Fluorescent dyes that are bright, stable, small, and biocompatible are needed for high-sensitivity two-photon imaging, but the combination of these traits has been elusive. We identified a class of ...squaraine derivatives with large two-photon action cross-sections (up to 10,000 GM) at near-infrared wavelengths critical for in vivo imaging. We demonstrate the biocompatibility and stability of a red-emitting squaraine-rotaxane (SeTau-647) by imaging dye-filled neurons in vivo over 5 days, and utility for sensitive subcellular imaging by synthesizing a specific peptide-conjugate label for the synaptic protein PSD-95.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Control over the strength of excitonic coupling in molecular dye aggregates is a substantial factor for the development of technologies such as light harvesting, optoelectronics, and quantum ...computing. According to the molecular exciton model, the strength of excitonic coupling is inversely proportional to the distance between dyes. Covalent DNA templating was proved to be a versatile tool to control dye spacing on a subnanometer scale. To further expand our ability to control photophysical properties of excitons, here, we investigated the influence of dye hydrophobicity on the strength of excitonic coupling in squaraine aggregates covalently templated by DNA Holliday Junction (DNA HJ). Indolenine squaraines were chosen for their excellent spectral properties, stability, and diversity of chemical modifications. Six squaraines of varying hydrophobicity from highly hydrophobic to highly hydrophilic were assembled in two dimer configurations and a tetramer. In general, the examined squaraines demonstrated a propensity toward face-to-face aggregation behavior observed via steady-state absorption, fluorescence, and circular dichroism spectroscopies. Modeling based on the Kühn–Renger–May approach quantified the strength of excitonic coupling in the squaraine aggregates. The strength of excitonic coupling strongly correlated with squaraine hydrophobic region. Dimer aggregates of dichloroindolenine squaraine were found to exhibit the strongest coupling strength of 132 meV (1065 cm–1). In addition, we identified the sites for dye attachment in the DNA HJ that promote the closest spacing between the dyes in their dimers. The extracted aggregate geometries, and the role of electrostatic and steric effects in squaraine aggregation are also discussed. Taken together, these findings provide a deeper insight into how dye structures influence excitonic coupling in dye aggregates covalently templated via DNA, and guidance in design rules for exciton-based materials and devices.
Novel pH-sensitive, water-soluble, long-wavelength fluorescent norsquaraine dyes and their barbituric and dicyanomethylene derivatives were synthesized and investigated. Some of these dyes contain a ...carboxylic functionality that was converted into an NHS ester to facilitate bio-conjugation. The absorption and emission spectra, fluorescence quantum yields, lifetimes, polarization, and photostabilities were measured free in solution and after binding to bovine serum albumin (BSA) and compared to those of conventional squaraines and the norcyanine dye CypHer5. Contrary to squaraines, norsquaraines are pH-sensitive but almost unaffected by the interaction with proteins. These dyes can potentially be used as fluorescent labels for biomedical applications, in particular for protein labeling, polarization-based assays, cell-based and pH-sensing measurements.
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•Novel pH-sensitive, water-soluble, fluorescent norsquaraine dyes were synthesized.•Squaric oxygen was substituted with a barbituric and dicyanomethylene group.•NHS esters of some of these dyes were synthesized to facilitate bio-conjugation.•Spectral and protolytic properties were investigated and compared to squaraines.•The dyes are suitable for protein labeling, polarization and pH-sensing assays.
Dye molecules that absorb light in the visible region are key components in many applications, including organic photovoltaics, biological fluorescent labeling, super-resolution microscopy, and ...energy transport. One family of dyes, known as squaraines, has received considerable attention recently due to their favorable electronic and photophysical properties. In addition, these dyes have a strong propensity for aggregation, which results in emergent materials properties, such as exciton delocalization. This will be of benefit in charge separation and energy transport along with fundamental studies in quantum information. Given the high structural tunability of squaraine dyes, it is possible that exciton delocalization could be tailored by modifying the substituents attached to the π-conjugated network. To date, limited theoretical studies have explored the role of substituent effects on the electronic and photophysical properties of squaraines in the context of DNA-templated dye aggregates and resultant excitonic behavior. We used
ab initio
theoretical methods to determine the effects of substituents on the electronic and photophysical properties for a series of nine different squaraine dyes. Solvation free energy was also investigated as an insight into changes in hydrophobic behavior from substituents. The role of molecular symmetry on these properties was also explored
via
conformation and substitution. We found that substituent effects are correlated with the empirical Hammett constant, which demonstrates their electron donating or electron withdrawing strength. Electron withdrawing groups were found to impact solvation free energy, transition dipole moment, static dipole difference, and absorbance more than electron donating groups. All substituents showed a redshift in absorption for the squaraine dye. In addition, solvation free energy increases with Hammett constant. This work represents a first step toward establishing design rules for dyes with desired properties for excitonic applications.
Squaraine dyes are candidates for DNA-templated excitonic interactions. This work presents substituent effects on the electronic and photophysicalproperties of squaraine dyes and a correlation between empirical Hammettconstant and those properties.
Molecular aggregates exhibit emergent properties, including the collective sharing of electronic excitation energy known as exciton delocalization, that can be leveraged in applications such as ...quantum computing, optical information processing, and light harvesting. In a previous study, we found unexpectedly large excitonic interactions (quantified by the excitonic hopping parameter
J
m
,
n
) in DNA-templated aggregates of squaraine (SQ) dyes with hydrophilic-imparting sulfo and butylsulfo substituents. Here, we characterize DNA Holliday junction (DNA-HJ) templated aggregates of an expanded set of SQs and evaluate their optical properties in the context of structural heterogeneity. Specifically, we characterized the orientation of and
J
m
,
n
between dyes in dimer aggregates of non-chlorinated and chlorinated SQs. Three new chlorinated SQs that feature a varying number of butylsulfo substituents were synthesized and attached to a DNA-HJ
via
a covalent linker to form adjacent and transverse dimers. Various characteristics of the dye, including its hydrophilicity (in terms of log
P
o/w
) and surface area, and of the substituents, including their local bulkiness and electron withdrawing capacity, were quantified computationally. The orientation of and
J
m
,
n
between the dyes were estimated using a model based on Kühn-Renger-May theory to fit the absorption and circular dichroism spectra. The results suggested that adjacent dimer aggregates of all the non-chlorinated and of the most hydrophilic chlorinated SQ dyes exhibit heterogeneity; that is, they form a mixture of dimers subpopulations. A key finding of this work is that dyes with a higher hydrophilicity (lower log
P
o/w
) formed dimers with smaller
J
m
,
n
and large center-to-center dye distance (
R
m
,
n
). Also, the results revealed that the position of the dye in the DNA-HJ template, that is, adjacent or transverse, impacted
J
m
,
n
. Lastly, we found that
J
m
,
n
between symmetrically substituted dyes was reduced by increasing the local bulkiness of the substituent. This work provides insights into how to maintain strong excitonic coupling and identifies challenges associated with heterogeneity, which will help to improve control of these dye aggregates and move forward their potential application as quantum information systems.
Molecular aggregates exhibit collective sharing of electronic excitation energy known as exciton delocalization, that can be leveraged in applications such as quantum computing, optical information processing, and light harvesting.
Molecular (dye) aggregates are a materials platform of interest in light harvesting, organic optoelectronics, and nanoscale computing, including quantum information science (QIS). Strong excitonic ...interactions between dyes are key to their use in QIS; critically, properties of the individual dyes govern the extent of these interactions. In this work, the electronic structure and excited-state dynamics of a series of indolenine-based squaraine dyes incorporating dimethylamino (electron donating) and/or nitro (electron withdrawing) substituents, so-called asymmetric dyes, were characterized. The dyes were covalently tethered to DNA Holliday junctions to suppress aggregation and permit characterization of their monomer photophysics. A combination of density functional theory and steady-state absorption spectroscopy shows that the difference static dipole moment (Δd) successively increases with the addition of these substituents while simultaneously maintaining a large transition dipole moment (μ). Steady-state fluorescence and time-resolved absorption and fluorescence spectroscopies uncover a significant nonradiative decay pathway in the asymmetrically substituted dyes that drastically reduces their excited-state lifetime (τ). This work indicates that Δd can indeed be increased by functionalizing dyes with electron donating and withdrawing substituents and that, in certain classes of dyes such as these asymmetric squaraines, strategies may be needed to ensure long τ, e.g., by rigidifying the π-conjugated network.
A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance ...energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.
We describe the photophysical properties of Seta-633, a commercially available near-infrared (NIR) dye, and its use as a fluorescent label to study the interaction between low-molecular-weight ...analytes and proteins using fluorescence lifetime as the readout parameter. In a model assay, we demonstrate that a biotinylated Seta-633 tracer binds to antibiotin with high specificity. Importantly, the lifetime of Seta-633−biotin increases about 1.8-fold upon binding to a specific antibody (antibiotin, MW = 160 kDa), while the titration with bovine serum albumin (BSA) or nonspecific antibody does not result in a noticeable change in lifetime. This behavior is contrary to that of fluorescent tracers like Cy5 or Alexa 647, which typically exhibit much smaller lifetime changes upon binding to antibodies.
The equilibrium between different tautomers that can be colored or colorless is an important feature for rhodamine dyes. Presently, this phenomenon is mostly discussed for rhodamine B. Herein, we ...studied the tautomerism and acid–base dissociation (HR+ ⇄ R + H+) of a set of rhodamines in organic media. Form R is an equilibrium mixture of the colored zwitterion R± and colorless lactone R0. Absorption spectra in 90 mass% aqueous acetone reflects the correlation between the dyes structure and the equilibrium constant, KT = R0/R±. Increase in the pKa value on transferring from water to organic solvents confirms the highly polar character of the R± tautomer. To reveal the role of the solvent nature, the tautomerism of an asymmetrical rhodamine, 2-(12-(diethyliminio)-2,3,5,6,7,12-hexahydro-1H-chromeno2,3-fpyrido3,2,1-ijquinolin-9-yl)benzoate, was examined in 14 media. This chain–ring tautomerism is an intramolecular acid–base reaction; the central carbon atom acts as a Lewis acid. The interaction with other Lewis acids, Li+, Ca2+, Mg2+, and La3+, results in rupture of lactone cycle. In polar solvents, lactones undergo photocleavage resulting in formation of highly fluorescent R±, whereas the blue fluorescence and abnormally high Stokes shift in low-polar media may be explained either by another photoreaction or by spiroconjugation and charge transfer in the exited state.
When molecules are aggregated such that their excited states form delocalized excitons, their spatial arrangement, or packing, can be coarsely controlled by templating and finely controlled by ...chemical substitution; however, challenges remain in controlling their packing on intermediate length scales. Here, we use an approach based on mechanically interlocked molecules to promote an elusive oblique packing arrangement in a series of three squaraine rotaxane dimers. We template the squaraine rotaxane dimers using DNA and observe two excitonically split bands of near‐equal intensity in their absorption spectra – a distinct signature of oblique packing, validated by theoretical modeling of the experimental results. Additional fine control of packing is demonstrated by fluorinating the macrocycle of the rotaxane, which promotes denser packing and stronger excitonic interactions.
Multiple levels of control: It is possible to consistently obtain an elusive oblique packing arrangement by using DNA to bring squaraine rotaxanes into close proximity. Further fine tuning excitonic interactions is possible via synthetic modifications of the squaraine rotaxanes.