Time-resolved luminescence microscopy of bimetallic lanthanide helicates in living cells

The cellular uptake mechanism and intracellular distribution of emissive lanthanide helicates have been elucidated by time-resolved luminescence microscopy (TRLM). The helicates are non-cytotoxic and taken up by normal (HaCat) and cancer (HeLa, MCF-7) cells by endocytosis and show a late endosomal–lysosomal cellular distribution. The lysosomes predominantly localize around the nucleus and co-localize with the endoplasmatic reticulum. The egress is slow and limited, around 30% after 24 h. The first bright luminescent images can be observed with an external concentration gradient of 5 mMof the EuIII helicate [Q = 0.21, t = 2.43 ms], compared to >10 mM when using conventional luminescence microscopy. Furthermore, multiplex labeling could be achieved with the TbIII [Q = 0.11, t = 0.65 ms], and SmIII [Q = 0.0038, t = 0.030 ms] analogues

Synthesis and cell localization of self-assembled dinuclear lanthanide bioprobes

Lanthanide bioprobes and bioconjugates are ideal luminescent stains in view of their low propensity to photobleaching, sharp emission lines and long excited state lifetimes permitting time-resolved detection for enhanced sensitivity. In this paper, we expand our previous work which demonstrated that self-assembled dinuclear triple-stranded helicates[Ln(2)(L-C2X)(3)] behave as excellent cell and tissue labels in immunocytochemical and immunohistochemical assays. The synthetic strategy of the hexadentate ditopic ligands incorporating dipicolinic acid, benzimidazole units and polyoxyethylene pendants is revisited in order to provide a more straightforward route and to give access to further functionalization of the polyoxyethylene arms by incorporating a terminal function X. Formation of the helicates [Ln(2)(L-C2X)(3)] (X= COOH, CH2OH, COEt, NH2, phthalimide) is ascertained by several experimental techniques and their stability tested against diethylenetriaminepentaacetate. Their photophysical properties (quantum yield, lifetime, radiative lifetime and sensitization efficiency) are presented and compared with those of the parent helicates [Ln(2)(L-C2)(3)]. Finally, the cellular uptake of five Eu-III helicates is monitored by time-resolved luminescence microscopy and their localization in HeLa cells established by co-staining experiments

Effect of the length of polyoxyethylene substituents on luminescent bimetallic lanthanide bioprobes

The new homoditopic ligand $H_2L^{C2'}$ self-assembles with lanthanide ions ($Ln^{III}$) to yield neutral bimetallic helicates of overall composition $[Ln_2(L^{C2'})_3]$; it is fitted with two hexakis(oxyethylene) chains to test their effects on the thermodynamic, photophysical and biochemical properties of these complexes, with particular emphasis on their uptake by living cells. At physiological pH and under stoichiometric conditions, the conditional stability constants $\log \beta_{23}$ are around 28 resulting in the speciation of the $Eu^{III}$ helicate being > 92% for a total ligand concentration of 1 mM. The ligand triplet state features adequate energy (0- phonon transition at $\approx 21 800 cm^{-1}$) for sensitising the luminescence of $Eu^{III}$ (Q = 19%) and $Tb^{III}$ (Q = 10%) in aerated water at pH 7.4. The $Eu(^5D_0)$ emission spectrum and lifetime (2.43 ms) are characteristic of a species with pseudo-$D_3$ symmetry and without bound water in the inner coordination sphere. The viability of HeLa cancerous cells is unaffected when incubated with up to 500 microM $[Eu_2(L^{C2'})_3]$ during 24 h. The $Eu^{III}$ helicate permeates into the cytoplasm of these cells by endocytosis and remains essentially undissociated, despite a low intracellular concentration of $0.28 \mu M$. In addition, the leakage of the $Eu^{III}$ helicate out of HeLa cells is very minimal over long periods of time. With respect to similar complexes with ligands bearing shorter tris(oxyethylene) chains, no substantial changes are observed, which opens the way for targeting experiments. This study also demonstrates that the $[Ln_2(L^{CX})_3]$ helicates are fairly robust entities since their core is unaffected by the substitution in the pyridine 4-position

A Versatile Ditopic Ligand System for Sensitizing the Luminescence of Bimetallic Lanthanide Bio-Imaging Probes

The homoditopic ligand 6,6’- [methylenebis(1-methyl-1H-benzimid- azole-5,2-diyl)]bis(4-{2-[2-(2-methoxy- ethoxy)ethoxy]ethoxy}pyridine-2-carboxylic acid) (H2LC2) has been tailored to self-assemble with lanthanide ions (LnIII), which results in the formation of neutral bimetallic helicates with the overall composition [Ln2(LC2)3] and also provides a versatile platform for further derivatization. The grafting of poly(oxyethylene) groups onto the pyridine units ensures water solubility, while maintaining sizeable thermodynamic stability and adequate antenna effects for the excitation of both visible- and NIR-emitting LnIII ions. The conditional stability constants (logb23) are close to 25 at physiological pH and under stoichiometric conditions. The ligand triplet state features adequate energy (0-phonon transition at ~21 900 cm-1) to sensitize the luminescence of EuIII (Q=21%) and TbIII (11%) in aerated water at pH 7.4. The emission of several other VIS- and NIR-emitting ions, such as SmIII (Q=0.38%) or YbIII (0.15%), for which in cellulo luminescence is evidenced for the first time, is also sensitized. The EuIII emission spectrum arises from a main species with pseudo-D3 symmetry and without coordinated water. The cell viability of several cancerous cell lines (MCF-7, HeLa, Jurkat and 5D10) is unaffected if incubated with up to 500 mm [Eu2(LC2)3] during 24 h. Bright EuIII emission is seen for incubation concentrations above 10 mm and after a 15-minute loading time; similar images are obtained with TbIII and SmIII. The helicates probably permeate into the cytoplasm of HeLa cells by endocytosis. The described luminescent helical stains are robust chemical species which remain undissociated in the cell medium and in presence of other complexing agents, such as edta, dtpa, citrate or l-ascorbate. Their derivatization, which would open the way to the sensing of targeted in cellulo phenomena, is currently under investigation

Increasing the efficiency of lanthanide luminescent bioprobes: bioconjugated silica nanoparticles as markers for cancerous cells

The lanthanide binuclear helicate [Ln(2)(L-C2)(3)] has been embedded into bare and NH2-functionalized silica nanoparticles (NPs) using water-in-oil microemulsion technique. TEM analysis reveals both [Ln(2)(L-C2)(3)]@SiO2 and [Ln(2)(L-C2)(3)]@SiO2/NH2 nanoparticles having a spherical morphology and being monodispersed with an average size of 55 +/- 5 and 90 +/- 10 nm, respectively. The energy of the ligand triplet state, similar to 21 800 cm(-1) ([Gd-2(L-C2)(3)]@NP), does not change upon incorporation into silica nanoparticles and is optimal for sensitizing Eu-III luminescence. As a consequence, [Eu-2(L-C2)(3)]@SiO2 and [Eu-2(L-C2)(3)]@SiO2/NH2 NPs display red emission due to characteristic D-5(0) > F-7(J) (J = 0-4) transitions with absolute quantum yield reaching 28% for the latter. NH2-functionalized NPs have then been conjugated with avidin (NP-avidin) or goat anti-mouse IgG antibody (NP-IgG) to test them as luminescent biomarkers. Time-resolved microscopy of immunocytochemical assays involving recognition of mucin-like proteins expressed on breast cancer MCF-7 cells by the 5D10 monoclonal antibody confirms that the NP-IgG bioprobe displays specific luminescent signal with signal-to-noise ratio approximate to 20% higher than the one obtained for the bioconjugate of molecular [Eu-2(L-C2(COOH))(3)] with IgG. In addition, immunoassays using a streptavidin-coated plate and the NP-IgG probe are able to detect 15 ng mL(-1) of the biotinylated 5D10 antibody with a signal-to-noise ratio of 100

Luminescent Bimetallic Lanthanide Bioprobes for Cellular Imaging with Excitation in the Visible-Light Range

A series of homoditopic ligands H2LCX (X=4–6) has been designed to self-assemble with lanthanide ions (LnIII), resulting in neutral bimetallic helicates of overall composition [Ln2ACHTUNGTRENUNG(LCX)3] with the aim of testing the influence of substituents on the photophysical properties, particularly the excitation wavelength. The complex species are thermodynamically stable in water (logb23 in the range 26–28 at pH 7.4) and display a metal-ion environment with pseudo-D3 symmetry and devoid of coordinated water molecules. The emission of EuIII, TbIII, and YbIII is sensitised to various extents, depending on the properties of the ligand donor levels. The best helicate is [Eu2ACHTUNGTRENUNG(LC5)3] with excitation maxima at 350 and 365 nm and a quantum yield of 9%. The viability of cervix cancer HeLa cells is unaffected when incubated with up to 500 mm of the chelate during 24 h. The helicate permeates into the cells by endocytosis and locates into lysosomes, which co-localise with the endoplasmatic reticulum, as demonstrated by counterstaining experiments. The relatively long excitation wavelength allows easy recording of bright luminescent images on a confocal microscope (lexc=405 nm). The new lanthanide bioprobe remains undissociated in the cell medium, and is amenable to facile derivatisation. Examination of data for seven EuIII and TbIII bimetallic helicates point to shortcomings in the phenomenological rules of thumb between the energy gap D(3pp*–5DJ) and the sensitisation efficiency of the ligands

Time-resolved lanthanide luminescence for lab-on-a-chip detection of biomarkers on cancerous tissues

PDMS-based microfluidic devices combined with lanthanide-based immunocomplexes have been successfully tested for the multiplex detection of biomarkers on cancerous tissues, revealing an enhanced sensitivity compared to classical organic dyes

Bioconjugated lanthanide luminescent helicates as multilabels for lab-on-a-chip detection of cancer biomarkers

The lanthanide binuclear helicate [Eu2(LC2(CO2H))3] is coupled to avidin to yield a luminescent bioconjugate EuB1 (Q ¼ 9.3%, s(5D0) ¼ 2.17 ms). MALDI/TOF mass spectrometry confirms the covalent binding of the Eu chelate and UV-visible spectroscopy allows one to determine a luminophore/protein ratio equal to 3.2. Bio-affinity assays involving the recognition of a mucin-like protein expressed on human breast cancer MCF-7 cells by a biotinylated monoclonal antibody 5D10 to which EuB1 is attached via avidin-biotin coupling demonstrate that (i) avidin activity is little affected by the coupling reaction and (ii) detection limits obtained by time-resolved (TR) luminescence with EuB1 and a commercial Eu-avidin conjugate are one order of magnitude lower than those of an organic conjugate (FITC-streptavidin). In the second part of the paper, conditions for growing MCF-7 cells in 100–200 mm wide microchannels engraved in PDMS are established; we demonstrate that EuB1 can be applied as effectively on this lab-on-a-chip device for the detection of tumour-associated antigens as on MCF-7 cells grown in normal culture vials. In order to exploit the versatility of the ligand used for selfassembling [Ln2(LC2(CO2H))3] helicates, which sensitizes the luminescence of both EuIII and TbIII ions, a dual on-chip assay is proposed in which estrogen receptors (ERs) and human epidermal growth factor receptors (Her2/neu) can be simultaneously detected on human breast cancer tissue sections. The Ln helicates are coupled to two secondary antibodies: ERs are visualized by red-emitting EuB4 using goat anti-mouse IgG and Her2/neu receptors by green-emitting TbB5 using goat anti-rabbit IgG. The fact that the assay is more than 6 times faster and requires 5 times less reactants than conventional immunohistochemical assays provides essential advantages over conventional immunohistochemistry for future clinical biomarker detection