Zhiqiang Ye

A europium(III) chelate as an efficient time-gated luminescent probe for nitric oxide

The first Eu(3+) chelate-based luminescent probe specific for nitric oxide (NO) has been designed and synthesized for highly sensitive and selective time-gated luminescence detection of NO. Based on the probe, a time-gated luminescence imaging technique was developed for imaging the endogenous NO production in living plant cells/tissues.

Novel fluorescent europium chelate-doped silica nanoparticles: preparation, characterization and time-resolved fluorometric application

Novel fluorescent europium(III) chelate-doped silica nanoparticles were prepared and characterized as a new type of fluorescence probe for quantitative bioassay. The preparation was carried out in a water-in-oil (w/o) microemulsion consisting of a strongly fluorescent Eu3+ chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-o-terphenyl–Eu3+ (BHHT–Eu3+), surfactant (Triton X-100), co-surfactant (n-hexanol, n-heptanol or n-octanol), aqueous phase (H2O or D2O) and oil phase (cyclohexane) by controlling the hydrolysis of tetraethylorthosilicate (TEOS). The effects of different co-surfactants and aqueous phases on the size and fluorescence lifetime of the nanoparticles were investigated. The results reveal that the size of the nanoparticles is decreased with a change of co-surfactants from n-hexanol to n-octanol, and the fluorescence lifetime of the nanoparticles is increased with a change of aqueous phase from H2O to D2O. A new method was established for the surface modification and bioconjugation of the nanoparticles. Nanoparticle-labeled streptavidin (SA) was used for the time-resolved fluoroimmunoassay of human hepatitis B surface antigen (HBsAg). The result shows that the new fluorescent europium(III) chelate-doped silica nanoparticles are suitable to be used as a fluorescence probe for highly sensitive bioassays.

Development of functionalized fluorescent europium nanoparticles for biolabeling and time-resolved fluorometric applications

A covalent binding-copolymerization method was developed to prepare silica-based fluorescent europium nanoparticles that can be used for biolabeling and highly sensitive time-resolved fluorescence bioassays. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion consisting of a conjugate of (3-aminopropyl)triethoxysilane bound to a fluorescent Eu3+ chelate, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl-Eu3+ (APS-BHHCT-Eu3+), free (3-aminopropyl)triethoxysilane (APS), tetraethyl orthosilicate (TEOS), Triton X-100, n-octanol, water, and cyclohexane by copolymerization of APS-BHHCT-Eu3+, APS, and TEOS with aqueous ammonia. Characterization by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are spherical and uniform in size, 36 ± 4 nm in diameter, highly photostable, and strongly fluorescent, having a fluorescence quantum yield of 50.6% and a long fluorescence lifetime of 384 µs. The amino groups directly introduced to the surface of the nanoparticles by using free (3-aminopropyl)triethoxysilane in the nanoparticle preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticles were used for streptavidin labeling, and the nanoparticle-labeled streptavidin was used in sandwich-type time-resolved fluoroimmunoassays (TR-FIA) of carcinoembryonic antigens (CEA) and hepatitis B surface antigens (HBsAg) in human sera. The methods give detection limits of 1.9 pg ml−1 for CEA, and 23 pg ml−1 for HBsAg. The concentrations of HBsAg in 30 human serum samples were determined, and the results were compared with those independently determined by an established TR-FIA method using the BHHCT-Eu3+-labeled streptavidin. A good correlation was obtained with a correlation coefficient of 0.993.

Visible-light-sensitized highly luminescent europium nanoparticles: preparation and application for time-gated luminescence bioimaging

Time-gated luminescence bioimaging based on microsecond-lifetime luminescent biolabels can provide complete background-free conditions for detecting target cells in an autofluorescence biosample matrix. However, a major drawback of the current lanthanide biolabels is the requirement for UV excitation (<370 nm), which leads to damage to many biological systems and greatly affects the improvement of time-gated luminescence instruments. Herein we describe luminescent europium nanoparticles that have an excitation peak around 406 nm with high quantum yield (∼66%) and fine monodispersity in aqueous solutions. The nanoparticles were prepared by copolymerization of a visible-light-sensitized Eu3+ complex 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)chlorosulfo-o-terphenyl-Eu3+-2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine conjugated with 3-aminopropyl(triethoxy)silane, free 3-aminopropyl(triethoxy)silane and tetraethyl orthosilicate in a water-in-oil reverse microemulsion. Characterization by transmission electron microscopy and luminescence spectroscopy indicates that the nanoparticles are monodisperse, spherical and uniform in size, <50 nm in diameter, and show strong visible-light-sensitized luminescence with a large quantum yield and a long luminescence lifetime. The new nanoparticles were successfully applied to distinguish an environmental pathogen, Giardia lamblia, within a concentrate of environmental water sample using a time-gated luminescence microscope with pulsed visible light excitation. The method resulted in highly specific and sensitive imaging for Giardia lamblia. These results suggest a broad range of potential bioimaging applications where both long time microscopy observation and high signal-to-background ratio are required for samples containing high concentrations of autofluorescence background.

A ruthenium(II) complex-based lysosome-targetable multisignal chemosensor for in vivo detection of hypochlorous acid.

Although considerable efforts have been made for the development of ruthenium(II) complex-based chemosensors and bioimaging reagents, the multisignal chemosensor using ruthenium(II) complexes as the reporter is scarce. In addition, the mechanisms of cellular uptake of ruthenium(II)-based chemosensors and their intracellular distribution are ill-defined. Herein, a new ruthenium(II) complex-based multisignal chemosensor, Ru-Fc, is reported for the highly sensitive and selective detection of lysosomal hypochlorous acid (HOCl). Ru-Fc is weakly luminescent because the MLCT (metal-to-ligand charge transfer) state is corrupted by the efficient PET (photoinduced electron transfer) process from Fc (ferrocene) moiety to Ru(II) center. The cleavage of Fc moiety by a HOCl-induced specific reaction leads to elimination of PET, which re-establishes the MLCT state of the Ru(II) complex, accompanied by remarkable photoluminescence (PL) and electrochemiluminescence (ECL) enhancements. The result of MTT assay showed that the proposed chemosensor, Ru-Fc, was low cytotoxicity. The applicability of Ru-Fc for the quantitative detection of HOCl in live cells was demonstrated by the confocal microscopy imaging and flow cytometry analysis. Dye colocalization studies confirmed very precise distribution of the Ru(II) complex in lysosomes, and inhibition studies revealed that the caveolae-mediated endocytosis played an important role during the cellular internalization of Ru-Fc. By using Ru-Fc as a chemosensor, the imaging of the endogenous HOCl generated in live macrophage cells during the stimulation was achieved. Furthermore, the practical applicability of Ru-Fc was demonstrated by the visualizing of HOCl in laboratory model animals, Daphnia magna and zebrafish.

A cell-membrane-permeable europium complex as an efficient luminescent probe for singlet oxygen

A unique cell-membrane-permeable europium complex has been developed as a probe for time-gated luminescence detection of singlet oxygen (1O2). Combined with the time-gated luminescence imaging technique, the probe was successfully used for investigating the time-dependent generation and distribution of 1O2 induced by the clinical drugs of photodynamic therapy in cancer cells.

Preparation, characterization and application of fluorescent terbium complex-doped zirconia nanoparticles.

Novel zirconia-based fluorescent terbium nanoparticles have been prepared as a fluorescent nanoprobe for time-resolved fluorescence bioassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion consisting of a strongly fluorescent Tb3+ complex, N,N,N1, N1-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-phenylpyridine]tetrakis(acetate)-Tb3+(BPTA-Tb3+), Triton X-100, hexanol, and cyclohexane by controlling co-condensation of Zr(OCH2CH3)4 and ZrOCl2. The characterizations by transmission electron microscopy and fluorometric methods indicate that the nanoparticles are uniform in size, 33± 4 nm in diameter, and have a fluorescence quantum yield of 8.9% and a long fluorescence lifetime of 2.0 ms. The zirconia-based fluorescent terbium nanoparticles show high stability against basic dissolution in a high pH aqueous buffer compared to the silica-based nanoparticles. A surface modification and bioconjugation method for the fluorescent nanoparticles was developed, and the nanoparticle-conjugated streptavidin (SA) was used for time-resolved floroimmunoassy (TR-FIA) of human prostate specific antigen (PSA). The result shows that the zirconia-based fluorescent terbium nanoparticles are useful as a fluorescent nanoprobe for time-resolved fluorescence bioassay.

Practical Implementation, Characterization and Applications of a Multi-Colour Time-Gated Luminescence Microscope

Time-gated luminescence microscopy using long-lifetime molecular probes can effectively eliminate autofluorescence to enable high contrast imaging. Here we investigate a new strategy of time-gated imaging for simultaneous visualisation of multiple species of microorganisms stained with long-lived complexes under low-background conditions. This is realized by imaging two pathogenic organisms (Giardia lamblia stained with a red europium probe and Cryptosporidium parvum with a green terbium probe) at UV wavelengths (320–400 nm) through synchronization of a flash lamp with high repetition rate (1 kHz) to a robust time-gating detection unit. This approach provides four times enhancement in signal-to-background ratio over non-time-gated imaging, while the average signal intensity also increases six-fold compared with that under UV LED excitation. The high sensitivity is further confirmed by imaging the single europium-doped Y2O2S nanocrystals (150 nm). We report technical details regarding the time-gating detection unit and demonstrate its compatibility with commercial epi-fluorescence microscopes, providing a valuable and convenient addition to standard laboratory equipment.

Artificial luminescent protein as a bioprobe for time-gated luminescence bioimaging

An artificial luminescent protein, apoferritin-encapsulated luminescent europium complex, has been designed/fabricated and displays good biocompatibility and long-lived luminescence, which means it can be used as a bioprobe to image living cells with a time-gated mode.

Preparation and time-gated luminescence bioimaging application of ruthenium complex covalently bound silica nanoparticles

Luminescent ruthenium(II) complex covalently bound silica nanoparticles have been prepared and used as a probe for time-gated luminescence bioimaging. The new nanoparticles were prepared by copolymerization of a luminescent Ru(II) complex tris(5-amino-1,10-phenanthroline)ruthenium(II) conjugated with 3-aminopropyl(triethoxy)silane (APS-Ru conjugate), free (3-aminopropyl)triethoxysilane (APS) and tetraethyl orthosilicate (TEOS) in a water-in-oil reverse microemulsion consisting of Triton X-100, n-octanol, cyclohexane and water in the presence of aqueous ammonia. Characterization by transmission electron microscopy indicates that the nanoparticles are monodisperse, spherical and uniform in size, 64+/-4 nm in diameter. Compared with the dye-doping nanoparticles, dye leakage of the new nanoparticles was remarkably decreased. In addition, it was found that the Ru(II) complex luminescence could be effectively enhanced with a longer luminescence lifetime (approximately 2.3 micros) after forming the nanoparticles, which enables the nanoparticles to be suitable as a bioprobe for time-gated luminescence bioimaging applications. The nanoparticle-labeled streptavidin was prepared and successfully used for time-gated luminescence imaging detection of an environmental pathogen, Giardia lamblia, with high specificity and sensitivity.