Solvatochromic Fluorescent Probes Research Articles

Effect of Divalent Cations on Polarity and Hydration at the Lipid/Water Interface Probed by 4-Aminophthalimide-Based Dyes.

The ion binding to the lipid/water interface can substantially influence the structural, functional, and dynamic properties of the cell membrane. Despite extensive research on ion-lipid interactions, the specific effects of ion binding on the polarity and hydration at the lipid/water interface remain poorly understood. This study explores the influence of three biologically relevant divalent cations─Mg2+, Ca2+, and Zn2+─on the depth-dependent interfacial polarity and hydration of zwitterionic DPPC lipid in its gel phase at room temperature. To measure these depth-dependent properties, we use a series of solvatochromic fluorescent probes synthesized based on 4-aminophthalimide with varying alkyl chain lengths (4AP-Cn; n = 5, 7, and 9). Employing steady-state fluorescence experiments and all-atom molecular dynamics (MD) simulations, we quantify changes in interfacial polarity and hydration induced by the cations binding to the lipid/water interface. Our results reveal that Zn2+ induces a significant blue shift in the fluorescence spectra of all 4AP-Cn dyes, indicating a marked decrease in local polarity (ETN ≤ 0.05) at the lipid/water interface compared to Mg2+ and Ca2+, which results in a higher polarity (ETN ≥ 0.2). The depth-dependent fluorescence spectra of dyes at the interface in the presence of Mg2+ and Ca2+ remain similar to those in the absence of cations, with only a minor red shift observed for Mg2+, implying a slight hydration effect. MD simulations show that cations primarily bind to the headgroup and glycerol regions of lipid. Simulations also reveal that Zn2+ causes substantial dehydration at the lipid/water interface, as detected by the 4AP-Cn dyes, while Mg2+ and Ca2+ have less pronounced effects, with only slight hydration induced by Mg2+. This study highlights the distinct positional effects of cations probed by 4AP-Cn probes at the lipid-water interface, underscoring the potential of 4AP-Cn dyes for monitoring depth-dependent changes in membrane properties induced by external agents or environmental conditions.

Coumarin 153, a solvatochromic fluorescent probe, for analyzing the biodiesel blends derived from various feedstocks.

Biodiesel is renewable energy source an alternative to conventional fossil fuels. The primary concern lies in detecting alcohol content in biodiesel, which can either be intentionally added by adulterants or remain in trace amounts from the refining process of biodiesel synthesis. In order to regulate the quality of biodiesel production, it is crucial to develop an analytical method for monitoring alcohol content in biodiesel. Present study identified Coumarin 153 (C-153) with outstanding solvatochromic characteristics in multiple biodiesel feedstocks (soybean, canola, sesame, and corn). Based on our spectroscopic investigations, this alcohol (methanol, ethanol, and propanol) sensing method has proven to be rapid, sensitive, ratiometric, and visually discernible, and the detection limit for ethanol in soybean biodiesel could reach 0.23% v/v. The percentage of alcohol (0-100% v/v) in the biodiesel determines significant changes in the lifetime values of the C-153 from 5.1ns to 0.35ns. Moreover, we depict explicit solvation models (ethanol) and implicit solvation models (biodiesel) from quantum chemical calculations to explain the experimental results. Based on our study, C-153 with alcohol sensing capabilities seems to have potential applications in biodiesel analysis. The present results will inspire future efforts to simplify and optimize the detection of alcohol in biodiesel by using optical methods.

Intramolecular Charge Transfer Inhibition Strategy toward a Desired Solvatochromic Fluorescent Platform: Visualization of Duple Organelles and Detection of Carbon Dioxide.

Solvatochromic fluorescent probes are crucial molecular tools to investigate and aggregate proteins' fold, visualize fine structures in biomembranes, and label different organelles in dual emission colors. However, solvatochromic fluorogens often displayed a weak emission at high polarity, hindering their bioimaging applications. To resolve this problem, herein, we propose an intramolecular charge transfer (ICT) inhibition strategy. The probe was designed with a single electronic donor and two acceptors in order to split and inhibit the ICT procedure. As a result, the probe displayed an intense emission at both low and high polarities and showed a large emission shift (84 nm) upon polarity change. Using the probe, we successfully imaged lipid droplets and the endoplasmic reticulum in different fluorescence colors. Moreover, the different degrees of lipid accumulation by oleic acid, stearic acid, and cholesterol (oleic acid > stearic acid > cholesterol) have been revealed. The lipid accumulation induced by the three lipids could be rapidly consumed under lipid-less conditions, and the lipids with stearic acid were the most difficult to be consumed. The biological results could facilitate the understanding and treatment of lipid accumulation and obesity. Furthermore, utilizing the polarity increase of diethylamine after the reaction with CO2, the ratiometric detection of CO2 has been achieved for the first time with the probe.

Integrating computational and experimental approaches in discovery and validation of MmpL3 pore domain inhibitors for specific labelling of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5 μs. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism.

A low-molecular-weight, long-wavelength emissive Coumarin: Green synthesis, spectral properties, fluorescence enhancement and solvatochromism application

In this study, a novel low-molecular-weight coumarin fluorescent dye, 3,4-dicyano-7-diethylaminocoumarin, was designed. This dye features a donor-π-bridge-acceptor (D-π-A) conjugated structure, which confers superior fluorescence properties.The dye was synthesized using a simple, efficient, and environmentally friendly two-step process, facilitating the direct conversion of the key intermediate into the final product via simultaneous addition and elimination reactions. A comprehensive structural characterization of the dye was undertaken, and its UV absorption and fluorescence emission properties were extensively analyzed using spectroscopic techniques, with structural explanations of the fluorescence activities provided by the Density Functional Theory (DFT) method.Experimental results revealed that this coumarin dye emits strong fluorescence, characterized by a long wavelength, a large Stokes shift, and high fluorescence intensity. It exhibits excellent fluorescent performance in various solvents. Despite experiencing fluorescence quenching in its solid state, the formation of an inclusion complex with β-cyclodextrin restores strong red fluorescence, presenting new possibilities for biofluorescence imaging. Notably, this coumarin dye also demonstrates a significant solvatochromic effect. This effect enables rapid visual discrimination among multiple sets of structurally and chemically similar organic solvents. Consequently, the dye holds promise as a versatile solvatochromic fluorescent probe for a broad spectrum of applications.

Fluorescent Solvatochromic Probes for Long-Term Imaging of Lipid Order in Living Cells.

High-resolution spatio-temporal monitoring of the cell membrane lipid order provides visual insights into the complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying the ordering of the microenvironment of plasma membrane microdomains. However, conventional probes, although capable of structural analysis, lack the necessary long-term photostability required for live imaging at the cellular level. Here, an ultra-high-light-resistant solvatochromic fluorescence probe, 2-N,N-diethylamino-7-(4-methoxycarbonylphenyl)-9,9-dimethylfluorene (FπCM) is reported, which enables live lipid order imaging of cell division. This probe and its derivatives exhibit sufficient fluorescence wavelengths, brightness, polarity responsiveness, low phototoxicity, and remarkable photostability under physiological conditions compared to conventional solvatochromic probes. Therefore, these probes have the potential to overcome the limitations of fluorescence microscopy, particularly those associated with photobleaching. FπCM probes can serve as valuable tools for elucidating mechanisms of cellular processes at the bio-membrane level.

Tetrazine-derived chromones as conditionally activated solvatochromic fluorescent probes for dual imaging of lipid droplets and mitochondria

Tetrazine-derived chromones were developed as conditionally activated solvatochromic fluorescent probes and applied for LD and mitochondrial imaging.

Push-pull intramolecular charge transfer solvatofluorochromic fluorophore for the selective and real-time detection of hydrazine

Hydrazine (N2H4) is a chemical molecule having a wide range of applications starting from its use in chemical reactions for the synthesis of pesticides, pharma and dye industry, rocket fuel etc. Along with its wide range of applications, it is also becoming a reason for environmental and health concerns, as a result wide ranging detection tools have been developed for the detection of hydrazine. Amongst various sensing tools fluorescence-based molecular probes that can be used for sensing chemical toxics are seeking attention due to their easy operability, high selectivity, sensitivity and their biocompatibility. In this work, we have synthesized and evaluated a solvatochromic fluorescent probe (MQM) that is capable for the rapid and selective detection of hydrazine in water samples with a limit of detection (0.58 µM). The most important application is the probe can be transformed into a portable analytical tool that can be used for the rapid on-site detection of hydrazine without the use of any sophisticated analytical instruments. The probe can be used for real-time detection of hydrazine in water, vapour phase, soil, and biological samples.

186 An application of the new solvatochromic fluorescent probe for the horizontal histopathological observation of skin tumor

186 An application of the new solvatochromic fluorescent probe for the horizontal histopathological observation of skin tumor

An ESIPT solvatochromic fluorescent and colorimetric probe for sensitive and selective detection of copper ions in environmental samples and cell lines.

Copper is one of the most important transition metals in our body for various physiological functions. An imbalance in the homeostasis of copper in our body can lead to various neurological disorders such as Alzheimer's disease, Parkinson's, and Menkes disease. As a result, there is a need for the detection of excess copper present in the environment and the human system. In this work, we have designed a quinoline-based fluorescent/colorimetric probe (QHS) for rapid and selective detection of copper ions via quenching of fluorescence/color change from yellow to peach which is visible to the naked eye. The probe displayed high selectivity towards copper(II), i.e., Cu(II) in the presence of different metal analytes in water samples. The sensing mechanism of the probe was confirmed by NMR, HRMS, IR spectroscopy, and SEM. The detection limit of Cu(II) ions was found to be 0.493 μM which is lower than the tolerable limit of copper (20 μM) in drinking water as per the EPA. The probe was also utilized for the visualization of Cu(II) in cell lines. The probe was also demonstrated for its application in real-time detection of aqueous samples using portable paper strips.

Influence of pH and low/high- methoxy pectin complexation on the hydrophobic binding sites of β-lactoglobulin studied by a fluorescent probe method

β-lactoglobulin(β-Lg)-polysaccharide soluble complexes formed in a specific pH range through electrostatic attraction have attracted a growing interest in the design of food-grade encapsulation systems for hydrophobic compounds, which is mainly ascribed to the ligand-binding properties of β-Lg. However, it remains unclear whether pH-induced conformational changes in β-Lg and its electrostatic complexation with anionic pectin affect their ability to bind hydrophobic compounds. Here, a fluorescent probe method was employed to provide useful insights into the field. Three solvatochromic fluorescent probes (i.e. Nile red, retinol and curcumin) were selected as representative models of hydrophobic compounds that were bound to the inner cavity or/and the outer surface of β-Lg. Binding with β-Lg or β-Lg/pectin complexes largely enhanced the fluorescence of the hydrophobic probes. Especially, the polarity difference of different binding sites on β-Lg was revealed by the fluorescence spectra of β-Lg-Nile red as a function of pH. Both Nile red and retinol were bound more favorably to β-Lg at neutral pH than at acidic pH, possibly due to the accessibility of the inner cavity in the former case. Upon acidification, the gradual reduction in fluorescence intensity of the pre-formed protein-ligands complexes (i.e. at pH 7.0) was ascribed to the dissociation between Nile red (or retinol) and the inner cavity of β-Lg. β-Lg-curcumin interactions were less affected by pH variations, suggesting curcumin mainly binding to the outer surface of β-Lg. For the three tested hydrophobic compounds, the formation of soluble complexes between β-Lg with pectin had no adverse effects on their interactions with the protein. These results may provide useful insights into the binding of hydrophobic compounds to β-Lg or β-Lg/pectin complexes. The methodology may also be extended to study the encapsulation performance of other biopolymers or particles.

Viscosity-Sensitive Solvatochromic Fluorescent Probes for Lipid Droplets Staining.

Lipid droplets (LDs) are simple intracellular storage sites for neutral lipids and exhibit important impact on many physiological processes. For example, the changes in the polar microenvironment inside LDs could affect physiological processes, such as lipid metabolism and storage, protein degradation, signal transduction, and enzyme catalysis. Herein, a new fluorescent chemo-sensor (Couoxo-LD) was formulated by our molecular design strategy. The probe could be applied to effectively label intracellular lipid droplets. Intriguingly, Couoxo-LD demonstrated positive sensitivity to both polarity and viscosity, which might be attributed to its D-π-A structure and the twisted rotational behavior of the carbon–carbon double bond (TICT). Additionally, Couoxo-LD was successfully implemented in cellular imaging due to its excellent selectivity, pH stability, and low biotoxicity. In HeLa cells, the co-localization curve between Couoxo-LD and commercial lipid droplet dyes overlapped at 0.93. The results indicated that the probe could selectively sense LDs in HeLa cells. Meanwhile, Couoxo-LD can be applied for in vivo imaging of zebrafish.

Structure Rigidification Promoted Ultrabright Solvatochromic Fluorescent Probes for Super-Resolution Imaging of Cytosolic and Nuclear Lipid Droplets.

Lipid droplets (LDs) containing cytosolic and nuclear LDs have recently received increasing attention because of their diverse biological roles in living systems. However, developing fluorescent probes for super-resolution visualization of these subcellular LDs still remains challenging due to insufficient fluorescence brightness and poor nuclear membrane permeability. Herein, we rationally synthesized a series of ultrabright solvatochromic fluorescent probes based on benzoboranils (BBAs) for LD-specific super-resolution imaging using structured illumination microscopy (SIM). The rigidly structured probes exhibit ultrahigh fluorescence quantum yields of up to 99.9% in low-polar solvents. They also show more significant fluorescence enhancements in lipid environments than commercial LD probes. Owing to these excellent merits, our lipophilic fluorescent probes can specifically light up subcellular LDs at ultralow concentrations down to 10 nM. Further use of BBA-CF3 for super-resolution SIM imaging of cytosolic and nuclear LDs and their fusion process was successfully achieved. The unprecedented spatial resolution for nuclear LDs with an FWHM value of 142 nm was also acquired. Collectively, our ultrabright fluorescent probes hold tremendous potential to unveil the mysterious roles of cytosolic and nuclear LDs in biological research using SIM.

A Solvatochromic Fluorescent Probe for Solvent Polarity Detection Using a Smartphone

AbstractA solvatochromic fluorescent probe (probe 1) with D‐π‐A molecular configuration was developed. Probe 1 exhibited excellent polarity sensitivity. The ET (30) value of solvents demonstrated a good linear relationship with the Stokes shift of probe 1 except for DMSO. Moreover, the linear correlation coefficient value reached 0.97. The probe demonstrated different colors based on the type of solvents. With the increase in solvent polarity, the color gradually changed from blue to earthy yellow. It has a linear functional relationship between the B value (Blue, the blue primary color of light in RGB) and ET (30) values of the solvent existed. It provided strong support for the rapid determination of solvent polarity. In addition, probe 1 can be used for the determination of alcohol levels in Baijiu by smartphone.

Ratiometric imaging of viral lipid order utilizing a solvatochromic fluorescent probe

The complex interplay between lipid phase structure of the host cells and nanoscale dynamics at the viral lipid envelope is considered critical for the pathogen’s ability to interact and fuse with the host-cell membrane. Despite decades of research, details about the exact role played by viral lipids in regulating its’ infectivity remain unknown. In particular, a clear mechanistic picture of viral lipid phase structure, and its impact on viral fusion is still lacking. This is partially owing to a dearth of techniques that reveal viral lipid organization in a holistic fashion.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells

AbstractWe report a crystallization‐induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely “AggRetina” probe, inherently binds to aggregated proteins and exhibits both a polarity‐dependent fluorescence emission wavelength shift and a viscosity‐dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein‐of‐interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro‐environment details inside misfolded and aggregated proteins may correlate to their bio‐chemical properties and pathogenicity.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells.

We report a crystallization-induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely "AggRetina" probe, inherently binds to aggregated proteins and exhibits both a polarity-dependent fluorescence emission wavelength shift and a viscosity-dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein-of-interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro-environment details inside misfolded and aggregated proteins may correlate to their bio-chemical properties and pathogenicity.

Fluorescence Aerosol Flow Tube Spectroscopy to Detect Liquid–Liquid Phase Separation

The phase behavior of atmospheric aerosol particles influences processes like gas-particle partitioning, solar light scattering, and cloud formation, ultimately affecting atmospheric air quality and climate. An important aspect of this phase behavior is whether an individual particle exists in a single homogeneous phase or undergoes liquid–liquid phase separation (LLPS). Herein, fluorescence aerosol flow tube (F-AFT) spectroscopy is developed to characterize LLPS in aerosolized submicron particles of 100–200 nm. A solvatochromic fluorescent probe molecule is incorporated into the particles. The link between its fluorescence emission and the local particle-phase chemical environment is used to determine the separation relative humidity (SRH) at which LLPS occurs. The LLPS behaviors of mixed organic/inorganic particles composed of polyethylene glycol (PEG), ammonium sulfate (AS), and sodium chloride (NaCl) are characterized. PEG/AS particles undergo LLPS at SRH values that vary with PEG composition. By comparison, PEG/NaCl particles continue as a single homogeneous phase to the RH of NaCl crystallization. The SRH values for the submicron particles are lower by >5% RH than those reported in the literature for supermicron particles deposited to substrate surfaces. Possible reasons for the differences are discussed, including kinetic and thermodynamic effects of system size and foreign substrate as well as observation time in the experimental apparatus.

Tracking lipid droplet dynamics for the discrimination of cancer cells by a solvatochromic fluorescent probe

Abstract High lipid metabolism is one of the fundamental characteristics of cancer cells different from normal cells due to lots of energy needed for the fast proliferation of cancer cells. As an important organelle to supply cellular energy, however, the difference of lipid droplets (LDs) in their dynamic features between cancer cells and normal cells remains unclear. A key difficulty is to develop a specific fluorescence-tunable probe with high signal-to-noise for rapidly and accurately identifying cancer cells via tracking lipid droplets’ dynamics. Here, we report a solvatochromic fluorescent probe, dansyl-morpholine (DNS-M), with high LDs-specificity to achieve the effective discrimination of cancer cells from normal cells by visually profiling the feature difference of LDs dynamics. Probe DNS-M is synthesized easily by one-step reaction, and possesses a large Stokes shift ∼200 nm, excellent photostablity and LDs-targeted capability with high overlap coefficient up to 0.97. Due to the remarkable biomembrance penetration, LDs-specificity and photostability, DNS-M has been applied to distinguish cancer cells from normal cells rapidly within 1 min in living cells and fixed cells, and real-time track dynamic behaviors of LDs in cancer cells, such as association, dissociation and fusion. More importantly, a significant difference in LDs behavior modes between cancer cells and normal cells is successfully visualized for the first time with our probe. We envision that our probe will become a rapid and easy-to-operate tool for cancer diagnosis and deeper understanding of LDs-related behaviors in cancer cells.

Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes.