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 [Ln2(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 [Ln2(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 [Ln2(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.

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

The lanthanide binuclear helicate [Eu(2)(L(C2(CO(2)H)))(3)] is coupled to avidin to yield a luminescent bioconjugate EuB1 (Q = 9.3%, tau((5)D(0)) = 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 microm 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 self-assembling [Ln(2)(L(C2(CO(2)H)))(3)] helicates, which sensitizes the luminescence of both Eu(III) and Tb(III) 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.

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.

Luminescent bimetallic lanthanide bioprobes for cellular imaging with excitation in the visible-light range.

A series of homoditopic ligands H(2)L(CX) (X=4-6) has been designed to self-assemble with lanthanide ions (Ln(III)), resulting in neutral bimetallic helicates of overall composition [Ln(2)(L(CX))(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 (log beta(23) in the range 26-28 at pH 7.4) and display a metal-ion environment with pseudo-D(3) symmetry and devoid of coordinated water molecules. The emission of Eu(III), Tb(III), and Yb(III) is sensitised to various extents, depending on the properties of the ligand donor levels. The best helicate is [Eu(2)(L(C5))(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 mum 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 (lambda(exc)=405 nm). The new lanthanide bioprobe remains undissociated in the cell medium, and is amenable to facile derivatisation. Examination of data for seven Eu(III) and Tb(III) bimetallic helicates point to shortcomings in the phenomenological rules of thumb between the energy gap DeltaE((3)pipi*-(5)D(J)) and the sensitisation efficiency of the ligands.

A versatile method for quantification of DNA and PCR products based on time-resolved EuIII luminescence.

A versatile and robust method for the determination of DNA and PCR products (<500 bp) is presented, based on a mix of an Eu(III) chelate and acridine orange (AO). The nucleic acid selective stains acridine orange (AO) and ethidium bromide (EB) quench the luminescence of the bimetallic [Eu(2)(L(C2))(3)] and of other monometallic chelates such as the macrocyclic complex [Eu(L(kel))], even at very low molar ratios. Stern-Volmer plots of the metal-centered emission intensities (F(0)/F) and Eu((5)D(0)) lifetimes (tau(0)/tau) show the AO quenching being purely dynamic with K(D) = 6.7 x 10(5) M(-1) for [Eu(2)(L(C2))(3)] and 1.6 x 10(6) M(-1) for [Eu(L(kel))], and bimolecular rate constants k(q) = 2.7 x 10(8) M(-1) s(-1) and 3.4 x 10(9) M(-1) s(-1), respectively. On the other hand, EB quenching is due to both dynamic and static mechanisms. In the presence of various types of DNA > 0.1 ng microL(-1) (dsDNA, ssDNA or circular DNA), the quenched luminescence is reinstated, AO and EB intercalating into DNA, which removes the interaction with the Eu(III) complexes. The best results are obtained with [Eu(2)(L(C2))(3)]/AO with detections limits in the range 0.18-0.66 ng microL(-1); detection limits for the [Eu(L(kel))]/AO system are slightly larger; simpler monometallic Eu(III) complexes with dipicolinate derivatives do not follow suit in that they decompose in the presence of DNA. The Eu(III)/AO method is shown to be pH insensitive in the range 3-10; furthermore it is essentially insensitive to 1000-fold excesses of potential interfering substances, e.g. BSA, glucose, chelating agents and anions, alkaline earth and transition metal cations, variations in luminescence intensity being < 5%, (10 analytes) or 5-10% (4 analytes); only Co(II) and Cu(II) interfere substantially.

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 microM of the Eu(III) helicate [Q = 0.21, tau = 2.43 ms], compared to >10 microM when using conventional luminescence microscopy. Furthermore, multiplex labeling could be achieved with the Tb(III) [Q = 0.11, tau = 0.65 ms], and Sm(III) [Q = 0.0038, tau = 0.030 ms] analogues.

Lanthanide bimetallic helicates for in vitro imaging and sensing.

As the need for targeting luminescent biolabels increases, for mapping selected analytes, imaging of cells and organs, and tracking in cellulo processes, lanthanide bimetallic helicates are emerging as versatile bioprobes. The wrapping of three ligand strands around two metallic centers by self-assembly affords robust molecular edifices with tunable chemical and photophysical properties. In addition, heterometallic helical chelates can be assembled leading to bioprobes with inherent chiral properties. In this paper, we review the literature demonstrating that neutral [Ln(2)(L(CX))(3)] (x=1-3) helicates represent a viable alternative to existing chelating agents for bio-analyses, while featuring specific enhanced properties. These bimetallic chelates self-assemble in water, and at physiological pH the 2:3 (Ln:L(CX)) complex is by far the dominant species, conditional stability constants logbeta(23) being in the range 23-30. The metal ions are 9-coordinate and lie in sites with slightly distorted D(3) symmetry. Efficient protection from water interaction by the tightly wrapped ligand strands results in sizeable photophysical properties, with quantum yields up to 24% for Eu(III) and 11% for Tb(III), while the luminescence of several other visible and/or near-infrared emitting Ln(III) ions is also sensitized. Noncytotoxicity for all the helicates is established for several living cell lines including HeLa, HaCat, MCF-7, 5D10, and Jurkat. We present new data pertaining to the live cell imaging ability of [Eu(2)(L(C1))(3)] and compare the three systems with x=1-3 with respect to thermodynamic stability, photophysics, cell-permeation ability, and targeting capability for sensing in cellulo processes. Prospects of derivatization for characterizing specific biological interactions are discussed.

A versatile ditopic ligand system for sensitizing the luminescence of bimetallic lanthanide bio-imaging probes.

The homoditopic ligand 6,6'-[methylenebis(1-methyl-1H-benzimidazole-5,2-diyl)]bis(4-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}pyridine-2-carboxylic acid) (H(2)L(C2)) has been tailored to self-assemble with lanthanide ions (Ln(III)), which results in the formation of neutral bimetallic helicates with the overall composition [Ln(2)(L(C2))(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 Ln(III) ions. The conditional stability constants (log beta(23)) are close to 25 at physiological pH and under stoichiometric conditions. The ligand triplet state features adequate energy (0-phonon transition at approximately 21 900 cm(-1)) to sensitize the luminescence of Eu(III) (Q=21 %) and Tb(III) (11 %) in aerated water at pH 7.4. The emission of several other VIS- and NIR-emitting ions, such as Sm(III) (Q=0.38 %) or Yb(III) (0.15 %), for which in cellulo luminescence is evidenced for the first time, is also sensitized. The Eu(III) emission spectrum arises from a main species with pseudo-D(3) 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 microM [Eu(2)(L(C2))(3)] during 24 h. Bright Eu(III) emission is seen for incubation concentrations above 10 microM and after a 15-minute loading time; similar images are obtained with Tb(III) and Sm(III). 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.

Non-cytotoxic, bifunctional EuIII and TbIII luminescent macrocyclic complexes for luminescence resonant energy-transfer experiments.

A new macrocyclic ligand, L3, has been synthesised, based on the cyclen framework grafted with three phenacyl light-harvesting groups and a C5-alkyl chain bearing a carboxylic acid function as a potential linker for biological material. Acidity constants are determined by spectrophotometric titrations, as well as conditional stability constants for the resulting 1:1 complexes with trivalent lanthanide ions. The complexes have stabilities comparable to 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (dtma) complexes, with pLn approximately 12-13. Photophysical properties of the ligand and of the EuL3 and TbL3 complexes have been determined for both microcrystalline samples and solutions in water and acetonitrile. They point to the metal ion being present in an environment with axial symmetry derived from the C4 point group. The hydration number determined for TbL3 decreases with increasing pH value and becomes fractional at pH 7.5, which points to an equilibrium between two differently solvated species and probably to the participation of the deprotonated carboxylic acid chain in the complexation. The quantum yields in water (1.9% for EuIII, 3.4% for TbIII) are smaller than those for complexes with the symmetrically substituted parent macrocycle, but efficient luminescence resonant energy transfer (LRET) was observed when Cy5 dye was added to the solutions. Finally, the influence of the TbL3 complex on cell viability is tested on both malignant (5D10 mouse hybridoma, Jurkat human T leukaemia, MCF-7 human breast carcinoma) and non-malignant (Hacat human keratinocyte) cell lines. Cell viability after 24 h incubation at 37 degrees C with 500 microM TbL3 was >90% for all cell lines, except Jurkat (>70%). All of these properties make LnL3 complexes interesting potential probes for bioanalyses.

A polyoxyethylene-substituted bimetallic europium helicate for luminescent staining of living cells.

The homoditopic ligand H2LC3 has been designed to form neutral triple-stranded bimetallic helicates of overall composition [Ln2(LC3)3]. The grafting of the polyoxyethylene fragments ensures water solubility and also favors cell penetration while being amenable to further derivatization. The ligand pKa values have been determined by spectrophotometric titration and range from 3.5 (sum of the first two) to 10.3. The thermodynamic stability of the helicates is large at physiological pH (logbeta23 in the range 22-23). The ligand triplet state has an adequate energy (0-phonon transition at approximately 20,800 cm(-1)) for sensitizing the luminescence of EuIII (Q=11%). Analysis of the EuIII emission spectrum points to an overall pseudo D3 symmetry for the metal environment. No significant effect of [Eu2(LC3)3] is observed on the viability of several cancerous cell lines (MCF-7, HeLa, Jurkat, and 5D10). The cell imaging properties of the EuIII helicate are demonstrated for the HeLa cell line by luminescence microscopy. Bright EuIII emission is seen for helicate concentration>50 microM and after 20-30 min loading time. The helicate stains the cytoplasm and the permeation mechanism is likely to be endocytosis.

Luminescent lanthanide bimetallic triple-stranded helicates as potential cellular imaging probes.

Water-soluble triple-stranded [Ln(2)(L)(3)] helicates have been successfully tested as imaging probes in human cervical adenocarcinoma cells (HeLa), the complex being not toxic and clearly staining their cytoplasm in a concentration-dependent manner.