BODIPY Dyes Research Articles

Meso-pyridinium substituted BODIPY dyes as mitochondria-targeted probes for the detection of cysteine in living cells and in vivo

Cysteine (Cys) is a crucial bio-thiol attributing to its significant role in many physiological and pathological processes and maintaining redox dynamic balance. In mitochondria, ROS can cause oxidative damage, and Cys can solve this problem by acting as an antioxidant. Herein, we designed and synthesized a series of meso-pyridin and meso-pyridinium substituted BODIPY dyes as fluorescent probes to distinguish Cys. Through screening the responsive behaviors of the all probes toward biothiols, we obtained an appropriate mitochondria-targeted probe BDP-S-o-Py+ for Cys-specific detection. Density functional theory (DFT) calculations were used to demonstrate the different reactivity of the probes. BDP-S-o-Py+ could detect Cys with fast response under biological environment. The probe BDP-S-o-Py+ had a low limit of detection (LOD, 72 nM) at the linear dynamic range from 0 to 50 μM. In particularly, BDP-S-o-Py+ could be used for imaging of mitochondrial Cys in living cells, and was capable of monitoring endogenous Cys in vivo, successfully.

Quantitative Spatiotemporal Mapping of Lipid and Protein Oxidation in Mayonnaise.

Lipid oxidation in food emulsions is mediated by emulsifiers in the water phase and at the oil–water interface. To unravel the physico-chemical mechanisms and to obtain local lipid and protein oxidation rates, we used confocal laser scanning microscopy (CLSM), thereby monitoring changes in both the fluorescence emission of a lipophilic dye BODIPY 665/676 and protein auto-fluorescence. Our data show that the removal of lipid-soluble antioxidants from mayonnaises promotes lipid oxidation within oil droplets as well as protein oxidation at the oil–water interface. Furthermore, we demonstrate that ascorbic acid acts as either a lipid antioxidant or pro-oxidant depending on the presence of lipid-soluble antioxidants. The effects of antioxidant formulation on local lipid and protein oxidation rates were all statistically significant (p < 0.0001). The observed protein oxidation at the oil–water interface was spatially heterogeneous, which is in line with the heterogeneous distribution of lipoprotein granules from the egg yolk used for emulsification. The impact of the droplet size on local lipid and protein oxidation rates was significant (p < 0.0001) but minor compared to the effects of ascorbic acid addition and lipid-soluble antioxidant depletion. The presented results demonstrate that CLSM can be applied for unraveling the roles of colloidal structure and transport in mediating lipid oxidation in complex food emulsions.

Mechanochemistry as a Sustainable Method for the Preparation of Fluorescent Ugi BODIPY Adducts

AbstractMechanochemistry, that is, the use of mechanical energy produced by milling or grinding to perform chemical transformations, was used as a green strategy using a minimal amount of solvent for the preparation of highly valuable formyl functionalized BODIPY dyes. The electronic properties of the meso‐aryl group play a key role in the fluorescence response of the adducts resulting from the Ugi reaction. Such an understanding of the molecular structure‐fluorescence interplay explains the brightness of these dyes in the cellular media monitored by fluorescence imaging. In the present report, we provide flow cytometry and fluorescence microscopy evidence that the novel dyes efficiently stain peripheral blood mononuclear cells (PBMCs) and Candida albicans cells without disturbing cell integrity. Moreover, it is demonstrated that by staining C. albicans cells, we can monitor the early and late steps of phagocytosis, using human monocyte‐derived macrophages. The dye series presented herein represent a novel application for formyl functionalized BODIPYs, expanding their applications in monitoring biological processes.

Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa.

In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

Rational molecular design of a reversible BODIPY-Based fluorescent probe for real-time imaging of GSH dynamics in living cells

Marring the reversible covalent chemistry with BODIPY dye, which is a superfamily of fluorophores with striking photophysical performances, would enable a panel of diverse dynamic fluorescent probes for biomedical applications. Herein we show that structural manipulation of BODIPY allows rational tuning of α-site or meso-site activation as well as the spectral response toward nucleophiles. By rational molecular design, we have obtained a highly specific and reversible GSH probe, αBD-GSH, which exhibits a tremendously fast and dynamic fluorescence response within the wide physiological GSH concentration range of 0–8 mM. We successfully applied αBD-GSH to real-time imaging of intracellular GSH dynamics in different cell lines. In light of the remarkable photophysical properties and synthesis flexibility of BODIPY dyes, the current findings will help to design more reversible BODIPY-based fluorescent probes targeting various bio-species.

Optimization of high-throughput lipid screening of the microalga Nannochloropsis oceanica using BODIPY 505/515

BackgroundMicroalgae are considered a promising platform for sustainable lipid production. Despite this, productivities need to be improved to facilitate an economically viable production process. This can be achieved through strain improvement, for instance by genetic engineering. Strain improvement strategies often deploy high-throughput screening platforms i.a. involving single-cell methodologies such as fluorescence-activated cell sorting (FACS) for the identification and isolation of better-performing strains. The heterokont microalga Nannochloropsis is a prospective candidate for the industrial production of lipids. Previous studies have reported the isolation of high lipid-producing Nannochloropsis strains by combining qualitative staining of lipid bodies using the fluorophoric dye BODIPY with FACS methodology. However, it has never been investigated how cellular physiology and different staining conditions hamper the reproducibility of this method as a quantitative screening procedure. ResultsHere we report the development of an optimized single cell lipid screening procedure for Nannochloropsis oceanica. Systematic assessment of different staining conditions revealed that treatment with 6% DMSO and 1.2 μg ml−1 BODIPY for 15 min is ideal for staining neutral lipids in an exponentially growing culture of N. oceanica. Cultures that are overproducing lipids, for example after exposure to external stimuli such as nutrient deprivation stress, require treatment with 10% DMSO and 1.2–1.6 μg ml−1 BODIPY for 36 min to facilitate complete staining of lipid bodies. We verify that DMSO is required to permeabilize the particularly tough cell barrier of Nannochloropsis and we show that exposure to 10% DMSO does not affect cell viability. Increasing concentrations of BODIPY, however, correlated with a decrease in viability when screening stressed cultures. Using the optimized protocol, reproductive viabilities can be expected to be ~91% and 83–82% for non-stressed and stressed cultures respectively. The optimized procedure allows for a quantitative prediction of cellular neutral lipids (R2= 0.981), as determined by comparison to results obtained through a reference procedure.

Insight into the Influence of the Chiral Molecular Symmetry on the Chiroptics of Fluorescent BINOL-Based Boron Chelates.

The prominent influence of the molecular symmetry, as defined by the symmetry point group, on the chiroptical behavior (electronic circular dichroism and, especially, circularly polarized luminescence) of simple fluorescent boron chelates (BODIPY and related BOPHY analogues) is studied and discussed. It is shown that increasing the dye symmetry by means of the D3 chiral symmetry group is a workable design option to enhance the level of differential emission of right- and left-circularly polarized light in BODIPY dyes and related emitters, and that the influence of the level of symmetry is stronger than the influence of the higher number of chiral moieties perturbing the acting achiral chromophore.

Probing the dye-semiconductor interface in dye-sensitized NiO solar cells.

The development of p-type dye-sensitized solar cells (p-DSSCs) offers an opportunity to assemble tandem photoelectrochemical solar cells with higher efficiencies than TiO2-based photoanodes, pioneered by O'Regan and Grätzel [Nature 353, 737-740 (1991)]. This paper describes an investigation into the behavior at the interfaces in p-DSSCs, using a series of BODIPY dyes, BOD1-3. The three dyes have different structural and electronic properties, which lead to different performances in p-DSSCs. We have applied photoelectron spectroscopy and transient absorption spectroscopy to rationalize these differences. The results show that the electronic orbitals of the dyes are appropriately aligned with the valence band of the NiO semiconductor to promote light-induced charge transfer, but charge-recombination is too fast for efficient dye regeneration by the electrolyte. We attribute this fast recombination, which limits the efficiency of the solar cells, to the electronic structure of the dye and the presence of Ni3+ recombination sites at the NiO surface.

Monitoring the formation of insulin oligomers using a NIR emitting glucose-conjugated BODIPY dye

Protein oligomers, which are formed due to the aggregation of protein molecules under physiological stress, are neurotoxic and responsible for several neurological diseases. Early detection of protein oligomers is essential for the timely intervention in the associated diseases. Although several probes have been developed for the detection of insoluble matured protein fibrils, fluorescent probes with emission in the near infrared (NIR) region for probing protein oligomers are very rare. In the present study we have designed and synthesized a glucose-conjugated BODIPY dye with emission in the NIR spectral range. Our detailed studies show that the new probe is not only capable of detecting matured fibrils but can also probe the formation of oligomers from the native protein. The new probe shows a large increase in its emission intensity upon association with oligomers and matured fibrils. Hence, the present probe has a great potential for the in vivo imaging of protein oligomers and matured fibrils. Detailed spectroscopic properties of the new probes in molecular solvents have been performed to understand its oligomers- and fibril- sensing mechanism.

A novel water-soluble BODIPY dye as red fluorescent probe for imaging hypoxic status of human cancer cells

The development of effective methods for real-time monitoring of nitroreductase (NTR) activity is of great significance for medical diagnosis and cancer research. Here, we present a novel water-soluble BODIPY-based chemodosimeter (NBB) for imaging the hypoxic status of human non-small-cell lung cancer A549 cells. We assume that ‘on–off’ response of NBB is activated by the NTRmediated reductive release of meso-phenol BODIPY dye resulting in unusual fluorescence.

Theoretical predictions of the spectroscopic properties of BODIPY dyes: Effects of the fused aromatic and heteroaromatic rings at the b, g bonds.

The fusion of aryl and heteroaryl moieties in the BODIPY skeleton is the important route to design BODIPYs exhibiting absorption and fluorescence in red or near infra-red regions. Despite the importance of this approach, little is known about the influence of aryl and heteroaryl rings on the position of the energy levels of the lower electronic states of such dyes. In this paper, the influence of benzene (B), thiophene (T) and furan (F) rings fused in the BODIPY core (b) at both b and g bonds on the optical properties of the symmetric BbB, TbT and FbF and asymmetric TbF, BbF and BbT dyes have been investigated by TD-CAM-B3LYP and RI-CC2 calculations. We have shown that in contrast to the locally excited (LE0-type) main vertical state, energy of which is nearly the same for all the substituents at the b and g bonds, energy of the next two states of the LE1- and LE2-type for the symmetric dyes or the charge transfer (CT1- and CT2-type) for the asymmetric dyes depends strongly on the nature of the rings at the b and g bonds. The benzene-fused dyes (BbF, BbT, BbB) have the lowest energy of the excited levels of the LE-type (or CT1-type) states, and inversion with LE0-type levels occurs. As a result, the dramatic decrease of the fluorescence quantum yield is expected in these cases. The identification of these effects provides a quality framework for understanding of how fused substituents regulate photophysical processes of BODIPYs.

Co-staining of KCa 3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence.

The Ca2+ activated potassium channel 3.1 (KCa3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole‐based BODIPY dye with a derivative of the KCa3.1 channel inhibitor senicapoc via linkers of different length. Patch‐clamp experiments revealed the inhibition of KCa3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa3.1 channels in non‐small‐cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre‐incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody‐based indirect immunofluorescence yielded identical or very similar densities of stained KCa3.1 channels. However, co‐staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.

Stealth and Bright Monomolecular Fluorescent Organic Nanoparticles Based on Folded Amphiphilic Polymer.

Fluorescent nanoparticles (NPs), owing to their superior brightness, are an attractive alternative to organic dyes. However, their cellular applications remain limited because of their large size, poor homogeneity, and nonspecific interactions in biological media. Herein, we propose a concept of monomolecular fluorescent organic nanoparticles of high brightness and very small size (10-14 nm) built of a single amphiphilic polymer bearing specially designed fluorescent dyes. We found that high PEGylation of poly(maleic anhydride-alt-1-octadecene (PMAO) favors a single-chain polymer folding into monomolecular stealth NPs with highly reduced nonspecific interactions with proteins and live cells. To ensure high stability of our NPs, the fluorophores (BODIPYs) are covalently linked to the polymer through an optimized linker. Among tested linkers of different lengths and polarity, a short medium-polar linker favoring location of the dyes at NPs interface ensures good fluorescence quantum yield and small particle size. The fluorescence brightness of these NPs has been dramatically enhanced by increasing the bulkiness of the BODIPY dyes that prevents their H-aggregation, reaching 2500000 M-1 cm-1 (extinction coefficient × quantum yield). Fluorescence microscopy revealed that the single-particle brightness of these NPs is ∼5-fold higher than that of QDot-585 using the same excitation wavelength (532 nm). Finally, when microinjected inside cells, these small and stealth NPs (10 nm diameter) distribute more evenly than 20 nm QDots inside the cytosol, showing similar spreading as a fluorescent protein. Thus, the developed monomolecular NPs, owing to their small size and stealth properties, are artificial analogues of fluorescent proteins, surpassing the latter >50-fold in terms of brightness.

Graphenic substrates as modifiers of the emission and vibrational responses of interacting molecules: The case of BODIPY dyes.

Graphenic substrates (GS), such as reduced graphene oxide (rGO) and graphene oxide (GO), are 2D materials known for their unique physicochemical properties such as their ability to enhance vibrational spectroscopic signals and quench the fluorescence of adsorbed molecules. These properties provide an opportunity to develop nanostructured GS-based systems for detecting and identifying different analytes with high sensitivity and reliability through molecular spectroscopic techniques. This work evaluated the capacities of different GS to interact with a highly fluorescent compound, thereby changing its optical emission response (fluorescence quenching) and amplifying its vibrational signal, which is the base of graphene-enhanced Raman scattering (GERS). To test these properties, we used a derivative of highly fluorescent BODIPY (BP) compounds, which cover a wide range of applications from solar energy conversion to photodynamic cancer therapy. GS prepared by using the Langmuir-Blodgett (LB) technique allowed us to quench the fluorescence emission of BP and improve its Raman spectroscopy detection limit due to the GERS effect. These results were interpreted in light of the π-π interactions taking place between the Csp2 domains of GS and the aromatic core of the BP fluorophore.

Modulating the Optical Properties of BODIPY Dyes by Noncovalent Dimerization within a Flexible Coordination Cage.

Aggregation of organic molecules can drastically affect their physicochemical properties. For instance, the optical properties of BODIPY dyes are inherently related to the degree of aggregation and the mutual orientation of BODIPY units within these aggregates. Whereas the noncovalent aggregation of various BODIPY dyes has been studied in diverse media, the ill-defined nature of these aggregates has made it difficult to elucidate the structure–property relationships. Here, we studied the encapsulation of three structurally simple BODIPY derivatives within the hydrophobic cavity of a water-soluble, flexible PdII6L4 coordination cage. The cavity size allowed for the selective encapsulation of two dye molecules, irrespective of the substitution pattern on the BODIPY core. Working with a model, a pentamethyl-substituted derivative, we found that the mutual orientation of two BODIPY units in the cage’s cavity was remarkably similar to that in the crystalline state of the free dye, allowing us to isolate and characterize the smallest possible noncovalent H-type BODIPY aggregate, namely, an H-dimer. Interestingly, a CF3-substituted BODIPY, known for forming J-type aggregates, was also encapsulated as an H-dimer. Taking advantage of the dynamic nature of encapsulation, we developed a system in which reversible switching between H- and J-aggregates can be induced for multiple cycles simply by addition and subsequent destruction of the cage. We expect that the ability to rapidly and reversibly manipulate the optical properties of supramolecular inclusion complexes in aqueous media will open up avenues for developing detection systems that operate within biological environments.

Photooxidation mediated by 11-cis and all-trans retinal in single isolated mouse rod photoreceptors.

Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.

A ratiometric electrochemiluminescence resonance energy transfer platform based on novel dye BODIPY derivatives for sensitive detection of lactoferrin

A ratiometric electrochemiluminescence resonance energy transfer (ECL-RET) platform depended on novel dye BODIPY derivatives was proposed for rapid detection of lactoferrin. This ECL-RET platform is composed of aptamer decorated BODIPY composites and C60@BSA, in which BODIPY derivative is the ECL probe and can generate significant resonance energy transfer with K2S2O8. BODIPY derivative and K2S2O8 are used as built-in reference signal and calibration signal respectively to eliminate background signal and abnormal change signal by double signal self-calibration process. At the same time, C60, as the accelerator of K2S2O8, can effectively increase the ECL signal and further transfer as much energy as possible to BODIPY derivative. Under optimal conditions, the constructed ECL-RET platform exhibited sensitive detection of lactoferrin in the wide linear range of 10-4- 850 ng/mL with a LOD of 42 fg/mL. Meanwhile, the proposed ECL-RET aptasensor demonstrated superior stability, specificity and reproducibility, displaying favorable application value in practical diagnosis of this method.

A Photochemical Dehydrogenative Strategy for Direct and Regioselective Dimerization of BODIPY Dyes.

By taking advantage of their intense visible-light absorptions, a photoinduced, AgI promoted oxidative dimerization of BODIPY dyes was developed to give structurally unprecedented α,α- and α,γ-linked dimers. In contrast to classical oxidative aromatic coupling, this methodology does not need a strong oxidant and relies on the singlet electron transfer process between excited-state BODIPYs and an electron acceptor to generate BODIPY based radical species.

Exploring the optical and nonlinear optical features of heteroleptic complexes with BODIPY and amido-BODIPY substitutions; A comparative theoretical study

In this study, special attention has been paid to understanding the structural effect on electronic structure, absorption spectra and nonlinear optical (NLO) response for a series of heteroleptic diimine dithiolate complexes [M(bpy)(bdt)] (M = Ni, Pd and Pt; bpy = 2,2′-bipyridine, bdt = 1,2-benzenedithiolate) attached to BODIPY and amido-BODIPY dyes through density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods. Natural bond orbital (NBO) analyses are also performed for scrutinizing the structural properties of the considered heteroleptic complexes. The calculated energy gaps show that the substitution of BODIPY or amido-BODIPY groups on the diimine ligand (bpy) leads to reduce of HLG. In contrast, these substitutions on the dithiolate ligand (bdt) are slightly increased energy gap. Moreover, the entitled substitutions at the diimine position can be red-shifted, and the expansion of absorption range to the near-IR region. It is interesting to note that the BODIPY and amido-BODIPY substitutions dramatically increase the static first hyperpolarizability (β0) value. The results suggest that structural modifications on the diimine and dithiolate ligands using BODIPY and amido-BODIPY dyes can finely tune their linear and nonlinear optical properties.

Lipid Peroxidation Assay Using BODIPY-Phenylbutadiene Probes: A Methodological Overview.

The assessment of reactive oxygen species has increasing importance in biomedical sciences, due to their biological role in signaling pathways and induction of cell damage at low and high concentrations, respectively. Detection of lipid peroxidation with sensing probes such as some BODIPY dyes has now wide application in studies using fluorescent microplate readers, flow cytometry, and fluorescence microscopy. Two phenylbutadiene derivatives of BODIPY are commonly used as peroxidation probes, non-oxidized probes and oxidized products giving red and green fluorescence, respectively. Peculiar features of lipoperoxidation and BODIPY dye properties make this assessment a rather complex process, not exempt of doubts and troubles. Color changes and fluorescence fading that are not due to lipid peroxidation must be taken into account to avoid misleading results. As a characteristic feature of lipoperoxidation is the propagation of peroxyl radicals, pitfalls and advantages of a delayed detection by BODIPY probes should be considered.