Project description:Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe(3+), Cu(2+), Zn(2+), and Hg(2+), illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems.
Project description:We reported the preparation of lifetime-tunable fluorescent metal nanoshells and used them as lifetime imaging agents for potential detection of multiple target molecules by a single cell imaging scan. These metal nanoshells were generated to have 40 nm silica cores and 10 nm silver shells. Three kinds of metal-ligand complexes tris(5-amino-1,10-phenanthroline)ruthenium(II) (Ru(NH(2)-Phen)(3) (2+)), tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)(3) (2+)), and tris(2,3-bis(2-pyridyl)pyrazine))ruthenium(II) (Ru(dpp)(3) (2+)) that have similar excitation and emission wavelengths but different lifetimes were respectively encapsulated in the cores of metal nanoshells for the purpose of fluorescence. Compared with the metal-free silica spheres, these metal nanoshells were found to display enhanced emission intensities and shortened lifetimes due to near-field interactions of Ru(II) complexes with the metal shells. The shortened lifetimes of these metal nanoshells were definitely unique relevant to the Ru(II) complexes: 10 ns for the Ru(Phen-NH(2))(3) (2+)-Ag nanoshells, 45 ns for the Ru(bpy)(3) (2+)-Ag nanoshells, and 200 ns for the Ru(dpp)(3) (2+)-Ag nanoshells. These lifetimes were longer than the lifetime of cellular autofluorescence (2 - 5 ns), so the emission signals of these metal nanoshells could be distinctly isolated from the cellular background on the lifetime cell images. Moreover, these lifetimes were also different from one another, resulting in the emission signals of three metal nanoshells could be distinguished from one another on the cell images. This feature may offer an opportunity to detect multiple target molecules in a single cell imaging scan when the metal nanoshells are bound with various targets in the cells.
Project description:We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis.
Project description:A new type of conjugated polybenzimidazole (CPBI) was synthesized through a simple polycondensation reaction without metal catalysis, and N-alkylation modification was carried out to solve the problems of solubility and fluorescence properties. A series of nano-microsphere polymers CPBIn with large conjugation, good solubility, and strong fluorescence has been successfully used as "turn-off" fluorescent probes for the first time. The results show that, under suitable N-alkylation conditions, the obtained CPBIn can be used as a highly sensitive and selective fluorescent probe for the detection of Cu2+ and Zn2+ at the same time, and their detection limits are both nM levels. In addition, CPBI2 can be designed as an ultra-sensitive IMPLICATION logic gate at the molecular level, cyclically detecting Cu2+. With the test paper containing CPBI2, easy and quick on-site detection can be achieved. This research provides a new idea for the brief synthesis of multifunctional materials.
Project description:Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions, measurements of their levels are of great importance to understand lysosome-associated biological events. However, appropriate probes to selectively detect Cl- ions within acidic lysosomes have not been developed to date. In this study, we prepared MQAE-MP, a lysosomal Cl--selective fluorescent probe, and applied it to gain information about biological processes associated with lysosomes. The fluorescence of MQAE-MP is pH-insensitive over physiological pH ranges and is quenched by Cl- with a Stern-Volmer constant of 204 M-1. Because MQAE-MP detects lysosomal Cl- selectively, it was employed to assess the effects of eleven substances on lysosomal Cl- concentrations. The results show that lysosomal Cl- concentrations decrease in cells treated with substances that inhibit proteins responsible for lysosomal membrane stabilization, induce lysosomal membrane permeabilization, and transport lysosomal Cl- to the cytosol. In addition, we investigated the effect of lysosomal chloride ions on the fusion of autophagosomes with lysosomes to generate autolysosomes during autophagy inhibition promoted by substances. It was found that changes in lysosomal Cl- concentrations did not affect the fusion of autophagosomes with lysosomes but an increase in the cytosolic Ca2+ concentration blocked the fusion process. We demonstrate from the current study that MQAE-MP has great potential as a lysosomal Cl--selective fluorescent probe for studies of biological events associated with lysosomes.
Project description:Metal ion probes are used to assess the accessibility of cysteine side chains in polypeptides lining the conductive pathways of ion channels and thereby determine the conformations of channel states. Despite the widespread use of this approach, the chemistry of metal ion-thiol interactions has not been fully elucidated. Here, we investigate the modification of cysteine residues within a protein pore by the commonly used Ag(+) and Cd(2+) probes at the single-molecule level, and provide rates and stoichiometries that will be useful for the design and interpretation of accessibility experiments.
Project description:Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.
Project description:Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.
Project description:We design and synthesize a novel 1,8-naphthalimide-based fluorescent probe MNP that features the dual capabilities of tracking lysosomes in living HeLa cells and sensitively detecting Fe3+ ions in aqueous solution. The MNP is obtained by modifying the morpholine group with a lysosomal targeting function and the piperazine group with an Fe3+ ion recognition function on the 1,8-naphthalimide matrix. In the presence of Fe3+ ions, the MNP acts as a recognition ligand to coordinate with the central Fe3+ ion, and the protonated [MNPH]+ is eventually generated, in which significant fluorescence enhancements are observed due to the intramolecular photo-induced electron transfer (PET) process being blocked. The limit of detection of Fe3+ ions is as low as 65.2 nM. A cell imaging experiment shows that the MNP has low cytotoxicity and excellent lysosomal targeting ability. Therefore, the MNP offers a promising tool for lysosomal tracking and relevant life process research.
Project description:This study presents the characterization of a novel multilayered three-dimensional (3D) polymer exhibiting aggregation-induced emission (AIE) properties when excited at a low wavelength of 280 nm. Utilizing fluorescence spectroscopy, we demonstrate that the polymer displays a marked enhancement in luminescence upon aggregation, a characteristic behavior that distinguishes AIE-active materials from conventional fluorophores. Furthermore, we explore the potential application of this multilayered 3D polymer as a fluorescent probe for the selective detection of specified metal ions. By incorporating chelating functional groups into the polymer matrix, we facilitate specific interactions with target metal ions, leading to significant fluorescence intensity changes that correlate with ion concentration. According to their cyclic voltammetry characteristics, the polymers have potential applications in cutting-edge electrical and optoelectronic systems. Our findings indicate that this multilayered 3D polymer serves as an effective fluorescent sensor and offers tunable optical properties, paving the way for innovative applications in environmental monitoring and biomedical diagnostics. The results underscore the utility of AIE-active polymers in developing advanced materials for sensitive and selective detection of metal ions, contributing to the growing field of smart sensing technologies.