Fengling Song

Fluorescence Ratiometry and Fluorescence Lifetime Imaging: Using a Single Molecular Sensor for Dual Mode Imaging of Cellular Viscosity

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.

Oxidation-Resistant Fluorogenic Probe for Mercury Based on Alkyne Oxymercuration

Mercury continues to be a major safety hazard to the general public. Therefore, a fluorescence method that has potential for on-site use can be very useful. Most fluorescent probes for mercury are based on the interaction between mercury ions and sulfur atoms, which may not be compatible with mercury samples prepared by oxidation processes. Herein we report a fluorogenic probe for mercury based on the unique ability of Hg2+ to convert an alkyne to a ketone. By this methodology, the probe, prepared in two steps in 80% overall yield, is converted to a very bright green fluorescent molecule by Hg2+ in pH 7 buffer. This fluorescence method is sensitive, showing a signal-to-background ratio of 3 at 8 ppb mercury level, and highly specific for this metal ion. Since this detection method does not rely on mercury−heteroatom interactions, the probe is resistant to oxidants and thus can be used against mercury samples prepared by oxidative procedures. We demonstrate that our method can be applied to fish and dental...

A Ratiometric Near-Infrared Fluorescent Probe for Hydrazine and Its in Vivo Applications

Based on modulation of the conjugated polymethine π-electron system of a cyanine dye derivative, a ratiometric near-infared fluorescent probe (Cy7A) for hydrazine (N2H4) has been designed and synthesized. Cy7A can be selectively hydrazinolysized with great changes in its fluorescent excitation/emission profiles, which makes it possible to detect N2H4 in water samples and living cells and, for the first time, visualize N2H4 in living mice.

Enhancement of a catalysis-based fluorometric detection method for palladium through rational fine-tuning of the palladium species.

Metal analyses in chemistry, materials science, and environmental science are currently performed using techniques such as inductively coupled plasma mass spectrometry and X-ray fluorescence, which require expensive instrumentation and are not high-throughput. Although fluorescent probes are known for their sensitivity and specificity and are amenable to high-throughput analyses, the robustness of such analyses are typically limited due to their binding-based nature. Herein we report an improvement of our previously reported catalysis-based fluorescent probe for palladium by rationally fine-tuning the redox and coordination chemistries of the palladium species involved in the O-deallylation reaction. This method now rivals current analytical methods with respect to sensitivity. We demonstrate palladium detection in various active pharmaceutical ingredients, spent catalytic converter materials, and a metal scavenger resin. Thus, fluorescent methods may have the potential for substituting the current instrument-intensive techniques.

A Fluorescent Ratiometric Chemodosimeter for Cu2+ Based on TBET and Its Application in Living Cells

Based on a through bond energy transfer (TBET) between Rhodamine and a naphthalimide fluorophore, a fluorescent ratiometric chemodosimeter RN1 was designed and prepared for single selective detection of Cu(2+) in aqueous solution and in living cells, as Cu(2+) acts as not only a selective recognizing guest but also a hydrolytic promoter.

Fluorescent Probes for Pd2+ Detection by Allylidene–Hydrazone Ligands with Excellent Selectivity and Large Fluorescence Enhancement

Because palladium is widely used in various catalysts and converters, which results in a high level of contamination of water systems and the soil by residual palladium, there is an urgent need for Pd(2+)-sensitive and -selective probes. Based on the special affinity of Pd(2+) to conjugated double-bond ligands, two fluorescence probes (RPd2 and RPd3) that contain conjugated allylidene-hydrazone ligands that link to colorless rhodamine-spirolactam have been developed. The results show that conjugated allylidene-hydrazones have a much better affinity toward Pd(2+), and consequently provide the probes with more acute color change and fluorescence enhancement (≈170-fold), and better selectivity over other metal ions (especially platinum-group elements, or PGEs) than the unconjugated allyl-hydrazine. With richer electron density and a more suitable stereo effect in the allylidene-hydrazone group, RPd2 displays the best specificity toward Pd(2+) and affords convenient detection by the naked eye. Its potential application for Pd(2+)-contaminated water and soil-sample analysis is revealed by proof-of-concept experiments.

Thermally activated delayed fluorescence of fluorescein derivative for time-resolved and confocal fluorescence imaging.

Compared with fluorescence imaging utilizing fluorophores whose lifetimes are in the order of nanoseconds, time-resolved fluorescence microscopy has more advantages in monitoring target fluorescence. In this work, compound DCF-MPYM, which is based on a fluorescein derivative, showed long-lived luminescence (22.11 μs in deaerated ethanol) and was used in time-resolved fluorescence imaging in living cells. Both nanosecond time-resolved transient difference absorption spectra and time-correlated single-photon counting (TCSPC) were employed to explain the long lifetime of the compound, which is rare in pure organic fluorophores without rare earth metals and heavy atoms. A mechanism of thermally activated delayed fluorescence (TADF) that considers the long wavelength fluorescence, large Stokes shift, and long-lived triplet state of DCF-MPYM was proposed. The energy gap (ΔEST) of DCF-MPYM between the singlet and triplet state was determined to be 28.36 meV by the decay rate of DF as a function of temperature. The ΔE(ST) was small enough to allow efficient intersystem crossing (ISC) and reverse ISC, leading to efficient TADF at room temperature. The straightforward synthesis of DCF-MPYM and wide availability of its starting materials contribute to the excellent potential of the compound to replace luminescent lanthanide complexes in future time-resolved imaging technologies.

An Effective Minor Groove Binder as a Red Fluorescent Marker for Live‐Cell DNA Imaging and Quantification

Since the understanding of DNA organization and structure in vivo is so important, fluorescent staining techniques using organic DNA-binding molecules are required for biological research and medical diagnosis, including cellular imaging and DNA quantification. However, membrane-permeable DNA-specific stains are uncommon. The minor groove binders 4’,6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 are presently used for DNA-specific staining, but they require ultraviolet excitation, which can lead to cellular damage due to lengthy irradiation. SYTO stains do provide cell-permeable dyes excitable by visible and near-infrared radiation. Unfortunately they are not specific nuclear stains, and moreover are of undisclosed chemical structures. Although the cell-permeant anthraquinone dye DRAQ5 shows red fluorescence emission and DNA-specific labeling, it is a DNA intercalator, which seriously interferes with the structure and function of nuclear DNA, in contrast to the minor groove binders such as SYTO17. Therefore, a pressing need exists to develop fluorescent dyes satisfying the multiple criteria of long-wavelength excitation/emission, high DNA selectivity, and live-cell permeability. Recently, a terbium complex [Tb·L] [2] was described which stained the nuclear DNA in mitotic cells using very low dye concentrations (< 1 mm). Unfortunately, intense irradiation of 350 nm is needed as excitation light. BENA435, with an N,N-dimethylpropane-1,3-diamine group, was reported to stain the nucleus in live cells, but still with excitation/emission located at the cyan region (lex(DNA) = 435 nm; lem(DNA) = 485 nm). Chang et al. [4] have discovered an excellent DNA-selective probe, C61, with relatively long emission wavelength (lem(DNA) = 540 nm) for live-cell nuclear imaging and DNA quantification. C61 shows a 19.9-fold fluorescence increase when bound to doublestranded (ds) DNA (FF DNA = 0.0675). Another promising finding has been reported by Thomas et al. , namely that a dinuclear ruthenium(II) polypyridyl complex (lem(DNA) = 680 nm) can be used as a nuclear DNA stain for both luminescence and transmission electron microscopy. Despite the high hydrophilicity and charge of the molecule, it was still taken up by live cells, but only if a relatively high concentration of more than 200 mm was used. In fluorescence microscopic imaging studies, it is desirable for DNA staining to involve large fluorescence enhancements, and for nuclei to stain rapidly even when low concentrations of dye are used, as this minimizes dye toxicity to live cells. Herein, we report a novel red fluorescent dye DEAB-TO3 (Scheme 1, DEAB = (diethylamino)butyl)), a TO-3 analogue with long-wavelength excitation and emission

Construction of Long-Wavelength Fluorescein Analogues and Their Application as Fluorescent Probes

Born to dye: Five fluorescein analogues were synthesised (see scheme). One analogue was found to emit in the NIR region with a high quantum yield, excellent photostability and good permeability. Three derivatives were found to specifically stain mitochondria and one dye responds to thiols with a strong turn-on NIR fluorescence signal and colorimetric change, in vitro and in vivo.

Studies Toward an Ideal Fluorescence Method to Measure Palladium in Functionalized Organic Molecules: Effects of Sodium Borohydride, Temperature, Phosphine Ligand, and Phosphate Ions on Kinetics

Residual metals in fine chemicals are currently detected by using inductively coupled plasma mass spectrometry, which requires expensive instrumentation and does not have high-throughput capabilities. Although fluorescent probes can be amenable to high-throughput analyses of metals, the utility of such analyses is limited due to the lack of generality. Herein, we report a significant improvement (≈19-fold) to our previously reported catalysis-based fluorescent probe for palladium. Specifically, we found that slightly elevated temperature dramatically improved the generality of the method and that the deallylation reaction of the nonfluorescent compound 1 was accelerated by phosphate ions in aqueous media. This method was capable of detecting 0.2 ppb palladium. We demonstrated reasonably accurate palladium detection in various active pharmaceutical ingredients and highly functionalized organic compounds.