Luminescent Lanthanide Chelates Research Articles

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization

BackgroundNeurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases.MethodsIn this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples.ResultsSignificant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model.ConclusionsAdvantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.

AMPED: a new platform for picolinate based luminescent lanthanide chelates.

The synthesis of a new nonacoordinating ligand based on an AMPED (6-amino-6-methylperhydro-1,4-diazepine) scaffold functionalized by three picolinate (6-carboxy-2-methylpyridine) arms is described. Coordination of lanthanide cations (Ln = Eu and Tb) was investigated by spectrophotometric titrations monitored by UV-Vis absorption and steady-state emission spectroscopy, showing the formation of [LnL] complexes in aqueous solutions. The corresponding Eu and Tb complexes were isolated and characterized, and their spectroscopic properties (luminescence quantum yields, excited state lifetimes) were determined in buffered water (TRIS/HCl, pH 7.4) and compared to the data reported in the literature for related systems. DFT modelling of the complexes showed the picolinate arms to be perfectly wrapped around the Ln(3+) cations, affording an excellent shielding of the metal as confirmed by the determination of the hydration number of q = 0 for both complexes. The high resolution emission spectrum was used to determine the radiative lifetime of Eu in the complex (τrad = 3.05 ms) and the metal-centred luminescence quantum yield (0.20). The modest 0.10 overall luminescence quantum yield of the Eu complex is a consequence of an energy transfer with medium efficiency (0.50) and a low metal centred luminescence efficiency attributed in part to the presence of numerous NH and CH bonds in close proximity to the metal centre.

ChemInform Abstract: Utilization of the Photophysical and Photochemical Properties of Phosphorescent Transition Metal Complexes in the Development of Photofunctional Cellular Sensors, Imaging Reagents, and Cytotoxic Agents

AbstractReview: [87 refs.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Labeling of Oligopeptides with Luminescent Lanthanide(III) Chelates on Solid Phase

Synthesis of a building block based on 6,6-(1H-pyrazole-1,3-diyl)bis(pyridine) (3) that allows introduction of photoluminescent lanthanide(III) chelates to the synthetic oligopeptides on solid phase using the standard Fmoc chemistry is described. Since the ligand based on (3) forms luminescent chelates with Eu3+, Sm3+, Tb3+ and Dy3+ ions, the ligand is highly useful for multiparametric homogenous assays simplifying considerably the preparation of chelates needed in these applications. Keywords: Lanthanide(III) chelates, oligopeptides, solid phase synthesis, labeling

Bioconjugation with Stable Luminescent Lanthanide(III) Chelates Comprising Pyridine Subunits

The use of long-lifetime emitting lanthanide(III) chelate labels or probes together with time-resolved fluorometry in detection provides a method to generate sensitive bioaffinity assays. However, the use of stable chelates demands very complicated optimization of the chelate structure. A great number of chelates have been synthesized, but usually, only the most prominent structures were then converted to corresponding biomolecule labeling reactants. This review covers the syntheses of luminescent lanthanide chelates comprising a pyridine subunit that allow solution and solid-phase bioconjugation.

New 9- or 10-Dentate Luminescent Lanthanide Chelates

Polyaminocarboxylate-based luminescent lanthanide complexes have unusual emission properties, including millisecond excited-state lifetimes and sharply spiked spectra compared to common organic fluorophores. There are three distinct sections in the structure of the luminescent lanthanide chelates: a polyaminocarboxylate backbone to bind the lanthanide ions tightly, an antenna molecule to sensitize the emission of lanthanide ions, and a reactive group to attach to biomolecules. We have previously reported the modifications on the chelates, on the antenna molecules (commonly cs124), and on the reactive sites. In searching for stronger binding chelates and better protection from solvent hydration, here we report the modification of the coordination number of the chelates. A series of 9- and 10-dentate chelates were synthesized. Among them, the 1-oxa-4,7-diazacyclononane (N2O)-containing chelate provides the best protection to the lanthanide ions from solvent molecule attack, and forms the most stable lanthanide coordination compounds. The TTHA-based chelate provides moderately good protection to the lanthanide ions.

Labeling of proteins and oligopeptides with luminescent lanthanide(III) chelates

Synthesis of a building block that allows introduction of photoluminescent europium(III) and samarium(III) chelates to synthetic oligopeptides on solid phase using standard Fmoc chemistry is described. Upon completion of the oligopeptide synthesis, these conjugates were converted to the corresponding lanthanide(III) chelates by treatment with appropriate lanthanide(III) salt. Also synthesis of a new terpyridine-based europium(III) chelate designed for solution phase protein labeling is demonstrated.

Progress in Lanthanides as Luminescent Probes

Lanthanides have recently found applications in different fields of biomolecular and medical research. Luminescent lanthanide chelates have created interest mainly due to their unique luminescent properties, such as their long Stokes' shift and exceptional decay times allowing efficient temporal discrimination of background interferences in the assays, such as immunoassays. Recently, new organometallic complexes have been developed giving opportunities to novel applications, in heterogeneous and homogeneous immunoassays, DNA hybridization assays, high-throughput screening as well as in imaging. In addition, encapsulating the chelates into suitable matrix in beads enables the use of new members of lanthanides extending the emission wavelength to micrometer range and decays from a few microseconds to milliseconds. As the luminescence is derived from complicated intra-chelate energy transfer, it also gives novel opportunities to exploit these levels in different types of energy transfer based applications. This review gives a short overview of recent development of lanthanide chelate-labels and discusses in more details of energy levels and their exploitation in new assay formats.

Solid-Phase Synthesis of Oligonucleotides Labeled with Luminescent Lanthanide(III) Chelates

The synthesis of phosphoramidite building blocks that allow introduction of luminescent europium(III), terbium(III), dysprosium(III), and samarium(III) chelates to oligonucleotides on the solid phase is described. Several labeled oligonucleotides using these building blocks were prepared, and the photophysical properties of these bioconjugates were investigated.

On-resin assembly of a linkerless lanthanide(III)-based luminescence label and its application to the total synthesis of site-specifically labeled mechanosensitive channels.

A synthesis strategy for the on-resin assembly of luminescent lanthanide chelates from commercially available compounds was developed. Advantages of the approach include the absence of spacers between the metal ion and the attachment site, and the compatibility with typical chemical protein synthesis protection schemes. Methoxycoumarin-labeled lysine and tris(tert-butyl)-DOTA were consecutively coupled with high efficiency to a free amino group in otherwise fully protected peptide segments using standard peptide synthesis methods. Addition of stoichiometric amounts of Tb(3+) to the modified, cleaved, and purified peptides yielded the desired lanthanide chelate. Incorporation of this label into a chemically synthesized, full-length mechanosensitive channel of large conductance (MscL) of E. coli and subsequent reconstitution into vesicles resulted in a functional mechanosensitive channel of comparable conductance to the wild-type channel. However, this channel required increased suction to gate. Excitation of the antenna molecule methoxycoumarin at 336 nm resulted in an emission spectrum typical for Tb(3+) and a luminescence lifetime of 0.67 ms. The location of the probe close to the backbone of this protein may provide precise information about conformational changes during channel opening from LRET studies.

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates.

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.

Derivatisation of microcystin with a redox-active label for high-performance liquid chromatography/electrochemical detection

Microcystins are a group of low molecular weight, cyclic peptide hepatotoxins. The most common detection and quantitation methods for these toxins are liquid chromatography with UV or mass spectrometric detections, phosphatase inhibition assays and enzyme-linked immunosorbent assays. In addition, derivatisation of these toxins with organic fluorophores followed by CE/laser induced fluorescence detection and HPLC/chemiluminescence detection; and with luminescent lanthanide chelates for competition assays have also been reported. However, the use of an electrochemical-active unit as a tag for microcystins has never been explored. Since the sulfhydryl group of 6-ferrocenylhexanethiol (Fc-C6-SH) can undergo a facile addition reaction with the α,β-unsaturated carbonyl group, this compound has been used as a redox-active labelling agent for a derivative of microcystins, microcystin-LR (MC-LR). The conjugate, Fc-MC-LR, has been isolated by high-performance liquid chromatography with electrochemical detection. The peak height-concentration curve was linear in the test range 20–400 ng of MC-LR (r value for linear regression >0.9987). The detection limit was determined to be ca. 18 ng MC-LR (S/N = 3). Meanwhile, the conjugate Fc-MC-LR has also been characterised by positive-ion electrospray-ionisation mass spectrometry. Electrochemical studies show that the adduct displays a reversible ferrocenium/ferrocene couple at ca. −0.040 V vs. SCE (scan rate = 50 mV s−1) in 0.1 M aqueous ammonium acetate–acetonitrile (55∶45 v/v).

Oligonucleotide conjugates based on acyclonucleosides and their use in DNA hybridization assays

Synthesis of two oligonucleotide building blocks based on acyclonucleosides ( 10 , 11 ) which enable the introduction of several nonluminescent and luminescent lanthanide(III) chelates to the oligonucleotide structure is described. They were used in an instrument-assisted DNA synthesis in a standard manner. A modified deprotection procedure was used to ensure metal complexation. Also the applicability of these oligonucleotide conjugates to DNA hybridization assays is demonstrated.

Introduction of Lanthanide(III) Chelates to Oligopeptides on Solid Phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).

Quantum yields of luminescent lanthanide chelates and far-red dyes measured by resonance energy transfer.

Luminescent lanthanide chelates have unusual spectroscopic characteristics that make them valuable alternative probes to conventional organic fluorophores. However, fundamental parameters such as their quantum yield, and radiative and nonradiative decay rates have been difficult or impossible to measure. We have developed a simple and robust method based on resonance energy transfer to accurately measure these parameters. In addition, the excitation/emission process in lanthanide chelates involves several steps, and we are able to quantify each step. These include excitation of an organic antenna, transfer of energy from the antenna to lanthanide, and then lanthanide emission. Overall, the parameters show that lanthanide chelates can be efficient long-lived emitters, making them sensitive detection reagents and excellent donors in resonance energy transfer. The method is also shown to be applicable to photophysical characterization of red-emitting dyes, which are difficult to characterize by conventional means.

Luminescent lanthanide chelates—a way to more sensitive diagnostic methods

The unique luminescence properties of certain lanthanides are valuable features in aiming at more sensitive label technologies for clinical diagnostic use. The use of these properties in bioanalytical assays requires development of specific chelate technologies for labelling, i.e. binding of the ion to bioaffinity reagents, carrying it through the assay procedure, creating the optimal environment for emission and so forth. So far the most successful lanthanide label technology is composed of sequential use of two different chelate systems: a bifunctional polyaminopolycarboxylate derivative used for labelling of bioaffinity reactants with the lanthanide ion and a β-diketone-TOPO chelate optimized for time-resolved fluorometric determinations. This technology has achieved an established position as a highly sensitive method in in vitro clinical immunodiagnostics. Subsequently, a number of alternative approaches have been developed, including stable and highly luminescent chelates, which can be directly coupled to bioaffinity reactants to form luminescent tracers. These chelates are invaluable in studies aiming at sensitive and quantifiable localizations of analytes directly in situ, e.g. on tissue sections or cell surfaces.

Effect of C-H bonds on the quenching of luminescent lanthanide chelates

The quenching of europium(III) and terbium(III) chelate luminescence by high-energy C-H vibrational manifolds was studied with two types of stable chelates, i.e., a seven-dentate phenylethynylpyridine derivative and a nine-dentate terpyridine derivative. The replacement of C-H bonds by C-D bonds in the chelating parts of the ligands had a clear positive effect on Eu(3+) luminescence but a negligible effect on Tb(2+) luminescence. In aqueous solution, however, the positive effect was undetectable, if the chelating ligand did not create complete shielding of the ion against aqueous quenching. In chelates, where the coordination of water molecules to the inner sphere is prevented, the residual quenching through C-H vibrational quanta can be avoided by replacement of all C-H bonds in the vicinity of the emitting ion by C-D bonds.