One Stone, Three Birds: pH Triggered Transformation of Aminopyronine and Iminopyronine Based Lysosome Targeting Viscosity Probe for Cancer Visualization.

The lysosomes of cancer cells have lower pH and higher viscosity than those of normal cells. These features can be used as sensitive and selective markers for cancer diagnosis. In this work, a pH and viscosity dual responsive lysosome targeting fluorescent probe 1 was designed based on the transformation of amino- and imino- forms of pyronine and the twisted intramolecular charge shuttle (TICS) sensing mechanism. Live cancer cells and tumors were effectively distinguished from normal cells and organs through fluorescence imaging of probe 1, which indicated that probe 1 could serve as an effective tool for visualization of tumors at organ level with high selectivity.

Three-Dimensional Microporous Hollow Fiber Membrane Microfluidic Device Integrated with Selective Separation and Capillary Self-Driven for Point-of-Care Testing.

The novel 3D microfluidic concept of "lab-on-hollow fiber membrane (HFM)" was presented for multifunctional and rapid biological assays, integrating sample size sieving and colorimetric quantification in an HFM. Herein, microporous HFMs with a gradient pore size and high hydrophilic flux were used as microfluidic device substrates. The membrane pores selectively trapped macromolecules within the inner surface, while allowing free diffusion of smaller molecules, including glucose and protein. The microfluidic flow rate in HFM closely followed the Lucas-Washburn and Laplace's models, indicating that the microfluidics facilitated the upward flow of the fluid by microcapillary action without external pumping. Subsequently, for sensing of different biomolecules, a highly sensitive fluorescent or optical chromogenic reagent was immobilized in HFM by an electrostatic interaction. Pyronin G fluorescence reagent was quenched by blood glucose, and the quenching efficiency showed a good linear correlation with glucose concentration (1-22 mM, R2 = 0.997). Moreover, this sensing platform was then further applied for the analysis of urine protein or glucose in the visible spectrum, with a wide testing range. Compared to traditional 2D flat membrane devices, this 3D-HFM microfluidic device exhibited excellent sensing versatility and color rendering uniformity with enhanced sensitivity. Target molecules screening, conditioning, enzymatic recognition, and signal readout of biomolecules have all been implemented on this device, which has paved the way to highly sensitive assays on point-of-care testing (POCT).

Development of a Next-Generation Fluorescent Turn-On Sensor to Simultaneously Detect and Detoxify Mercury in Living Samples.

Strategies for simultaneous detection and detoxification of Hg2+ using a single sensor from biological and environmental samples are limited and have not been realized in living organisms so far. We report a highly selective, small molecule "turn-on" fluorescent sensor, PYDMSA, based on the cationic dye Pyronin Y (PY) and chelating agent meso-2,3-dimercaptosuccinic acid (DMSA) for the simultaneous detection and detoxification of inorganic mercury (Hg2+). After Hg2+ detection, concomitant detoxification was carried out with sufficient efficacy in living samples, which makes the sensor unique. PYDMSA exhibits high selectivity for Hg2+ over other competing metal ions with an experimental detection limit of ∼300 pM in aqueous buffer solution. When PYDMSA reacts with Hg2+, the CS-C9 bond in the sensor gets cleaved. This results in the "turn-on" response of the fluorescence probe with a concomitant release of one equivalent of water-soluble Hg2+-DMSA complex which leads to a synchronous detoxifying effect. The sensor by itself is nontoxic to cells in culture and has been used to monitor the real-time uptake of Hg2+ in live cells and zebrafish larvae. Thus, PYDMSA is a unique sensor which can be used to detect and detoxify mercury at the same time in living samples.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

Photophysical and photodynamic properties of Pyronin Y in micellar media at different temperatures.

In this study, photophysics and photodynamical properties of Pyronin Y (PyY) in different liquid media were investigated. Interactions of PyY, which is a positively charged pigment compound pertaining to the xanthene derivatives with surfactants possessing distinct charges, were determined by using the molecular absorption and fluorescence spectroscopy techniques. It was observed that band intensities of absorption and fluorescence spectra belonging to PyY increase in proportion to the water when compared to three micelle systems, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X-100 (TX-100). This suggests that interactions in micelle systems are different from those in deionized water, and solvation and surface interactions modify. It is determined that the strongest interaction occurs between PyY dye and SDS, anionic surfactant, and this interaction arises from the electrostatic forces. Calculated photophysical parameters indicated that the microenvironment of PyY in SDS micelle is different to that of other systems. In temperature studies, it was reported that increasing the temperature of the samples increased non-radiative transitions. Steady-state fluorescence anisotropy values were calculated by using fluorescence intensities of PyY compound in pre-micellar, micellar and post-micellar systems. Once the PyY fluorescence probe is added to the surfactant containing solutions below the critical micelle concentrations, the measured anisotropy values were found to be low because the probe remains in the deionized water phase. When the surfactant concentration of the medium becomes closer to the critical micelle concentrations, the steady-state anisotropy value prominently increases. This is because of the restrictions on the rotational diffusion of the probe in micellar solution. It is observed that positively charged PyY shows a higher affinity to the negatively charged SDS compared with the positively charged CTAB and neutral TX-100 surfactants. This can be explained by Coulombic interactions.

Photophysics and photodynamics of Pyronin Y in n-alcohols.

The photophysical properties and photodynamics of Pyronin Y (PyY) dye compound in seven polar protic solvents (n-alcohols) were examined as a function of temperature by using UV-visible, steady-state and time-resolved fluorescence spectroscopy techniques. To understand dye-solvent interactions, photophysical parameters including Stokes' shifts, fluorescence quantum yields and fluorescence lifetimes were determined. To examine the effect of solvent polarity, the difference between the ground state dipole moment and the excited state dipole moment was determined. For this purpose, the multiple regression analysis and the Kamlet-Taft technique were used. Moreover, photodynamic parameters, rotational relaxation times and steady-state anisotropy were calculated. The result showed that the specific interactions of PyY with the solvent molecules take place through hydrogen bonding. As the hydrocarbon chain of the alcohols gets longer, photophysical parameters diminish, probably because of weaker hydrogen bonding. Furthermore, it was found out that the dipole moment of excited states (µe ) is higher than that of the ground state (µg ). In addition, Brownian motions increased with the increasing temperature that weakened the fluorescence character of PyY. It was also revealed that the rotation of PyY increased with a prolonged hydrocarbon chain of alcohol series, due to the lesser extent of hydrogen bonding.

In situ formation of pyronin dyes for fluorescence protease sensing.

We report a reaction-based strategy for the fluorogenic detection of protease activity. Based on the "covalent-assembly" probe design principle recently put forward by the Yang group for detection of Sarin related threats (J. Am. Chem. Soc., 2014, 136, 6594-6597), we have designed two unusual non-fluorescent caged precursors (mixed bis-aryl ethers) which are readily converted into a fluorescent unsymmetrical pyronin dye through a domino cyclisation-aromatisation reaction triggered by penicillin G acylase (PGA) or leucine aminopeptidase (LAP). Fluorescence-based in vitro assays and HPLC-fluorescence/-MS analyses support the claimed activation mechanism whose the further implementation to "smart" imaging agents for the study of protease function in vivo is expected.

The pH-influenced PET processes between pyronine and different heterocycles.

The OFF-ON and ON-OFF type pH probes based on rosamine were designed by using the relative electron densities between pyronine and various linked heterocycles. Probe 1a with an indole-pyronine skeleton gave an OFF-ON pH response (pKa = 1.41) with decreasing pH, and the relative fluorescence intensity increased 15-fold, while probe 1b with an imidazole-pyronine skeleton did not give an ON-OFF response to different pH values. When pyronine was connected with a quinolinyl group, i.e., probes 1c-d, the red emission (around 575-800 nm) gave a monotonous ON-OFF pH response (pKa = 3.26 and 2.62, respectively) with decreasing pH. The relative fluorescence intensities decreased 263- and 46-fold, respectively. Changes in the electron donating abilities of the nitrogen containing heterocycles were used to explain variations in PET processes within the probes, and their pH-dependent PET mechanisms were verified using time-dependent density functional theory calculations. Confocal fluorescence imaging was also used to evaluate the potential biomedical application of probes 1a-d. Ultimately, probe 1d with an appropriate pKa value and good biocompatibility showed lysosome targeting ability.

Prediction of Rate Constant for Supramolecular Systems with Multiconfigurations.

The control of supramolecular systems requires a thorough understanding of their dynamics, especially on a molecular level. It is extremely difficult to determine the thermokinetic parameters of supramolecular systems, such as drug-cyclodextrin complexes with fast association/dissociation processes by experimental techniques. In this paper, molecular modeling combined with novel mathematical relationships integrating the thermodynamic/thermokinetic parameters of a series of isomeric multiconfigurations to predict the overall parameters in a range of pH values have been employed to study supramolecular dynamics at the molecular level. A suitable form of Eyring's equation was derived and a two-stage model was introduced. The new approach enabled accurate prediction of the apparent dissociation/association (k(off)/k(on)) and unbinding/binding (k-r/kr) rate constants of the ubiquitous multiconfiguration complexes of the supramolecular system. The pyronine Y (PY) was used as a model system for the validation of the presented method. Interestingly, the predicted k(off) value ((40 ± 1) × 10(5) s(-1), 298 K) of PY is largely in agreement with that previously determined by fluorescence correlation spectroscopy ((5 ± 3) × 10(5) s(-1), 298 K). Moreover, the k(off)/k(on) and k-r/kr for flurbiprofen-ß-cylcodextrin and ibuprofen-ß-cyclodextrin systems were also predicted and suggested that the association processes are diffusion-controlled. The methodology is considered to be especially useful in the design and selection of excipients for a supramolecular system with preferred association and dissociation rate constants and understanding their mechanisms. It is believed that this new approach could be applicable to a wide range of ligand-receptor supramolecular systems and will surely help in understanding their complex mechanism.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 µm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

The Fluorescence Properties of Three Rhodamine Dye Analogues: Acridine Red, Pyronin Y and Pyronin B.

The fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime of Acridine Red (AR), Pyronin Y (PYY), and Pyronin B (PYB) in aqueous and organic solvents were measured by steady state fluorescence, time-correlated single photon counting, and electronic absorption methods. The rate constants of radiation and non radiation process (kf and kic) were calculated to elucidate the structural effect on the fluorescence mechanism. The data for each compound are compared with that of the corresponding rhodamine dye. AR showed significant longer lifetime and higher quantum yield than PYY and PYB, because the alkyls on N atom enhance the internal conversion (IC), the longer the alkyl the faster the IC. However, the structural variation does not alter the rate constant of radiation process (kf) but does change kic significantly. The phenyl in Rhodamine B or Rhodamine 6G shows only a slight effect on the fluorescence properties. Ethanol is the solvent in which all five compounds exhibit longest lifetime and highest fluorescence quantum yield.

Near-infrared fluorescent 9-phenylethynylpyronin analogues for bioimaging.

The syntheses and biological applications of two novel fluorescent 9-phenylethynylpyronin analogues containing either carbon or silicon at the position 10 are reported. Both fluorescent probes exhibited a relatively strong fluorescence in methanol and phosphate buffer saline in the near-infrared region (705-738 nm) upon irradiation of either of their absorption maxima in the blue and red regions. The compounds showed high selectivity toward mitochondria in myeloma cells in vivo and allowed their visualization in a favored tissue-transparent window, which makes them promising NIR fluorescent tags for applications in bioimaging.

Thermodynamics and kinetics of adaptive binding in the malachite green RNA aptamer.

Adaptive binding, the ability of molecules to fold themselves around the structure of a ligand and thereby incorporating it into their three-dimensional fold, is a key feature of most RNA aptamers. The malachite green aptamer (MGA) has been shown to bind several closely related triphenyl dyes with planar and nonplanar structures in this manner. Competitive binding studies using isothermal titration calorimetry and stopped flow kinetics have been conducted with the aim of understanding the adaptive nature of RNA-ligand interaction. The results of these studies reveal that binding of one ligand can reduce the ability of the aptamer pocket to adapt to another ligand, even if this second ligand has a significantly higher affinity to the free aptamer. A similar effect is observed in the presence of Mg(2+) ions which stabilize the binding pocket in a more ligand bound-like conformation.

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group.

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.

Quantification of arsenic in dialysate solution and scalp hair samples of kidney failure patients by cloud point extraction and electrothermal atomic absorption spectroscopy.

A method has been developed for the determination of arsenic (As) in pharmaceutical and scalp hair samples of kidney failure patients by cloud point extraction (CPE). The scalp hair samples were subjected to microwave-assisted digestion in nitric acid-hydrogen peroxide (2 + 1, v/v). Then, dialysate and digested scalp hair solutions were preconcentrated by CPE using pyronine B as a complexing agent. The resulting complex was entrapped in nonionic surfactant (Triton X-114) prior to its determination by electrothermal atomic absorption spectroscopy. The validity of the CPE method for As was checked by analysis of a certified reference material of human hair and a standard addition method for dialysate solution. The chemical variables affecting the analytical performance of the CPE methods were studied and optimized. After optimization of the complexation and extraction conditions, a preconcentration factor of 52 was obtained for As in 10 mL of dialysate solution and acid digested samples of scalp hair. Under the optimum experimental conditions, the LOD and LOQ of As for the preconcentration of 10 mL of solution were 0.022 and 0.073 microg/L, respectively. The level of As in scalp hair samples of kidney failure patients was higher than in healthy controls.

Improving the Brightness of Pyronin Fluorophore Systems through Quantum-Mechanical Predictions.

The pyronin class of fluorophores serves a critical role in numerous imaging applications, particularly involving preferential staining of RNA through base pair intercalation. Despite this important role in molecular staining applications, the same set of century-old pyronins (i.e., pyronin Y (PY) and pyronin B (PB)), which possess relatively low fluorophore brightness, are still predominantly being used due to the lack of methodology for generating enhanced variants. Here, we use TD-DFT calculations of interconversion energies between structures on the S1 surface as a preliminary means to evaluate fluorophore brightness for a proposed set of pyronins containing variable substitution patterns at the 2, 3, 6, and 7 positions. Using a nucleophilic aromatic substitution/hydride addition approach, we synthesized the same set of pyronins and demonstrate that quantum-mechanical computations are useful for predicting fluorophore performance. We produced the brightest series of pyronin fluorophores described to date, which possess considerable gains over PY and PB.

Assaying Cell Cycle Status Using Flow Cytometry.

In this chapter, four methods are described that can be used to assess cell cycle status in flow cytometry. The first method is based on the simultaneous analysis of cellular DNA content using a fluorescent DNA dye (propidium iodide) and of a nuclear proliferation marker (Ki-67). The second is based on the differential staining of DNA and RNA using Hoechst 33342 and Pyronin Y: this method is particularly useful to distinguish quiescent cells in G0 phase from G1 cells. Finally, two methods are described based on DNA incorporation of the synthetic nucleosides BrdU and EdU.

9-Cyanopyronin probe palette for super-multiplexed vibrational imaging.

Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.

Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access.

A synthesis of the carbopyronine dye Carboxy ATTO 647N from simple materials is reported. This route proceeds in 11 forward steps from 3-bromoaniline with the key xanthone intermediate formed using a new oxidation methodology. The step utilizes an oxidation cycle with base, water, iodine, and more than doubles the yield of the standard permanganate oxidation methodology, accessing gram-scale quantities of this late-stage product. From this, Carboxy ATTO 647N was prepared in only four additional steps. This facile route to a complex fluorophore is expected to enable further studies in fluorescence imaging.

Host-assisted guest self-assembly: enhancement of the dimerization of pyronines Y and B by gamma-cyclodextrin.

Buckle up! The dimerization of small fluorescent guests is strongly enhanced in presence of a cyclodextrin host. The host cavity acts like a belt to assist the self-assembly of guests (see picture). Small variations in the guest structure have significant influence on the stability and geometry of the aggregates.The role of small variations in the structural properties of host and guest molecules on the stoichiometry and strength of supramolecular associations is analyzed. Earlier we found that a change in substituents from pyronine B to pyronine Y has a dramatic effect on both the stability and the dynamics of the association of these guests with beta-cyclodextrin as host. Now we study the association between these two pyronines and a cyclodextrin with a bigger cavity (gamma-cyclodextrin) using UV/Vis absorption and fluorescence spectroscopy. The absorption spectra of the pyronines show complex variations with cyclodextrin concentration indicating that pyronine dimerization is strongly enhanced inside the cavity of the cyclodextrin. A full model is proposed and the equilibrium constants of the involved processes and the absorption and emission spectra of the different species are estimated. The equilibrium constants of the formation of complexed dimers are much higher than those for free dimerization or for the inclusion of a single guest. The gamma-cyclodextrin host acts like a belt to assist the guest self-assembly. The differences in the stability of pyronine B and pyronine Y dimers are explained on the basis of their structure and geometry.