Solvatochromic Dyes Research Articles

Optical Detection of Heparin in Whole Blood Samples Using Nanosensors Embedded in an Agarose Hydrogel.

Point-of-care quantification of the anticoagulant heparin still remains a significant clinical challenge as the reference method (colorimetric anti-factor Xa assay) cannot be performed in whole blood. Our group recently put forth the novel optical nanosensing principle using an ionic solvatochromic dye as a signal transducer. These nanosensors demonstrated significantly improved selectivity and sensitivity compared to ion-exchange-type polyion nanosensors and enabled protamine/heparin quantification in blood plasma samples. However, because the readout is absorbance-based, they are still not suitable for whole blood measurements. To overcome the background absorbance of blood, the nanosensors were here embedded in an agarose hydrogel capable of filtering out red blood cells while allowing plasma components to diffuse into the gel. Calibration curves for both protamine and heparin were successfully obtained in buffer, undiluted plasma, and undiluted whole blood using different colorimetric image analysis methods and a simple experimental setup.

Extending the π-system of inverted solvatochromic phenolate-based dyes shifts their inversion point due to an increment in their sensitivity to solvent polarizability

Two pairs of phenolate-based dyes with a π-connecting system of different lengths were synthesized, and their spectral variations were investigated in pure solvents of variable polarity and methanol-dichloromethane solvent mixtures. All the dyes studied exhibited inverted solvatochromism, and multiparametric analysis of their solvatochromic absorption bands of these dyes revealed that the solvent acidity and polarizability are mainly responsible for this behavior. The extension of the π-connecting system brings out of the shifts of the spectral behavior exhibited by the dyes towards shorter wavelengths with a concomitant displacement of the position where their solvatochromic inversion takes place towards higher solvent polarity due to an increment in the ability of the extended dyes to sense the solvent polarizability, without modifying the specific solute–solvent interactions in the first or the second solvation layer, a result confirmed by theoretical calculations.

Optimised generalized polarisation analysis of C-laurdan reveals clear order differences between T cell membrane compartments

Heterogenous packing of plasma membrane lipids is important for cellular processes like signalling, adhesion and sorting of membrane components. Solvatochromic membrane fluorophores that respond to changes from liquid-ordered (lo) phase to liquid-disordered (ld) by red shifts in their emission spectra are often used to assess lipid packing. Their response can be quantified using generalized polarisation (GP) using fluorescence microscopy images from two emission ranges, preferably from a region of interest (ROI) limited to a specific membrane compartment. However, image quality is limited by Poisson noise and convolution by the point spread function of the imaging system. Examining GP-analysis of C-laurdan labelled T cells using the image restoration procedure deconvolution, we demonstrate that deconvolution substantially improves the image resolution by making the plasma membrane clearly discernible and facilitating plasma membrane ROI selection. We conclude that automatic ROI selection has advantages over manual ROI selection when it comes to reproducibility and speed, but reliable GP-measurements can also be obtained by manually demarcated ROIs. We find that deconvolution enhances the difference in GP-values between the plasma and intracellular membranes and demonstrate that moving an intensity defined plasma membrane ROI outwards from the cell further improves this differentiation. By systematically changing the key deconvolution regularization parameter signal to noise, we establish a protocol for deconvolution optimisation applicable to any solvatochromic dye and imaging system. The image processing and ROI selection protocol presented improves both the resolution and precision of GP-measurement and will enable detection of smaller changes in membrane order than is currently achievable.

Alginic Acid Beads Containing Fluorescent Solvatochromic Dyes Display an Emission Color Response to a Cationic Surfactant.

Lipophilic fluorescent dyes can be employed as sensors for surfactants present in concentrations above the critical micellar concentration (CMC) where the dyes are monodispersed in micelles. However, the surfactant concentration range over which these dyes are effective is narrowed because by the sigmoidal nature of their responses. To overcome this limitation, we developed a novel sensor material comprised of a labeled fluorescent solvatochromic dye covalently bonded to alginate gel, which is known to strongly adsorb cationic surfactants. We hypothesized that the dye-alginate conjugate would undergo fluorescent color changes in response to binding of surfactants which alter the polarity of the surrounding environment. Indeed, addition of the representative cationic surfactant, cetylpyridinium chloride (CPC), to an aqueous solution of the alginate conjugated fluorescent solvatochromic dye leads to a visible fluorescent color change when the concentration of CPC is below the CMC. The average values of the color appearance parameter, referred to as a hue, of light emitted from gels, calculated by analysis of fluorescence microscopy images using ImageJ software, were found to be approximately linearly dependent on the concentration of CPC encapsulated in the alginate-fluorescent dye complex. This finding shows that absorbed CPC can be quantitatively determined over a wide concentration range in the form of simple fluorescence wavelength or visible responses.

Poly (Vinylidene Difluoride) Polymer in 1-Ethyl-3-methylimidazolium Acetate and Acetic Acid Containing Solvents: Tunable and Recoverable Solvent Media to Induce Crystalline Phase Transition and Porosity

In this report, 1-ethyl-3-methylimidazolium acetate, [EMIM][AcO] ionic liquid (IL) and acetic acid (AA) comprised solvents were used for the thermal treatment of poly (vinylidene difluoride), PVDF. Here, besides the various combinations of IL and AA in solvents, the pure IL and AA were also applied as a solvent upon thermal treatments. The samples obtained after the treatment were analysed for structural and crystalline phase changes, porosity, and molecular weight distribution with various analytical techniques. The Kamlet-Taft parameters measurement of the IL and AA containing solvents with different solvatochromic dyes was also performed to examine their solvent properties and correlate with the properties of the treated PVDF materials. The treatment of PVDF with pure IL results in the formation of highly carbonaceous material due to extensive dehydroflurination (DHF) as well as possibly successive cross-linking in the polymer chains. Upon IL and AA combined solvent treatment, the neat PVDF which composed of both α- and β crystalline phases was transformed to porous and β-phase rich material whereas in case of pure AA the non-porous and pure α-phase polymeric entity was obtained. A combined mixture of IL and AA resulted in a limited the DHF process and subsequent cross-linking in the polymer chains of PVDF allowed the formation of a porous material. It was observed that the porosity of the thermally treated materials was steadily decreasing with increase in the amount of AA in solvents composition and solvent with an AA:IL mole ratio of 2:1 resulted in a PVDF material with the highest porosity amongst the applied solvents. A recovery method for the IL and AA combined solvent after the thermal treatment of PVDF was also established. Hence, with varying the type of solvents in terms of composition, the highly carbonaceous materials as well as materials with different porosities as well as crystalline phases can be obtained. Most importantly here, we introduced new IL and AA containing recoverable solvents for the synthesis of porous PVDF material with the electroactive β-phase.

Ultrafast photoinduced dynamics of a donor-(mathrm pi)bridge-acceptor based merocyanine dye

Merocyanine dyes are of great interest amongst researchers due to their nonlinear optical (NLO) properties and solvatochromism. Molecular structure of these dyes constitutes conjugated pathway between the donor and acceptor substituents, with lowest energy transition of mathrm pi–mathrm pi* character. To rationalize the design of these dyes and deduce structure-property relationship, it is eminent to unravel the excited state dynamics in these complex molecular structures in different solvents. Here we have studied excited state dynamics of a merocyanine dye known as HB194, which has shown commendable efficiency in small molecule based bulk heterojuction solar cells. We have employed femtosecond transient absorption in combination with the quantum chemistry calculations to unravel the solvent dependent charge transfer dynamics of HB194. The excited state decays of the HB194 in different solvents show multi-exponential components. The analysis of the time-resolved data reveals that the polar solvents induce conformationally relaxed intramolecular charge transfer state. In non-polar solvent cyclohexane, only solvent-stabilized ICT state is observed. Additionally, we observe an anomalously red-shifted emission in ethylene glycol centred at sim 750 nm. Our computational calculations suggest the presence of molecular dimers resulting into observed red-shifted emission band. Our work therefore underscores the importance of gathering molecular-level insight into the system-bath interactions for designing next generation merocynanine-based solvatochromic dyes.

A miniaturized all-in-one optical nose based on 3D printing technology and patterned paper substrate for discrimination of volatile organic compounds

Herein, an integrated design of a paper-based colorimetric sensor array, which includes both sensor array and sampling vessel is described. The paper substrate was patterned for providing hydrophobic barriers to deposit sensing elements, while a hydrophilic regions were embedded on the paper for sample solution. In this way, the paper substrate works as a sensing device and sampling vessel, simultaneously. A miniaturized cubic reaction chamber sizing (30 × 30 × 0.02) mm, equipped with a small aperture for sample injection was fabricated using 3D printing technology to hold the paper substrate. The required sample volume was lowered down dramatically (13.0 μL) due to the all-in-one sampling and sensing device. This miniaturized sensor array concept was employed to detect and discriminate volatile organic compounds (VOCs) relating to different solvent families that are known as cancer biomarkers. A colorimetric sensor array was fabricated by spotting 12 indicators belonging to three families of solvatochromic dyes, pH indicators and metallochromic dyes. This simple proposed device was able to discriminate the studied solvents into three chemical families including polar protic, dipolar aprotic and nonpolar with 93% accuracy. In addition, this sensor showed potential for quantitative and semi-quantitative analysis of volatile compounds in their mixtures. In total, the designed device is portable, rapid, and cost-effective and requires low volumes of samples that can be considered a good candidate for various VOC analysis with limited volume.

Synthesis and Characterization of Newly Designed and Highly Solvatochromic Double Squaraine Dye for Sensitive and Selective Recognition towards Cu2.

Synthesis and characterization of a novel and zwitterionic double squaraine dye (DSQ) with a unique D-A-A-D structure is being reported. Contrary to the conventional mono and bis-squaraine dyes with D-A-D and D-A-D-A molecular frameworks reported so far, DSQ dye demonstrated strong solvatochromism allowing for the multiple ion sensing using a single probe by judicious selection of the suitable solvent system. The DSQ dye exhibited a large solvatochromic shift of about 200 nm with color changes from the visible to NIR region with metal ion sensitivity. Utilization of a binary solvent consisted of dimethylformamide and acetonitrile (1:99, v/v), highly selective detection of Cu2+ ions with the linearity range from 50 μM to 1 nM and a detection limit of 6.5 × 10−10 M has been successfully demonstrated. Results of the Benesi–Hildebrand and Jobs plot analysis revealed that DSQ and Cu2+ ions interact in the 2:1 molecular stoichiometry with appreciably good association constant of 2.32 × 104 M−1. Considering the allowed limit of Cu2+ ions intake by human body as recommended by WHO to be 30 μM, the proposed dye can be conveniently used for the simple and naked eye colorimetric monitoring of the drinking water quality.

Immobilization of Solvatochromic Dyes on Transparent Cellulose Films: an Improved Method for the Study of Homeopathic Potencies.

Highly diluted and succussed solutions (homeopathic potencies) have been shown to interact with a wide range of solvatochromic dyes based on changes in their UV-visible spectra. Studies so far have involved free dyes in solution, but there is a pressing need to find ways to investigate the potency-dye interaction using isolated dye molecules to ask more searching physico-chemical questions regarding the fundamental nature of potencies. The aims of the present study have been to look for ways to covalently immobilize solvatochromic dyes onto transparent cellulose films and hence be in a position to investigate dye-potency interactions without the complication of dye-dye interactions, including dye aggregation, which can occur with free dyes in solution. To date, a total of nine different dyes have been immobilized on cellulose films using epoxide activation of hydroxyl groups on the cellulose surface. Using this method, studies have begun looking at the time course of potency action on one of these immobilized dyes, Brooker's merocyanine. Results show that the interaction of Arsenicum 10M with this dye consists of three phases-an initial growth phase, a sustained plateau of interaction, and a final decline phase lasting several days. A method has been developed that successfully immobilizes solvatochromic dyes onto transparent cellulose film. These films can then be used in a spectrophotometer to study at a much more detailed level how potencies interact with dyes compared with using free dyes in solution. Results indicate that the information gained in this way provides new insights regarding the fundamental nature of potencies. Specifically, studies using immobilized Brooker's merocyanine with Arsenicum 10M reveal that the lifetime of the potency is much longer than expected and that its action consists of three distinct phases, suggesting a resonant interaction with the dye. How resonant interaction might help to explain the clinical action of potencies is discussed.

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection—Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

Silicea terra 200cH evaluated by two different spectroscopy methods: a pilot study

Abstract
 Spectroscopy has been shown to be a useful method to study the physicochemical properties of homeopathic preparations. In this pilot study, the aim was to compare two methods (photon scattering and visible-light spectroscopy) in the physical evaluation of Silicea terra 200cH. Two samples have been examined and compared: Silicea terra 200cH and Sac lac 200cH, both prepared in aqueous solution (Cemon Lab, Italy). Lactose was included because the first 3 potencies of Silicea terra are made by trituration in lactose. In the first test, performed in Italy, slight variations in chrominance and luminance due to micro-vibrational 3D phenomena were analyzed. The principle of the experimental procedure is to modify and analyse the incidence and refraction angles of the light hitting the bottles of liquid under examination. Measurements were made using an innovative camera device developed by Daniele Gullà, called MIRA/CORA (proprietary name). The image sensor is a Charge-Coupled Device (CCD)/ Complementary Metal Oxide Semiconductor (CMOS) with interferential matrix filter. A red laser beam, at wavelength 652 nm, hits the bottle and the variations of scattered light are recorded and measured by the device. All measurements have been performed in a dark room at a constant temperature of 22°C +/- 0,2°C using a filter with very narrow spectral bands (10 nm). Proprietary software analyses the incoming frames showing the spectral variations in amplitude and frequency and then interpolates variation in the measured data within a vibrational range between 0.01 Hz and 10 Hz. The measurements have been repeated three times on both Silicea terra 200cH and Sac lac 200cH within a few minutes of each other. Measurements of the mean entropy of the signals, statistically elaborated with T Student test, yielded a two tailed p value < 0.05, where the entropy of the signal recorded from the Silicea terra 200cH sample was statistically lower than the 200cH Sac lac sample. In the second test, performed in Brazil, variations in absorbance were used to identify Silicea terra 200cH compared with Sac lac 200cH and a control solution of non-succussed 30% alcohol, using six solvatochromic dyes, following the method developed by Cartwright [1,2]. Both homeopathic samples were imported from Italy, diluted 1:100 in 30% hydro-alcoholic solution, and submitted to 100 succussions using an automatic mechanical arm (Denise, AUTIC, Brazil) prior to being tested. Samples were inserted into dyes solutions in a 1:60 ratio, and evaluated by visible spectroscopy (FEMTO Spectrophotometer, Brazil). Dyes were prepared in ethanol P.A., according to previous established methods [3]. Samples and dyes were protected from light during the manipulation and environmental conditions of the laboratory (temperature, humidity, and magnetic flux) were monitored during the tests. Three series in triplicate were performed and the results were analyzed by ANOVA / Tukey, comparing both samples and the unsuccussed 30% hydroalcoholic control solution. Among all tested dyes, only BDN showed an interaction with Silicea terra 200cH, reproducing the conclusions obtained in [3]. We conclude that two different spectroscopic methods were able to differentiate Silicea terra 200cH from Sac lac 200cH, suggesting that changes in solvent organization could be involved in the homeopathic signaling process, along with changes in dipole moments of solvent and dyes. The results are potentially in line with a recent published paper [4], that supports the proposition that the lower entropy of the verum signal compared with controls could be explained by increased coherent vibrations of the verum sample, modulated by Nambu-Goldstone bosons. According to Del Giudice and colleagues [5], coherent behaviors are correlated with massless quasi-particles, coding ordering information.

Immobilisation of Solvatochromic Dyes on Transparent Cellulose Films; an Improved Methodology for the Study of Homeopathic Potencies.

Abstract
 Background Highly diluted and succussed solutions (homeopathic potencies) have been shown to interact with a wide range of solvatochromic dyes based on changes in their UV-visible spectra. Studies so far have involved free dyes in solution, but there is a pressing need to find ways to investigate the potency-dye interaction using isolated dye molecules in order to ask more searching physico-chemical questions regarding the fundamental nature of potencies.
 Aims and Methods The aims of the present study have been to look for ways to covalently immobilise solvatochromic dyes onto transparent cellulose films and hence be in a position to investigate dye-potency interactions without the complication of dye-dye interactions, including dye aggregation, which can occur with free dyes in solution.
 Results To date a total of nine different dyes have been immobilised on cellulose films using epoxide activation of hydroxyl groups on the cellulose surface. Using this methodology studies have begun looking at the time course of potency action on one of these immobilised dyes, Brooker’s merocyanine. Results show that the interaction of Arsenicum 10M with this dye consists of three phases – an initial growth phase, a sustained plateau of interaction and a final decline phase lasting several days. 
 Conclusions A methodology has been developed that successfully immobilises solvatochromic dyes onto transparent cellulose film. These films can then be used in a spectrophotometer to study at a much more detailed level how potencies interact with dyes compared with using free dyes in solution. Results indicate that the information gained in this way provides new insights regarding the fundamental nature of potencies. Specifically, studies using immobilised Brooker’s merocyanine with Arsenicum 10M reveal that the lifetime of the potency is much longer than expected and that its action consists of three distinct phases, suggesting a resonant interaction with the dye.

Mercury chloride toxicity attenuation of the Brine Shrimp Artemia Salina after treatment with Mercurius corrosivus as isotherapic


 Isotherapics prepared from toxic substances have been described as attenuation factors for heavy metal intoxication in aquatic animals. Herein, Artemia salina and mercury chloride were used as a model to identify treatment-related bioresilience. The aim was to describe the effects of Mercurius corrosivus (MC) in different potencies on Artemia salina cyst hatching and on mercury bioavailability. Artemia salina cysts were exposed to 5.0 µg/mL of mercury chloride during the hatching phase. MC 6cH, 30cH, and 200cH were prepared and poured into artificial seawater. Different controls were used (nonchallenged cysts and challenged cysts treated with water, succussed water, and Ethilicum 1cH). Four series of nine experiments were performed for 4 weeks to evaluate the percentage of cyst hatching considering all moon phases. Soluble total mercury (THg) levels and precipitated mercury content were also evaluated. Solvatochromic dyes were used to check for eventual physicochemical markers of MC biological activity. Two-way analysis of variance (ANOVA) with mixed models was used for evaluating the effect of different treatments and the simultaneous influence of the moon phases on the cysts hatching rate, at both observation times (24 and 48 hours). When necessary, outliers were removed, using the Tukey criterion. The level of significance α was set at 5%. Significant delay (p<0.0001) in cyst hatching was observed after treatment with MC 30cH, compared with the controls. An increase in THg concentration in seawater (p<0.0018) and of chlorine/oxygen ratio (p<0.0001) in suspended micro-aggregates was also seen, with possible relation with mercury bioavailability. Specific interaction of MC 30cH with the solvatochromic dye ET33 (p<0.0017) was found. The other observed potencies of Mercurius corrosivus 6 and 200 cH were not significant in relation to the observed groups. The results were postulated as being protective effects of MC 30cH on Artemia salina, by improving its bioresilience.

Unitary Response of Solvatochromic Dye to Pulse Excitation in Lipid and Cell Membranes

The existence of acoustic pulse propagation in lipid monolayers at the air-water interface is well known. These pulses are controlled by the thermodynamic state of the lipid membrane. Nevertheless, the role of acoustic pulses for intra- and inter-cellular communication is still a matter of debate. Herein, we used the dye di-4-ANEPPDHQ, which is known to be sensitive to the physical state and transmembrane potential of membranes, in order to gain insights into compression waves in lipid-based membrane interfaces. The dye was incorporated into lipid monolayers made of phosphatidylserine or phosphatidylcholine at the air-water-interface. A significant blue shift of the emission spectrum was detected when the state of the monolayer was changed from the liquid-expanded (LE) to the liquid-condensed (LC) phase. This "transition sensitivity" of di-4-ANEPPDHQ was generalized in experiments with the bulk solvent dimethyl sulfoxide (DMSO). Upon crystallization of solvent, the emission spectrum also underwent a blue shift. During compression pulses in lipid monolayers, a significant fluorescence response was only observed when the main transition is crossed. The optical signature of these waves─in terms of sign and magnitude─was identical to the response of di-4-ANEPPDHQ during action potentials in neurons and excitable plant cells. These findings corroborated the suggestion that action potentials are nonlinear state changes that propagate in the cell membrane.

Probing molecular interactions in the system {water + 2-(diethylamino) ethanol} using solvatochromic dyes and excess partial molar enthalpies, at 298.15 K

The polarity of the binary {water + 2-(diethylamino)ethanol (DEEA)} mixtures was investigated, across the whole composition range using solvatochromic dyes, expressed in terms of the Kamlet-Abboud-Taft (π*, α, β) and ETN Reichardt parameters, at 298.15 K. The preferential solvation model of Bosch and co-workers was applied to each solvatochromic dye. Direct excess partial molar enthalpies, HiE, for the same system, at the same temperature and atmospheric pressure, were obtained at both ends of the composition range using a semi-adiabatic isoperibol solution calorimeter and an expeditious method pioneered by Yoshikata Koga. Infinite dilution values of this same thermodynamic property were calculated for the DEEA in water and for the water in DEEA and compared with the literature values for the systems {water + 2-(dimethylamino)ethanol (DMAE)} and {water + 2-(ethylamino)ethanol (EEA)}, to infer the effect of branching and chain length on this property. Values for the interaction enthalpy between pairs of molecules of the same species, HiiE, were also estimated, in the same composition ranges studied, revealing endothermic processes, at both ends. A successful multiparametric correlation between excess partial molar enthalpy of DEEA and the solvent polarity parameters, π*, α and β allowed to quantitatively discriminate the relative relevance of the different types of the DEEA-solvent interactions.

Proximity-induced surfactant aggregation

We describe surfactant aggregation (micellization) that is induced by the proximity between two interfaces. Under conditions where surface aggregation does not occur for a single interface, we observe surfactant aggregation between two glass solids that are separated by a thin aqueous film that is <30 nm thick. The surfactant is dodecyltrimethylammonium bromide (DTAB). Interfacial aggregation occurs at 1/100th of the critical micelle concentration (cmc) and is likely an electrostatic effect of the introduction of a second interface. Nile red, a solvatochromic dye, is used to indicate aggregation.

Characterization of Phase Separated Planar Lipid Bilayer Membrane by Fluorescence Ratio Imaging and Scanning Probe Microscope

The lipid membrane forms nanodomains (rafts) and shows heterogeneous properties. These nanodomains relate to significant roles in various cell functions, and thus the analysis of the nanodomains in phase-separated lipid membranes is important to clarify the function and role of the nanodomains. However, the lipid membrane possesses small-sized nanodomains and shows a small height difference between the nanodomains and their surroundings at certain lipid compositions. In addition, nanodomain analysis sometimes requires highly sensitive and expensive apparatus, such as a two-photon microscope. These have prevented the analysis by the conventional fluorescence microscope and by the topography of the scanning probe microscope (SPM), even though these are promising methods in macroscale and microscale analysis, respectively. Therefore, this study aimed to overcome these problems in nanodomain analysis. We successfully demonstrated that solvatochromic dye, LipiORDER, could analyze the phase state of the lipid membrane at the macroscale with low magnification lenses. Furthermore, we could prove that the phase mode of SPM was effective in the visualization of specific nanodomains by properties difference as well as topographic images of SPM. Hence, this combination method successfully gave much information on the phase state at the micro/macro scale, and thus this would be applied to the analysis of heterogeneous lipid membranes.

Mapping the solute–solvent interactions for the interpretation of the three types of solvatochromism exhibited by phenolate-based dyes

• The solvatochromism of phenolate-based dyes is rationalized from their solute–solvent interactions in solution. • Multiparametric analysis with Catalán’s solvent scales provided a differentiation of the three types of solvatochromism. • Each type of solvatochromism exhibit different sensitivities to solvent properties. • The pass from negative to positive solvatochromism is governed by the increase of the solvent polarizability by the dyes. • Three strategies for controlling the solvatochromism observed by structural modifications of phenolate dyes are presented. We report the correlation between the hydrogen-bond donor acidity SA, the hydrogen-bond acceptor basicity SB, the solvent polarizability SP, and solvent dipolarity SdP with the type of solvatochromism exhibited by phenolates dyes. We use Catalán’s multiparametric regression analysis of three sets of dyes exhibiting negative, inverted, and positive solvatochromism. We show that these three types of behavior can be differentiated by the solute–solvent interaction these dyes established in solution. Negative solvatochromic dyes exhibit a more robust response to SA than SdP, whereas SP is an essential solvent property for positive and inverted solvatochromic dyes. Our results can be rationalized for the sake of the structural features required for a particular kind of solvatochromism to take place; this is illustrated with selected examples where the introduction of atoms with a + M mesomeric effect or the increase in annulation of the dyes, decrease the SA and SdP sensitivity and increase the SP sensitivity of the dyes, and ultimately control the solvatochromism exhibited from a negative to an inverted or from an inverted to positive behavior.

Critical evaluation of fluorescent dyes to evaluate the stability and ligand binding properties of an anti-cocaine mAb, h2E2

Monoclonal antibodies have become a mainstay of modern drug development. However, unlike small molecule drugs, mAbs are large proteins that need to be characterized for their stability, heterogeneity, and tendency to aggregate. Many different extrinsic fluorescent dyes have been used to monitor the thermal stability, aggregation, and ligand binding characteristics of many different proteins. Some of these dyes change their fluorescence when bound to proteins due to changes in the hydrophobicity of their microenvironment (solvatochromic dyes such as Sypro Orange), while others respond to differences in rotational mobilities (rotor dyes such as DASPMI), and others have been used to detect fibrils and amyloid-like protein aggregation (amyloid dyes such as Thioflavin T). Previously, we used DASPMI dye and differential scanning fluorimetry to quantitate the binding of cocaine and cocaine metabolites to a humanized anti-cocaine h2E2 mAb under development for the treatment of cocaine use disorders. In the present study, we evaluated six dyes in these three classes for their ability to monitor domain denaturation and cocaine binding of the h2E2 mAb, both in its clinical formulation buffer and in PBS buffer. We noted that the Thioflavin T dye commonly used to assess amyloid formation was also capable of monitoring h2E2 mAb thermal denaturation and ligand binding using differential scanning calorimetry. However, unlike the DASPMI dye, the Thioflavin T dye caused a dose-dependent stabilization of the unliganded (apo) mAb, and when using the methodology developed with the DASPMI dye, decreased the apparent affinity of the mAb for cocaine as a function of dye concentration. Thus, although Thioflavin T differential fluorimetry data appears to be suitable for measuring cocaine affinity for this h2E2 mAb, the apparent mAb Kd values for cocaine are dependent on Thioflavin T dye concentration, reinforcing and extending the unique use of the DASPMI dye for this purpose.

En Route to Stabilized Compact Conformations of Single-Chain Polymeric Nanoparticles in Complex Media.

Precise control over the folding pathways of polypeptides using a combination of noncovalent and covalent interactions has evolved into a wide range of functional proteins with a perfectly defined 3D conformation. Inspired hereby, we develop a series of amphiphilic copolymers designed to form compact, stable, and structured single-chain polymeric nanoparticles (SCPNs) of defined size, even in competitive conditions. The SCPNs are formed through a combination of noncovalent interactions (hydrophobic and hydrogen-bonding interactions) and covalent intramolecular cross-linking using a light-induced [2 + 2] cycloaddition. By comparing different self-assembly pathways of the nanoparticles, we show that, like for proteins in nature, the order of events matters. When covalent cross-links are formed prior to the folding via hydrophobic and supramolecular interactions, larger particles with less structured interiors are formed. In contrast, when the copolymers first fold via hydrophobic and hydrogen-bonding interactions into compact conformations, followed by covalent cross-links, good control over the size of the SCPNs and microstructure of the hydrophobic interior is achieved. Such a structured SCPN can stabilize the solvatochromic dye benzene-1,3,5-tricarboxamide–Nile Red via molecular recognition for short periods of time in complex media, while showing slow exchange dynamics with the surrounding complex media at longer time scales. The SCPNs show good biocompatibility with cells and can carry cargo into the lysosomal compartments of the cells. Our study highlights the importance of control over the folding pathway in the design of stable SCPNs, which is an important step forward in their application as noncovalent drug or catalyst carriers in biological settings.