The lipophilic nature of organic dyes complicates their effectiveness in aqueous solutions. In this work we investigate three different strategies for achieving water‐solubility of the ...diazaoxatriangulenium (DAOTA+) chromophore: hydrophilic counter ions, aromatic sulfonation of the chromophore, and attachment of charged side chains. The long fluorescence lifetime (FLT, τf=20 ns) of DAOTA+ makes it a sensitive probe to analyze solvation and aggregation effects. Direct sulfonation of the chromophore was found to increase solubility drastically, but at the cost of greatly reduced quantum yields (QYs) due to enhanced non‐radiative deactivation processes. The introduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τf=18 ns) and QY (ϕf=0.56) of the dye to the same level as the parent chromophore in acetonitrile. Time‐resolved fluorescence spectroscopy also reveals a high resistance to aggregation and non‐specific binding in a high loading of bovine serum albumin (BSA). The results clearly show that addition of charged flexible side chains is preferable to direct sulfonation of the chromophore core.
Aqueous solvation of fluorescent triangulenium dyes was made possible by functionalization with charged side chains and pairing with hydrophilic counterions. Mono‐exponential fluorescence decays with fluorescence lifetimes over 18 ns in aqueous solution resulted. Fluorescence properties were largely unaffected by the presence of 600 μm bovine serum albumin in buffered solutions.
The design of aqueous probes and binders for complex, biologically relevant anions presents a key challenge in supramolecular chemistry. Herein, a tetrahedral assembly with cationic faces and corners ...is reported that is capable of discriminating between anionic and neutral guests in water. Electrostatic repulsion between subcomponents can be overcome by the addition of an anionic template, or generating a robust covalent framework by incorporating tris(2‐aminoethyl)amine (TREN). The resultant TREN‐capped, water‐soluble, fluorescent cage binds mono‐ and poly‐phosphoric esters, including nucleotides. Its covalent skeleton renders it stable at micromolar concentrations in water, enabling the fluorometric detection of biologically relevant guests in an aqueous environment. Selective supramolecular encapsulants, such as 1, could enable new sensing applications, such as recognition of toxins and drugs, under biological conditions.
Think positive: A fluorescent 12+ metal–organic cage is reported that binds guests in water at low concentrations, selectively binding anions over their neutral congeners. A variety of biologically relevant anions can be bound in aqueous environments, providing a platform for the design and use of large, hollow cages under biological conditions.
Fluorescent nanoparticles have become attractive for bioanalysis and imaging, due to their high brightness and photostability. Many different optical materials have been applied in fluorescent ...nanoparticles with a broad range of properties and characteristics. One appealing approach is the incorporation of molecular organic fluorophores in nanoparticles with the intention of transferring their known attractive solution-state properties directly to the nanoparticles. However, as molecular dyes are packed closely together in the nanoparticles their interactions most often result in fluorescence quenching and change in spectral properties making this approach challenging. In this perspective we will first discuss the origins of quenching and spectral shifts observed in dye based nanoparticles. On this background, we will then describe various designs of dye based NPs and how they address the challenges of dye-dye interactions and quenching. Our aim is to provide a general framework for understanding the supramolecular mechanisms that determine the photophysics of dye based nanoparticles. This framework of molecular photophysics and its relation to the internal structure of dye based nanoparticles can hopefully serve to assist rational design and optimization of new and improved dye based nanoparticles.
Fluorescent dye based nanoparticles have high potential for many applications. Here we highlight key photophysical challenges and design principles to keep in mind in the search for new brightly fluorescent nanoparticles.
The synthesis of an antiaromatic tetraoxa8circulene annulated with four perylene diimides (PDI), giving a dynamic non‐planar π‐conjugated system, is described. The molecule contains 32 aromatic rings ...surrounding one formally antiaromatic planarized cyclooctatetraene (COT). The intense absorption (ϵ=3.35×105 M−1 cm−1 in CH2Cl2) and emission bands are assigned to internal charge‐transfer transitions in the combined PDI‐circulene π‐system. The spectroscopic data is supported by density functional theory calculations, and nuclear independent chemical shift calculation indicate that the antiaromatic COT has increased aromaticity in the reduced state. Electrochemical studies show that the compound can reversibly reach the tetra‐ and octa‐anionic states by reduction of the four PDI units, and the deca‐anionic state by reduction of the central COT ring. The material functions effectively in bulk hetero junction solar cells as a non‐fullerene acceptor, reaching a power conversion efficiency of 6.4 %.
Annelating the antiaromatic tetraoxa8circulene with four perylenediimide units yields an intensely absorbing and emissive charge‐transfer molecule. The aromatic/antiaromatic character of neutral and charged species and the incorporation in bulk hetero junction solar cell (power conversion efficiency of 6.4 %) is described.
Donor‐Acceptor Stenhouse Adduct (DASA), a class of push‐pull negative photochrome, has received large interest lately owing to its versatile synthesis, modularity and excellent photoswitching in ...solutions. From a technological perspective, it is imperative for this class of photoswitches to work robustly in solid state, e. g. thin films. We feature a molecular framework for the optimized design of DASAs by introducing a new thioindoline donor (D3) and assessing its performance against known 2nd generation indoline‐based donors. The systematic structure‐function investigations suggest that to achieve robust reversible photoswitching, a ground state with low charge separation is desired. DASAs with stronger electron donors and a larger charge separation in the ground state result in a low population of the photothermalstationary state (PTSS) and reduced photostability. The DASA with thioindoline donor (D3A3) seems to be a special case among the donor series as it causes a red shift (ca. 15 nm), however with less polarization of the ground state and marginally better photostability as compared to the unsubstituted 2‐methyl indoline (D1A3). We also emphasize the consideration of the key additional factors that can modulate the red‐light photoswitching properties of DASA chromophores in polymer thin films, which might not be dominant in homogenous solution state.
Structure‐property guidelines are disclosed for creating robust donor‐acceptor Stenhouse adduct (DASA) photochromes. A comprehensive investigation has revealed that a DASA photochrome with a low degree of charge separation is desired to achieve a robust, reversible photoswitching. Other indirect factors that could modulate the DASA photoswitching in polymer thin films are also discussed in detail.
Raman probes have received growing attention for their potential use in super-multiplex biological imaging and flow cytometry applications that cannot be achieved using fluorescent probes. However, ...obtaining strong Raman scattering signals from small Raman probes has posed a challenge that holds back their practical implementation. Here, we present new types of Raman-active nanoparticles (Rdots) that incorporate ionophore macrocycles, known as cyanostars, to act as ion-driven and structure-directing spacers to address this problem. These macrocycle-enhanced Rdots (MERdots) exhibit sharper and higher electronic absorption peaks than Rdots. When combined with resonant broadband time-domain Raman spectroscopy, these MERdots show a ∼3-fold increase in Raman intensity compared to conventional Rdots under the same particle concentration. Additionally, the detection limit on the concentration of MERdots is improved by a factor of 2.5 compared to that of Rdots and a factor of 430 compared to that of Raman dye molecules in solution. The compact size of MERdots (26 nm in diameter) and their increased Raman signal intensity, along with the broadband capabilities of time-domain resonant Raman spectroscopy, make them promising candidates for a wide range of biological applications.
Functionalization of new sites on the triangulenium structure has been achieved by early-stage chlorination with N-chlorosuccinimide (NCS), giving rise to two new triangulenium dyes (1 and 3). By ...introducing the chlorine functionalities in the acridinium precursor, positions complementary to those previously obtained by electrophilic aromatic substitution on the final dyes are accessed. The chlorination is selective, giving only one regioisomer for both mono- and dichlorination products. For the monochlorinated acridinium compound, a highly selective ring-closing reaction was discovered, generating a single regioisomer of the cationic 4helicene product. Further investigations into the mechanism of the 4helicene formation lead to the first isolation of the previously proposed intermediate of the two-step SNAr reaction, key to all aza-bridged triangulenium and helicenium systems. Late-stage functionalization of DAOTA+ with NCS gave rise to a different dichlorinated compound (2). The fully ring closed chlorinated triangulenium dyes 1, 2, and 3 show a redshift in absorption and emission, while maintaining relatively high fluorescence quantum yields of 36%, 26%, and 41% and long fluorescence lifetimes of 15, 12.5, and 16 ns, respectively. Cyclic voltammetry shows that chlorination of the triangulenium dyes significantly lowers reduction potentials and thus allows for efficient tuning of redox and photoredox properties.
Increasing demand for detecting single molecules in challenging environments has raised the bar for the fluorophores used. To achieve better resolution and/or contrast in fluorescence microscopy, it ...is now essential to use bright and stable dyes with tailored photophysical properties. While long fluorescence lifetime fluorophores offer many advantages in time-resolved imaging, their inherently lower molar absorption coefficient has limited applications in single molecule imaging. Here we propose a generic approach to prepare bright, long fluorescence lifetime dyad fluorophores comprising an absorbing antenna chromophore with high absorption coefficient linked to an acceptor emitter with a long fluorescence lifetime. We introduce a dyad consisting of a perylene antenna and a triangulenium emitter with 100% energy transfer from donor to acceptor. The dyad retained the long fluorescence lifetime (∼17 ns) and high quantum yield (75%) of the triangulenium emitter, while the perylene antenna increased the molar absorption coefficient (up to 5 times) in comparison to the free triangulenium dye. These triangulenium based dyads with significantly improved brightness can now be detected at the single molecule level and easily discriminated from bright autofluorescence by time-gated and other lifetime-based detection schemes.
Inhibitor protonation of azasugars of the isofagomine type when bound to enzyme can be investigated using photon induced electron transfer (PET) quenching of an attached fluorophore. For this ...purpose, Isofagomine, iso‐d‐galacto‐fagomine, and iso‐l‐gulo‐fagomine were converted to N‐(10‐chloroanthracenenyl‐9‐alkyl) derivatives where the alkyl group contained one, two, or three methylene groups. The new derivatives displayed pH dependent fluorescence; as the ammonium forms they were fluorescent, while 90–99 % of the fluorescence was quenched in the amine forms. The 3 isofagomine derivatives were competitive inhibitors of T. Maritima Ι‐glucosidase with Ki values from 0.37–4.6ΤM. Similarly, the iso‐d‐galacto‐fagomines inhibited A. Niger Ι‐galactosidase with Ki values from 63–2000 nm. When bound to the enzymes the inhibitors displayed between 1–15 % fluorescence.
To check the pH conditions in the active site of an enzyme an inhibitor with on/off fluorescence upon protonation is required. Herein, the potent glycosidase inhibitor isofagomine is converted into “glycosidase fluorescence indicator”.
Fluorescent dye based nanoparticles (NPs) have received increased interest due to their high brightness and stability. In fluorescence microscopy and assays, high signal to background ratios and ...multiple channels of detection are highly coveted. To this end, time-resolved imaging offers suppression of background and temporal separation of spectrally overlapping signals. Although dye based NPs and time-resolved imaging are widely used individually, the combination of the two is uncommon. This is likely due to that dye based NPs in general display shortened and non-mono-exponential lifetimes. The lower quality of the lifetime signal from dyes in NPs is caused by aggregation caused quenching (ACQ) and energy migration to dark states in NPs. Here, we report a solution to this problem by the use of the small-molecule ionic isolation lattices (SMILES) concept to prevent ACQ. Additionally, incorporation of FRET pairs of dyes locks the exciton on the FRET acceptor providing control of the fluorescence lifetime. We demonstrate how SMILES NPs with a few percent rhodamine and diazaoxatriangulenium FRET acceptors imbedded with a cyanine donor dye give identical emission spectra and high quantum yields but very different fluorescence lifetimes of 3 ns and 26 ns, respectively. The two spectrally identical NPs are easily distinguished at the single particle level in fluorescence lifetime imaging. The doping approach for dye based NPs provides predictable fluorescence lifetimes and allows for these bright imaging reagents to be used in time-resolved imaging detection modalities.
FRET nanoparticles assembled with small-molecule ionic isolation lattices (SMILES) provide control of both emission wavelength and lifetime. This allows simple design of dye based nanoparticles for fluorescence lifetime microscopy.
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