Solvatochromic Dyes Research Articles

Emerging solvatochromic push-pull dyes for monitoring the lipid order of biomembranes in live cells.

Solvatochromic dyes have emerged as a new class of fluorescent probes in the field of lipid membranes due to their ability to identify the lipid organization of biomembranes in live cells by changing the colour of their fluorescence. This type of solvatochromic function is useful for studying the heterogeneous features of biomembranes caused by the uneven distribution of lipids and cholesterols in live cells and related cellular processes. Therefore, a variety of advanced solvatochromic dyes have been rapidly developed over the last decade. To provide an overview of the works recently developed solvatochromic dyes have enabled, we herein present some solvatochromic dyes, with a particular focus on those based on pyrene and Nile red. As these dyes exhibit preferable photophysical properties in terms of fluorescence microscopy applications and unique distribution/localization in cellular compartments, some have already found applications in cell biological and biophysical studies. The goal of this review is to provide information to researchers who have never used solvatochromic dyes or who have not discovered applications of such dyes in biological studies.

Investigating homeopathic potencies with membrane bound solvatochromic dyes

Background Solvatochromic dyes have demonstrated themselves to be useful probes in the molecular-level study of homeopathic potencies. The range of dyes available, and their broad division into two main electronic structural groups, is permitting a detailed investigation of the fundamental nature of potencies. The immobilisation of solvatochromic dyes on transparent membranes takes these investigations to a new level of investigative precision. Aims To attach as many different solvatochromic dyes as possible to triacetylcellulose membranes, and where successful, to observe in detail the spectroscopic changes that occur on exposure to potencies. Immobilisation of dyes onto membranes de-couples the initial dye-potency interaction from later aggregation processes, thereby allowing a host of questions to be asked about what exactly potencies are doing to solvatochromic dyes and in turn indicating what the identities of potencies are likely to be. Methodology A number of methods have been explored for covalently linking dyes to membranes, which at the same time maintain the integrity of the solvatochromic chromophore. Immobilised dyes, in contrast to dyes in solution, behave differently, which means water-insoluble dyes for instance, can be used under a range of solution conditions that were not possible previously. Results and discussion Results from three positively solvatochromic dyes show that potencies cause a bathochromic shift in their spectra, confirming what has been seen previously with encapsulated dyes but with greater clarity. Results can be explained by invoking a polarising effect of potencies and differential stabilisation of the dyes’ ground and excited electronic states. The kinetics of the dye-potency interaction indicate the effect of potency reaches a maximum and then declines. Conclusion Immobilisation of solvatochromic dyes presents a significant step forward in the ability to investigate homeopathic potencies at the physico-chemical level.

Proceedings of the XXXIII GIRI meeting

It’s being quite a long journey until here. Although it seens we are not so far from the top, there is so much still to be done. Be invited by Drs. Leoni Bonamin and Francisco Eizayaga to be part of IJHDR editorial board team is a honor and a duty and i am happy to collaborate in this task, mainly in this issue, in which the Proceedings of the XXXIII GIRI meeting are presented.
 
 The search for the understanding of how high dilutions can restart steady patterns in biological systems have never been so clear and direct face the high-quality level results described on the XXXIII GIRI meeting on last June, helded in London.
 
 Through physicochemical and biological models, this new issue highlights basic research on isopathics, immunostimulatory effects and also physical studies of homeopathic potencies, using solvatochromic dyes, micro-evaporation and other methods as tools. Theoretical approaches were also considered, such as the search for the vital force evidences, as well as clinical studies concerning the Zika virus and dental caries in Brazil and anti-age therapy in Russia.
 
 So let us feel delighted in this present and celebrate our learning, because the high dilutions provides endless possibilities.

Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics

Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.

Solvent Effects in Supramolecular Chemistry: Linear Free Energy Relationships for Common Intermolecular Interactions.

The proper choice of solvent is of major importance for all studies in supramolecular chemistry, including molecular recognition in host-guest systems, intramolecular folding, self-assembly, and supramolecular polymerization. In this Perspective, the usefulness of linear free energy relationships (LFERs) is highlighted to unravel the effect of solvents on coordinate bonding (e.g., cation-crown ether), hydrogen bonding, halogen bonding, dipolar aggregation, and π-π-stacking. For all of these intermolecular interactions widely applied in supramolecular systems, LFER relationships between the Gibbs binding energies and common solvent polarity scales including ET(30), π*, α or β based on solvatochromic dyes, scales derived from binding processes such as Gutmann donor and acceptor numbers or hydrogen bond donor and acceptor scales, or physical functions like the Kirkwood-Onsager or the Liptay-Onsager functions could be demonstrated. These relationships can now be applied toward a better understanding of the prevailing intermolecular forces for supramolecular interactions. They further enable a rational selection of the most suitable solvent for the preparation of self-assembled materials and the estimation of binding constants without the need for time-consuming comprehensive investigations of solvents.

Semi-interpenetrating Polyurethane Network Foams Containing Highly Branched Poly(N-isopropyl acrylamide) with Vancomycin Functionality.

Highly branched poly(N-isopropylacrylamide) (HB-PNIPAM), functionalized with vancomycin at the chain ends, acted as a bacterial adhesive and was incorporated into polyurethane foams to form semi-interpenetrating networks. PNIPAM was labeled with a solvatochromic dye, Nile red. It was found that the thermal response of the polymer was dependent on the architecture, and temperature-dependent color changes were observed within the foam. The foams had open pore structures, and the presence of HB-PNIPAM substantially reduced the shrinkage of the foam as the temperature was increased up to 20 °C. The foams were selectively adhesive for Staphylococcus aureus (Gram-positive bacteria) compared to Pseudomonas aeruginosa (Gram-negative bacteria), and the presence of S. aureus was indicated by increased fluorescence intensity (590-800 nm).

Core-Shell Pluronic-Organosilica Nanoparticles with Controlled Polarity and Oxygen Permeability.

Nanostructured systems constitute versatile carriers with multiple functions engineered in a nanometric space. Yet, such multimodality often requires adapting the chemistry of the nanostructure to the properties of the hosted functional molecules. Here, we show the preparation of core–shell Pluronic-organosilica “PluOS” nanoparticles with the use of a library of organosilane precursors. The precursors are obtained via a fast and quantitative click reaction, starting from cost-effective reagents such as diamines and an isocyanate silane derivative, and they condensate in building blocks characterized by a balance between hydrophobic and H-bond-rich domains. As nanoscopic probes for local polarity, oxygen permeability, and solvating properties, we use, respectively, solvatochromic, phosphorescent, and excimer-forming dyes covalently linked to the organosilica matrix during synthesis. The results obtained here clearly show that the use of these organosilane precursors allows for finely tuning polarity, oxygen permeability, and solvating properties of the resulting organosilica core, expanding the toolbox for precise engineering of the particle properties.

Synthesis and Solvatochromic Behavior of Zwitterionic Donor–Bridge–Acceptor Systems with Oligo(p-phenylene) Spacers

Oligo(p-phenylene)s with a donor phenol group and an acceptor pyridinium moiety separated by one and two p-phenylene units were synthesized by the linear iterative Suzuki–Miyaura coupling method using aryl nonaflates as effective coupling reagents. Zwitterionic forms of these push–pull molecules were generated upon deprotonation of the phenol leading to large redshifts in absorbance maxima. UV-vis absorbance studies also revealed strong dependence of the band position on solvent polarity: a smooth bathochromic shift can be observed with the decrease of the solvent polarity. The molecule with one p-phenylene bridging unit showed the strongest solvatochromic characteristics in the series, spanning the range of 167 nm while moving from polar water to less polar N,N-dimethylformamide. The magnitude of this shift was close to Reichardt's dye — one of the most solvatochromic organic dyes known.

The use of a preferential–solvation index (PSI) for the comparison of halochromic systems

The study of the influence of salts on the spectral data of solvatochromic probes in solution is often a subject of difficult interpretation due to the puzzling nature of the species involved (solute, solvent, and salt). This ternary system can be understood in a similar way to the studies of binary solvent mixtures in the presence of solvatochromic dyes. Recently, a preferential–solvation index (PSI) was introduced for the study of binary solvent mixtures. The index is calculated as a ratio of two areas from a typical experimental PS plot and is not based on any theoretical model nor fitting parameters of questionable meaning. In this paper, the PSI approach was defined and used to understand salt–dependent processes in electrolyte solutions. The PSI values were calculated for fifty examples of halochromism available in the literature involving six solvatochromic probes. It was applied to systems comprising different solvents, salts, and probes, where one of these three variables was systematically varied. The values of PSI for the systems were compared with solvent (Reichardt’s ET (30) and Gutmann’s donor number) and cation (Klopman’s softness character and ionic radii) parameters. The observed trends shed light on probe–salt and solute–solvent interactions in solution, allowing a deeper understanding of halochromic and other salt–dependent systems.

Characterization of Brazilian palygorskite (Guadalupe region) and adsorptive behaviour for solvatochromic dyes

AbstractThis work presents the results of the physical characterization of palygorskite and its adsorptive behaviour for three solvatochromic dyes (Nile blue chloride (NBC), methylene blue (MTB) and dithizone (DTZ)). Adsorption isotherms were used to determine the maximum adsorption of the solvatochromic dyes on the palygorskite. The characterization of palygorskite was carried out via mineralogical and chemical analysis with X-ray diffraction, X-ray fluorescence, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy, surface-charge measurement (ζ-potential), thermogravimetric analysis, textural analysis and cation-exchange capacity analysis. The material consists of palygorskite and quartz and its chemistry is dominated by SiO2, MgO and Fe2O3. The specific surface area and cation-exchange capacity of the palygorskite are 142 m2 g–1 and 41 cmol(+) kg–1, respectively. The SEM and TEM analyses showed a fibrous structure with fibres 20–100 nm long. The thermogravimetric analysis showed three endothermic events at 57.3°C, 171.8°C and 439.6°C. The adsorption capacities of the palygorskite for NBC (basic pH), MTB (basic pH) and DTZ (neutral pH) were 0.082, 0.013 and 0.102 g g–1, respectively. The adsorptions of NBC and MTB were fitted with the Langmuir isotherm model and the adsorption of DTZ was fitted with the Sips model.

Modulating solvation interactions of deep eutectic solvents formed by ammonium salts and carboxylic acids through varying the molar ratio of hydrogen bond donor and acceptor

Deep eutectic solvents (DESs) have gained increasing popularity in separation science due to the fact that their physico-chemical properties can be easily fine-tuned by varying the type or ratio of hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD). While it is well-known that the molar ratio of HBA/HBD affects the melting point of a eutectic mixture, much less is understood regarding its effect on the magnitude of individual solvation interactions. This is largely due to the fact that established solvatochromic dye methods lack sensitivity when the HBA/HBD ratio is varied slightly in a eutectic mixture. Herein, this study is the first to measure the variation of DES solvation interactions with small changes in the molar ratio of HBA/HBD using inverse gas chromatography (IGC). Solute-solvent interactions of three different DES systems comprised of ammonium salts and organic acids were examined. The probe molecules were studied for 18 eutectic mixtures of varied HBA and HBD composition. DES hydrogen bond basicity, hydrogen bond acidity, and dispersive-type interactions exhibited the greatest change when the molar ratio of HBA/HBD was varied in the eutectic mixture. Results from this study demonstrate that the HBA/HBD ratio can be used to modulate the solvation characteristics for this class of DESs in separations and that the stoichiometric ratio of the HBA/HBD is important in ensuring their reproducible preparation.

Quantitative Examination of Changes in Fluidity and Cholesterol Content in Bovine Aortic Endothelial Cells using Phasor Analysis of Laurdan Fluorescence Lifetime

Cholesterol is important for maintaining cell membrane integrity and is known to have an effect on the function of ion channels in endothelial cells. Here, we use the properties of Laurdan to investigate how various cellular cholesterol manipulations, altering membrane fluidity and adding a cholesterol analog affect physical properties of bovine aortic endothelial cells (BAEC). Laurdan, a solvatochromic dye, has a spectral red shift in its emission when in a more liquid disordered (Ld) phase compared to a liquid ordered phase (LO). BAECs labeled with Laurdan were treated with methyl-ß-cyclodextrin (MßCD) to remove cholesterol, MßCD saturated with cholesterol to enrich cholesterol content, the cholesterol analog epicholesterol, or fluidizing alcohols benzyl alcohol (BA) or dodecanol and compared to BAECs treated with PBS+Ca2+ as a control. Two-photon excitation was used to excite Laurdan and lifetime measurements were compared at two emission channels (‘blue channel’ and ‘green channel’) in order to differentiate between changes in membrane fluidity and cholesterol content using two methods: traditional phasor analysis and a multiparametric distance analysis using phasor data. Treatment with MßCD, BA and dodecanol resulted in a shift to shorter lifetimes in the blue channel, indicative a more polar (Ld) environment, while enriching cells with cholesterol shifted phasors to longer lifetimes. In the green channel, the phasors of cholesterol enriched cells were shifted in the direction that corresponds to an increase in dipolar relaxation. Only treatment with a higher concentration of MßCD caused a shift in the opposite direction. Using Laurdan and phasor analysis to distinguish changes in fluidity from changes in cholesterol gives us the ability to manipulate these parameters to assist in determining the mechanisms by which cholesterol can impair cell signaling processes.

Sub-Diffraction Limited Imaging of Lipid Domains using Ratiometric STED

Cellular membranes are comprised of heterogeneously distributed lipid domains that are important for cellular functions. While recent literature has greatly augmented the importance of the spatial separation and the structural features of membrane rafts, the biophysical techniques available for studying membrane nanodomains still remain sparse. Studying lipid clustering and domain formation at a sub-diffraction level continues to be a challenge despite great advances in the field of super-resolution microscopy. In this study, we report the use of a commercially available solvatochromic dye (Nile Red) as a lipid domain sensor in conjunction with stimulated emission depletion (STED) microscopy. By maintaining a fixed concentration of constantly exchanging fluorophores in bulk, we all but eliminate photobleaching from the STED depletion laser, an issue that is commonly associated with STED microscopy. We further leverage the solvatochromic nature of the dye, by monitoring its spectral shift to report on the lipid order changes as a result of changes in lipid composition. As a proof of concept, we performed ratio metric STED imaging on surface-immobilized large unilamellar vesicles (LUVs) prepared with varying lipid compositions to test the validity of our approach. Our results demonstrate that our simple, yet novel approach provides spatial resolution at a <100nm level, while simultaneously reporting on the lipid order of the immobilized LUVs. We anticipate that this technique will be applicable to a wide range of biological systems, and will yield valuable insights into the spatial and chemical organization of lipid rafts in cell membranes. This work was supported by the NIH R01 AI150453 grant to GBM.

Preferential solvation index as a tool in the analysis of the behavior of solvatochromic probes in binary solvent mixtures

The analysis of the solvation of a given solute is a puzzling phenomenon if the solute is placed in binary solvent mixtures. This is due to the preferential solvation (PS) of the solute by one of the solvents of the mixture. Solvatochromic dyes are used to describe binary solvent mixtures with different proportions of the components, from plots of molar transition energy (ET) values of a solvatochromic dye as a function of the mole fraction of the most polar solvent (X2) in the mixture. The quantitative analysis of these plots is commonly made by fitting the data using a quantitative solvent exchange model, with considerable success. However, although the fits are generally good, the results obtained are of difficult interpretation. Recently, a PS (or non–ideality) index (PSI) was introduced in an attempt to develop a simple tool for the qualitative and quantitative analysis of deviations from linearity verified in ET (dye) with X2 in mixtures of water with alcohols. Thus, it is necessary to extend the study related to the PSI to different probes and other solvent mixtures, to verify the advantages and limitations regarding the applications of this tool. Herein the solvatochromic behavior of five solvatochromic dyes in sixty–two binary solvent mixtures of a hydrogen–bond acceptor solvent (DMSO or acetonitrile) and a hydrogen–bond donor solvent (water and alcohols) was investigated. The PSI values were calculated and interpreted for each binary mixture. The comparison of PSIs for structurally different dyes in the binary mixtures provided interesting information about the solvation of the dyes. Thus, the analysis of the PSI values for the mixtures allow to interpret the phenomenon of PS not only in terms of a commitment of solute–solvent and solvent–solvent interactions, but also in terms of the intrinsic properties of the probe.

Complexes of carboxylato pillar[6]arene with Brooker-type merocyanines: Spectral properties, pKa shifts and the design of a displacement assay for trimethyl lysine

The supramolecular complexes of three strongly solvatochromic dyes, Brooker’s merocyanine (M1) and its two derivatives (M2, M3) with carboxylato pillar[6]arene (WP6) were studied in aqueous solutions. The dye-WP6 mixtures were described in terms of four equilibrium reactions: the acidic dissociations of the pyridinium phenols into the zwitterionic phenolates, the acidic dissociations of the complexed phenols, the bindings of the phenol form dyes to WP6 and the bindings of the phenolates to WP6. The equilibrium constants were determined by an analysis of the absorption spectra. It was found that the acidity of the phenol form merocyanines were largely reduced on complexation, pKa shifts of 1.1–1.6 units were observed. In neutral solutions, the complexes of the phenol forms of M1 and M2 were dominant, in contrast to the more acidic M3 (a dibromo derivative), of which the phenolate complex was more stable. Comparing the spectral properties, the binding constants and the pKa-s of the dye-WP6 complexes, the complex M3⋅WP6 was chosen to be tested as a displacement assay. It was demonstrated that this complex functioned as a colorimetric indicator displacement assay which discriminated trimethyl lysine from other lysine derivatives.

Protamine/heparin optical nanosensors based on solvatochromism.

Optical nanosensors for the detection of polyions, including protamine and heparin, have to date relied upon ion-exchange reactions involving an analyte and an optical transducer. Unfortunately, due to the limited selectivity of the available ionophores for polyions, this mechanism has suffered from severe interference in complex sample matrices. To date no optical polyion nanosensors have demonstrated acceptable performance in serum, plasma or blood. Herein we describe a new type of nanosensor based on our discovery of a “hyper-polarizing lipophilic phase” in which dinonylnaphthalenesulfonate (DNNS−) polarizes a solvatochromic dye much more than even an aqueous environment. We have found that the apparent polarity of the organic phase is only modulated when DNNS− binds to large polyions such as protamine, unlike singly charged ions that lack the cooperative binding required to cause a significant shift in the distribution of the polarizing DNNS− ions. Our new sensing mechanism allows solvatochromic signal transduction without the transducer undergoing ion exchange. The result is significantly improved sensitivity and selectivity, enabling for the first time the quantification of protamine and heparin in human plasma using optical nanosensors that correlates with the current gold standard analysis method, the anti-Xa factor assay.

Ion-Selective optodes: Alternative approaches for simplified fabrication and signaling

This review highlights recent advances in ion-selective optodes (ISOs), an ionophore-based optical chemical sensor. Progress in the development of new optode components, fabrication on various novel substrate materials, simplified signaling methods are summarized. Paper-based ISOs, hydrogels and other integrated sensing devices have enabled cost-effective sensing by reading from colorimetric response and distance change. Signal transduction based on photoactivation, upconverting, chemiluminescence and delayed luminescence have offered solutions to potential background optical interference. The intrinsic pH cross-response from chromoionophore-based ISOs have been overcome with the exhaustive sensing mode, solvatochromic dyes and other charged molecules. Various applications of ISOs including cellular and in vivo monitoring with nano-optodes are also discussed.

Single‐Molecule Sensing by Grating‐based Spectrally Resolved Super‐Resolution Microscopy

Recent advances in single‐molecule spectroscopic imaging techniques, such as spectrally resolved stochastic optical reconstruction microscopy (SR‐STORM), have been effective for obtaining detailed spectral information at the molecular level. However, its application for single‐molecule sensing is highly challenging owing to its complicated configuration and limited spectral information. In this study, we demonstrated single‐molecule polarity sensing by combining grating‐based SR‐STORM with the solvatochromic dye, Nile red. The spatial and spectral resolutions of the custom‐built grating‐based SR‐STORM were examined for various color ranges of fluorescent dyes, and this approach was successfully applied for sensing nanoscale local polarity of various organic solvent molecules and surfactant molecules. Furthermore, we demonstrated that the proposed method effectively distinguished the different polarities of functional groups within surfactant molecules at the single‐molecule level. We anticipate that the proposed approach can be combined with other chromic molecules for application to many other chemical systems.

Binding Modes of a Phenylpyridinium Styryl Fluorescent Dye with Cucurbiturils.

In order to explore how cucurbituril hosts accommodate an N-phenyl-pyridinium derivative guest, the complexation of the solvatochromic dye, 4-(4-(dimethylamino)styryl)-1-phenylpyridinium iodide (PhSt) with α,α′,δ,δ′-tetramethyl-cucurbit[6]uril (Me4CB6) and cucurbit[7]uril (CB7) was investigated by absorption spectroscopic, fluorescence and NMR experiments. In aqueous solutions, PhSt forms 1:1 complexes with both cucurbiturils, the complex with CB7 has a higher stability constant (Ka = 6.0 × 106 M−1) than the complex with Me4CB6 (Ka = 1.1 × 106 M−1). As revealed by NMR experiments and confirmed by theoretical calculations, CB7 encapsulates the whole phenylpyridinium entity of the PhSt cation guest, whereas the cavity of Me4CB6 includes only the phenyl ring, the pyridinium ring is bound to the carbonyl rim of the host. The binding of PhSt to cucurbiturils is accompanied by a strong enhancement of the fluorescence quantum yield due to the blocking of the deactivation through a twisted intramolecular charge transfer (TICT) state. The TICT mechanism in PhSt was characterized by fluorescence experiments in polyethylene glycol (PEG) solvents of different viscosities. The PhSt-CB7 system was tested as a fluorescence indicator displacement (FID) assay, and it recognized trimethyl-lysine selectively over other lysine derivatives.

Effect of donor and acceptor end-groups on electronic structure and spectral-fluorescent properties of merocyanines in frozen ethanol

UV–vis absorption and fluorescence spectra of a series of merocyanines with varying heterocyclic donor and carbonyl-comprising acceptor groups have been studied in frozen ethanol at 77 K. Due to a greater polarity of a frozen solvent matrix in comparison with the liquid solvent, the electronic structure of donor–π–acceptor molecules in the former becomes much more dipolar. The resulting spectral effects depend on the donor–acceptor abilities of the terminal groups. In the case of positively solvatochromic indole-based dyes comprising the 1,3-indandione or barbituric acceptors, going from 293 to 77 K results in a narrowing and bathochromic shifts of absorption bands. Vice versa, for more dipolar reversely and negatively solvatochromic merocyanines comprising the thiobarbituric acceptor-group, the solvent freezing leads to a broadening and hypsochromic shifts of the absorption bands, which is very unique, taking into account that the solute vibrations/rotations should decrease in a frozen solvent. In all cases, the fluorescence bands are shifted hypsochromically at cryo-temperature and are closely mirror-like to the absorption ones, which, along with the smaller Stokes shifts, has been explained by the inhibited solvent rearrangement in the frozen matrix, resulting in a greater similarity of the ground state S0 and the fluorescent excited state S1. The deductions about the electronic structure of the studied dyes in the ground and excited states and its dependence on the donor–acceptor properties of the end-groups correlate well with the results of the PCM/(TD)-DFT calculations performed within the four-level diagram of electronic transitions.