Highly sensitive and selective light-up fluorescent probe for monitoring gallium and chromium ions in vitro and in vivo.

Over the past decades, gallium (Ga) compounds have gained importance in the field of cancer treatment. Gallium acts as an iron mimic and disturbs iron-dependent propagation and other processes in tumor cells. However, the toxicity of gallium was also well documented in vitro and in vivo in animals. Though the oral administration of gallium in humans is less toxic, it has also been shown that a long period of administration could induce tumor fibrosis. Chromium (Cr), a naturally occurring heavy metal, is commonly used in industrial processes and can cause severe health problems in humans. It has been found to be closely involved in the metabolism of nucleic acids, proteins and fats in humans. Cr(iii) salts can be used as micronutrients and dietary supplements. However, similar to gallium (Ga3+), chromium (Cr3+) can build up to an excessive degree that is harmful to the human body. Therefore, it would be of great interest to develop chemosensing for the selective and sensitive detection of gallium and chromium ions in vitro and in vivo. Herein, we reported that an NBD-based (4-chloro-7-nitrobenzo-2-oxa-1,3-diazole) fluorescent probe (NBDT) was fabricated with demonstrated extraordinary specificity and sensitivity. A swift response toward Ga3+ and Cr3+ ions was discovered using fluorescence enhancement over a wide pH range and with cycle stability. Furthermore, lighted up by Ga3+ and Cr3+ ions in vitro, this NBDT sensor was successfully applied to detect exogenous Ga3+ and Cr3+ ions in MDA-MB-231 and HepG2 cells. Additionally, using zebrafish as the in vivo model, we demonstrated the capability of this NBDT for detecting and imaging Ga3+ and Cr3+ ions in zebrafish. Taken together, this NBDT has indicated great potential for detecting and monitoring Ga3+ and Cr3+ ions in vitro and in vivo.

Molecular Glue that Spatiotemporally Turns on Protein-Protein Interactions.

We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells.

An approach of high sensitivity and selectivity for hydrogen peroxide (H2O2) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent "turn off" probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H2O2-reactive group, aryl boronate, for sensitive and selective detection of H2O2. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H2O2 detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H2O2 in living cells by confocal microscopy, supporting its future applications to selectively monitor H2O2 levels and identify H2O2-related physiological or pathological responses from live cells or tissues in the near future.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)