional ensemble techniques like TCSPC, flash photolysis, transient absorption, stop-flow, etc. The basic premises of such unified global analysis and sanctity of extracted parameters critically depends on the minimum but precise description of reaction scheme, for which careful inspection of ensemble spectroscopy data for photo-physical behaviour is very important. Though in this contribution we discussed and demonstrated the merits of photon antibunching and bunching spectroscopy for dye-quencher interaction in cationic cetyltrimethylammonium bromide (CTAB) micellar solution by photo-induced electron transfer mechanism and the influence of micellar charge and microenvironment on the interaction kinetics, but in principal similar arguments are equally applicable to any other interaction mechanisms which alter fluorescence photon correlations, like Förster resonance energy transfer (FRET), proton transfer, isomerisation, etc.

Toward Understanding the Binding Synergy of Trastuzumab and Pertuzumab to Human Epidermal Growth Factor Receptor 2.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancer. Combination therapy using trastuzumab and pertuzumab antibodies targeting HER2 has shown better survival outcomes in breast cancer patients. In the quest to understand the synergistic effect observed due to combination therapy, trastuzumab, pertuzumab, and their F(ab')2 fragments were labeled with radioisotope and fluorescent probes. Detailed in vitro studies to understand binding synergism in HER2 overexpressing cell lines were done. Antibodies and their F(ab')2 fragments prepared by enzyme digestion with pepsin were radiolabeled with iodine-125. In vitro binding studies to evaluate immunoreactivity, specificity, affinity, and binding synergism between radiolabeled trastuzumab, pertuzumab, and their F(ab')2 fragments were carried out. Synergism was observed by 20-30% enhanced uptake of radiolabeled pertuzumab and its F(ab')2 fragments in the presence of excess of unlabeled trastuzumab or F(ab')2-trastuzumab. However, uptake of radiolabeled trastuzumab was not enhanced in the presence of excess pertuzumab or its fragments; rather inhibition or competition in binding to HER2 was observed. Studies using fluorescent antibodies by flow cytometry confirmed enhanced binding of pertuzumab in the presence of trastuzumab. Live cell tracking was done to give insights into the binding synergy and fate of fluorescent antibodies . Colocalization of antibodies on HER2 followed by internalization in the cells was observed. The radiolabeled immunoconjugates served as an important tool for experimental characterization of interaction between pertuzumab and trastuzumab to HER2. Studies with fluorescent antibodies corroborated the binding data and provided evidence of colocalization and internalization of both the antibodies in HER2-positive cells.

Picosecond to Second Fluorescence Correlation Spectroscopy for Studying Solute Exchange and Quenching Dynamics in Micellar Media.

Numerous studies have been devoted to understand the reaction kinetics in micelles, where the accessible kinetic time window is often limited by the dynamic range of the employed spectroscopic technique. This is usually accompanied by a selection of probes that comfortably explore time scales where slow solute exchange kinetics is negligible, as compared to the fast excited state reactions. This has led to an undervaluation of the role played by dynamic partitioning of hydrophilic solutes in microheterogeneous media. Here, we employ fluorescence correlation spectroscopy (FCS) and the zwitterionic dye Rhodamine 110 to quantitatively explore the impact of solute exchange on the photoinduced electron transfer between this dye and N,N-dimethylaniline in micellar media. Our study elucidates the coupling and interplay between the kinetics of photophysics, quenching, and solute exchange through a quantitative unified molecular-state quenching-kinetic model that describes the steady-state ensemble and FCS data from subnanosecond photon antibunching to millisecond diffusions.

Binding Constant Determined from the Angstrom-Scale Change in Hydrodynamic Radius of Transferrin upon Binding with Europium Using Dual-Focus Fluorescence Correlation Spectroscopy.

Monitoring the binding of a large fluorescently tagged molecule to a small solute by fluorescence correlation spectroscopy (FCS) is rather uncommon because the binding-related change in diffusion coefficient is very small. Here, we use a high-precision variant of FCS, namely, dual-focus FCS (2fFCS), for measuring the angstrom-scale change of the hydrodynamic radius of the bilobal metal transport protein transferrin (Tf) upon binding europium ions. Applying a sequential 1:2 complexation model, we use these measurements for determining the binding constants (K). Our results show a 0.7 Å change of the protein's hydrodynamic radius upon 1:1 Tf-Eu complex formation and a second change of 1.8 Å upon subsequent binding of a second europium ion. More than one unit variation in logK indicates an intrinsic dissimilarity in metal affinity of the C- and N-lobes of Tf, which agrees well with earlier reported ensemble spectroscopy results.

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M-1 , with dissociation constants faster or equal to 103  s-1 , which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104  M-1 . In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241 Am3+ , where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.

Photon Antibunching Reveals Static and Dynamic Quenching Interaction of Tryptophan with Atto-655.

Fluorescence correlation spectroscopy (FCS) of photoinduced electron transfer (PET) between the dye Atto-655 and the amino acid tryptophan has been extensively used for studying fast conformational dynamics of small disordered peptides and proteins. However, a precise understanding of the quenching mechanism and its exact rates that would explain ensemble as well as single-molecule spectroscopy results is still lacking. In this contribution, a general unified model for intermolecular PET between Atto-655 and tryptophan is developed, which involves ground-state complex formation, quenching sphere of action, and dynamic quenching at the single-molecule level. We present measurements of fluorescence antibunching, fluorescence lifetime, and steady-state fluorescence intensity and absorbance and demonstrate that our model is capable to describe all results in a global and coherent manner.

Molecular Origin and Self-Assembly of Fluorescent Carbon Nanodots in Polar Solvents.

Despite numerous efforts, there are several fundamental ambiguities regarding the photoluminescence of carbon dots (CDs). Spectral shift measurements display characteristic of both π-π* and n-π* transitions for the main absorption or excitation band at ∼350 nm, contrary to common assignment of exclusive n-π* transition. Additionally, the generally perceived core-state transition at ∼250 nm, involving sp2-networked carbogenic domains shielded from external environments, needs to be reassessed because it fails to explain the observed fluorescence quenching and spectral shift. These results have been explained based on the molecular origin of PL in CDs invoking the similarity between CD and citrazinic acid. Fluorescent derivatives of the latter are recognized to be produced during citric-acid-based CD synthesis. Concentration-dependent spectral splitting of the main excitation band in combination with the temperature-dependent PL results has been envisioned assuming self-assembly of CDs into various H-aggregates.

Origin of Excitation Dependent Fluorescence in Carbon Nanodots.

The fascinating aspect of excitation dependent fluorescence in carbon nanodots has led to several hypotheses, starting from particle size distribution to the presence of different emissive states and even to sluggish solvent relaxation around nanodot. In this contribution we provide definitive evidence for the involvement of discrete multiple electronic states for the excitation dependent emission in carbon nanodots. The presence of different types of aggregates even at very dilute solutions used in ensemble fluorescence spectroscopy, where fluorescence intensity shows linear dependence with absorbance, is the origin of these multiple electronic states. Inhomogeneous broadening due to slow solvent relaxation leading to excitation dependent spectral shift has negligible influence in conventional solvents.

Photon Antibunching in Complex Intermolecular Fluorescence Quenching Kinetics.

We present a novel fluorescence spectroscopic method, which combines fluorescence antibunching, time-correlated single-photon counting (TCSPC), and steady-state emission spectroscopy, to study chemical reactions at the single molecule level. We exemplify our method on investigating intermolecular fluorescence quenching of Rhodamine110 by aniline. We demonstrate that the combination of measurements of fluorescence antibunching, fluorescence lifetime, and fluorescence steady state intensity, captures the full picture of the complex quenching kinetics, which involves static and dynamics quenching, and which cannot be seen by steady-state or lifetime measurements alone.

Observation of the Marcus inverted region for bimolecular photoinduced electron-transfer reactions in viscous media.

The general observation of Marcus inverted region (MIR) for bimolecular electron-transfer (ET) reactions in different viscous media, e.g., micelles, reverse micelles, vesicles, ionic liquids, DNA scaffold, etc. has been doubted in some recent publications arguing limitations in Stern-Volmer (SV) analysis to account for the static and transient stages of quenching in these slow diffusing media. Thus, following a theoretical treatment based on a spherically symmetric diffusion equation coupled with conventional Marcus ET description, it has been suggested that the MIR observed in viscous media arises due to the inadequate consideration of different quenching regimes and also due to the differential excited-state lifetimes of the fluorophores used than a genuine one (J. Am. Chem. Soc. 2012, 134, 11396). However, the overall treatment in this study is severely compromised by setting the minimum solvent reorganization energy (λs) to ∼0.96 eV while fitting the experimental data, which unambiguously suggests that the inversion in ET rate will never appear in the exergonicity (-ΔG(0)) range of 0.16 to 0.71 eV, as is the case for the studied ET systems. Besides, the applicability of the conventional Marcus ET model (instead of Sumi-Marcus two-dimensional ET model) in such extremely viscous media with exceptionally slow solvent response is highly debatable and perhaps is the main cause of the failure in fitting the experimental data quite satisfactorily. In the present study involving ultrafast ET quenching for coumarin derivatives by dimethylaniline donor in viscous ionic liquid media, we demonstrate clear MIR for the intrinsic ET rates, directly obtained from the ultrafast decay components of 1-10 ps, a time scale in which diffusion of reactants is negligible and the ET rates are either faster than or, at the most, competitive with the solvent reorganization. The appearance of MIR at ΔG(0) ∼ -0.5 eV, significantly lower than expected from the λs value, further substantiate the nonapplicability of conventional ET description but certainly advocate for the applicability of the Sumi-Marcus two-dimensional ET model in such media. Moreover, no obvious correlation has experimentally been observed between the excited-state lifetimes of the coumarin derivatives and the ET rates for a large number of dyes used in the present study. On the basis of the present results and drawing inferences from reported literatures in viscous media, we conclude that not only is the appearance of MIR very genuine but also the mechanistic model necessary to account the observed facts for the bimolecular ET reactions in a viscous medium is the two-dimensional ET description, which deals with an extremely slow relaxing solvent coordinate and a fast relaxing intramolecular coordinate to describe the ET reactions.

A nano-confined charged layer defies the principle of electrostatic interaction.

The reactivity between two charged molecules and the activity of charged biomolecules are mainly governed by the principle of electrostatic interaction, i.e., like charges repel and opposite charges attract. In the present study it is shown that the principle of electrostatic interaction is violated in the nano-confined biomimetic environment. Thus a positively charged molecule shows more preference to a positively charged surface compared to a negatively charged surface.

Confined ultrafast torsional dynamics of Thioflavin-T in a nanocavity.

The influence of confinement in the supramolecular ß-cyclodextrin nanocavity on the excited state torsional dynamics of the amyloid fibril sensor, Thioflavin-T, is explored using subpicosecond fluorescence up-conversion spectroscopy. In the presence of ß-cyclodextrin, the emission intensity and the fluorescence lifetime of Thioflavin-T significantly increases, indicating the confinement effect of the nanocage on the photophysical behaviour of the dye. Detailed time-resolved fluorescence studies show an appreciable dynamic Stokes' shift for the dye in the ß-cyclodextrin nanocavity. Analysis of the time-resolved area normalized emission spectra (TRANES) indicates the formation of an emissive TICT state. The rate of formation of the TICT state, as calculated from the time dependent changes in the peak frequency and the width of the emission spectra, is found to be substantially slower in the ß-cyclodextrin nanocavity compared to that in bulk water. Present results indicate that ultrafast torsional motion in Thioflavin-T is significantly retarded due to confinement by the ß-cyclodextrin nanocavity.

Tuning of intermolecular electron transfer reaction by modulating the microenvironment inside copolymer-surfactant supramolecular assemblies.

Photoinduced intermolecular electron transfer (ET) dynamics between various 7-aminocoumarin acceptors and N,N-dimethylaniline (DMAN) donor has been studied in copolymer-surfactant supramolecular assemblies prepared in aqueous 1% P123 triblock copolymer micellar solution with varying concentration of surfactants (sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium chloride (CTAC), and triton-X-100 (TX100)). The aim of the present study is to modulate the reaction environment, especially the degree of micellar hydration inside the P123 micelle by the addition of the surfactants, which can modulate the ET reaction through the changes in the ET rates and the reaction exergonicity. Within the limited surfactant to copolymer molar ratios (n) used in the present study, fluorescence spectroscopy, dynamic light scattering (DLS), and small-angle neutron scattering (SANS) investigations indicate that the copolymer-surfactant supramolecular assemblies retain their micellar structure, although the micellar size gradually decreases with n. The redox potentials of the electron donor and acceptors are also found to change with n, although the extent of the effect is different for SDS, CTAC, and TX100 cosurfactants. In the presence of CTAC, the estimated exergonicity (-ΔG(0)) of the ET reaction is found to increase with an increase in n compared with that in pure P123, whereas it decreases marginally with SDS and remains almost the same for TX100. Substantial quenching of coumarin fluorescence is observed in the presence of DMAN in all copolymer-surfactant micellar aggregates because of ET reaction. The ET rate is seen to increase gradually with an increase in SDS and CTAC concentration in the supramolecular assembly, although it remains unaffected on the addition of TX100. The increased ionic strength in the Corona region of the copolymer-surfactant supramolecular aggregates due to the addition of the ionic surfactants has been envisaged for the increase in the ET rates. A correlation of the quenching rate constants with the free-energy changes (ΔG(0)) of the ET reactions shows the typical bell-shaped curve as predicted by Marcus outersphere ET theory. A substantial shift along the exergonicity axis (~0.3 eV) for the appearance of the Marcus correlation is observed in some cases, although the extent of such shift depends on both the nature of the cosurfactant and the amount of cosurfactant used in the copolymer-surfactant supramolecular assembly. Therefore, these preliminary results suggest a possibility of not only modulating the ET rates but also tuning the appearance of Marcus inversion along the exergonicity scale by suitably tuning the reaction environment inside the copolymer-surfactant supramolecular assemblies with a relatively more hydrophilic cosurfactant.

Ultrafast electron transfer dynamics in micellar media using surfactant as the intrinsic electron acceptor.

Ultrafast photoinduced intermolecular electron transfer (ET) dynamics involving 7-aminocoumarin derivatives as electron donor and pyridinium moiety of surfactant molecules in cetylpyridinium chloride (CPC) micelle as electron acceptor has been investigated to understand the role of separation and orientation of reactants on micellar ET reactions. Unlike in noninteracting micelles (like Triton-X-100, sodium dodecyl sulfate, dodecyltrimethylammonium bromide, etc.), where surfactant-separated donor-acceptor pairs are understood to give the ultrafast ET component with the shortest time constant in the range of approximately 4 ps, in CPC micelles with pyridinium moiety as the intrinsic acceptor the ultrafast ET component is found to be in the subpicosecond time scale (of around 240 fs). This time scale is very similar to the values reported in the cases of ultrafast ET reactions involving coumarin dyes in electron-donating solvents. The ultrafast ET times in CPC micelles are significantly faster than the diffusive solvation dynamics in the micellar media. Correlation of the observed ET rates in the present cases with the free-energy changes of the reactions shows the inverse-bell-shaped correlation, predicted by Marcus ET theory. Interestingly, the onset of the Marcus inversion appears at a relatively lower exergonicity, which is attributed to the nonequilibrium solvent configuration during the ultrafast ET reaction, as envisaged from two-dimensional ET (2DET) model. Along with the ultrafast ET component, there are also slower ET components in these systems, which are attributed to those close-contact donor-acceptor populations in the micelles that have relatively weaker electronic coupling due to improper orientation of the interacting donor-acceptor pairs. The present results suggest that, along with the shifting of Marcus inversion at lower exergonicity, the ET rates can also be maximized in a micellar media by using surfactant molecule as an intrinsic reactant.

Identifying the bond responsible for the fluorescence modulation in an amyloid fibril sensor.

An ultrafast intramolecular bond twisting process is known to be the responsible mechanism for the sensing activity of the extensively used amyloid fibril sensor thioflavin T (ThT). However, it is not yet known which one of the two possible single bonds in ThT is actually involved in the twisting process. To resolve this fundamental issue, two derivatives of ThT have been designed and synthesized and subsequently their photophysical properties have been studied in different solvents. It is understood from the present study that the rotation around the central C-C single bond, and not that around the C-N single bond, is primarily responsible for the sensor activity of ThT. Detailed viscosity-dependent fluorescence studies revealed that the ThT derivative with restricted C-N bond rotation acts as a better sensor than the derivative with free C-N bond rotation. The better sensory activity is directly correlated with a shorter excited-state lifetime. Results obtained from the photophysical studies of the ThT derivatives have also been supported by the results obtained from quantum chemical calculations.

Viscosity effect on the ultrafast bond twisting dynamics in an amyloid fibril sensor: thioflavin-T.

The femtosecond fluorescence upconversion technique is used to study the effect of viscosity on the excited state relaxation dynamics of an amyloid fibril sensor, thioflavin-T, in different solvent media. The excited state decay in all of the solvents is seen to be dependent on the emission wavelength. From the constructed time-resolved emission spectra, it is seen that the present system shows dynamic Stokes' shift as well as an appreciable increase in the spectral width with time. These temporal spectral characteristics of time-resolved emission spectra have been assigned to the formation of a new emissive species from the locally excited state of the thioflavin-T molecule. The formation of the new emissive state from the locally excited state is also supported by the fact that an iso-emissive point appears in the time-resolved area normalized emission spectra. From the detailed study on the excited state dynamics of thioflavin-T as a function of solvent viscosity, it is concluded that the new emissive state is formed due to the twisting around the central C-C single bond in the excited state of thioflavin-T. The formation rate of the twisted emissive state from the locally excited state is found to be nicely correlated with the viscosity of the medium.

Time-resolved fluorescence and small angle neutron scattering study in pluronics-surfactant supramolecular assemblies.

Interaction of cationic surfactant, cetyl trimethyl ammonium bromide (CTAB), with pluronics F88 (EO(103)-PO(39)-EO(103)) and P105 (EO(37)-PO(56)-EO(37)) micelles and its effect on modulating the location of an anionic solute in the mixed micelles have been investigated using time-resolved fluorescence and small angle neutron scattering (SANS) studies. SANS results indicate the formation of pluronic-CTAB supramolecular assemblies, in which the hydrophobic chains of CTAB occupy the hydrophobic core of the pluronic micelle while the positively charged head groups reside at the micellar core-corona interface. Rotational correlation time of the anionic probe in these supramolecular assemblies increases with an increase in the CTAB concentration, and the observed results are explained on the basis of the probe movement from the surface to the interior of the micelle due to the increased electrostatic attraction. Dynamic Stokes' shift measurements also support the movement of the probe due to the addition of the surfactant to the supramolecular assemblies. From the studies with different pluronics, it is indicated that the concentration of CTAB required to drag the probe molecule into the interior of the micelles is linearly correlated to the thickness of the corona region of the respective micelles.