Advantages of Lanthanides   |    Uses of Technology   |    Additional Applications

ADDITIONAL APPLICATIONS

Lumiphore’s technology can be adapted for use in a wide variety of applications. The only modifications required are minor changes in the linker in order to optimize the labeling of a wide range of substrates.

Radiopharmaceuticals: Targeted radioisotopes are deployed as imaging agents in the context of single-photon emission computed tomography and positron emission tomography. Such diagnostic agents are also used as a companion in targeted radioisotope therapy wherein a radionuclide which emits therapeutically useful ionizing radiation is similarly localized within specific biological sites by attachment to an accessory molecule that imparts appropriate biodistribution and pharmacokinetic properties. Metallic radioisotopes offer versatile imaging and therapeutic properties, but loss of metallic radioisotopes from their site-directing molecules can lead to deleterious side-effects or reduced contrast and efficacy. There is therefore a recognized, compelling need for improved chelating groups for use in radiopharmaceuticals. Such chelating groups must rapidly bind radioisotopes, so that they are compatible with the practicalities of clinical laboratory preparation. They must also stably bind the cation so that none is released in vivo, at least prior to its decay. Ideal chelating groups would stably coordinate metal cations currently used for radioisotope-based diagnosis and therapy, display facile complexation kinetics, and provide a convenient synthetic handle for attachment to targeting moieties.

To meet these needs, Lumiphore is developing a platform of novel macrocyclic chelating groups we term aromatic macrocyclic bifunctional chelators (AMBFC’s). These novel “caged” macrocyclic chelating groups display faster and more stable binding as compared to acyclic and mono-macrocyclic chelators currently used. AMBFC’s coordinate not only traditional metallic radioisotopes such as Y+3, but also more exotic cations such as Zr+4 and Th+4 whose isotopes have hitherto remained undeveloped but possess intriguing radiochemical characteristics. By means of this approach we aim both to improve the utility of existing radiopharmaceuticals and to expand the scope of this technology to radionuclides that are at present underdeveloped in the clinic.

In-Vitro Diagnostics: Diagnostic assays have relied on heterogeneous formats where at least one component is immobilized and analytical components are added in several stages with numerous washing steps integrated into the protocols. Often, these assays incorporate enzymatic tags such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) that are able to detect molecules at reasonably low concentrations because of the amplification of the signal conferred by the enzyme’s action on a chemiluminescent or colorimetric substrate. Unfortunately, these enzymes are mostly limited to the study of single molecular interactions and are not easily adaptable to multiplexing applications where one might be evaluating the presence of several molecules or interactions simultaneously. Also, heterogeneous assay formats are inherently slow due to limiting rates of diffusion between solution and solid phases.

Luminescent lanthanide complexes are ideally suited for homogeneous assay formats which are preferred for High Throughput Screening (HTS) applications. The small size, multiplexing potential, gated detection and favorable energy transfer properties of lanthanide complexes offer a high level of flexibility in assay design. In addition, the option to accumulate signal through multiple reads amplifies the signal relative to the background, further enhancing the limits of detection.

Fluorescence resonance energy transfer (FRET) between lanthanide donors and organic acceptors has been well studied. The degree of energy transfer between a donor and acceptor can be monitored by exciting the donor and observing emission of the acceptor. The extent of energy transfer (brightness of acceptor) correlates with the relative distance between the donor and acceptor. 

The binding of two different antibodies to a molecule possessing complementary antigens in close proximity can be evaluated by monitoring only the acceptor fluorescence. The transfer only works when the donor and acceptor are in close proximity, so the acceptor emission serves as a measure of the interaction under investigation. If the haptens are monovalent, only the binding event involving the lanthanide is detectable unless the organic acceptor is separately excited. Even then, the lifetime of the acceptor’s fluorescence is short preventing the use of time gated detection and reducing sensitivity.

Appropriately positioning donors and acceptors on two potentially interacting molecules yields information about binding and offers numerous advantages over single label fluorescence assays. No wash steps are required and the long-lived fluorescence of the lanthanide is transferred from the donor to the acceptor eliminating background fluorescence.

Lumiphore’s luminescent reagents offer greatly increased sensitivity and specificity in the biological assay market.

Our Lumi4®-Tb complex was featured recently in the Proceedings of the National Academy of Sciences (PNAS) in a study by Professor Lawrence W. Miller of the Department of Chemistry, University of Illinois, Chicago. The Lumi4-Tb complex linked to the antibiotic trimethoprim, (Lumi-4-Tb-trimethoprim), was utilized as a time-resolved luminescence imaging agent for microscopy of living cells. Trimethoprim binds to the protein Escherichia coli dihydrofolate reductase (eDHFR), which can be expressed as a fusion protein with proteins of interest using recombinant methods. Selective binding to eDHFR by Lumi4-Tb-trimethoprim thus provides a convenient method to attach Lumi4-Tb to proteins. In this paper, the authors showed that protein-protein interactions can be probed in live cells, using luminescence resonance energy transfer (LRET similar to FRET). For example, upon binding of Lumi4-Tb-trimethoprim to eDHFR expressed as a fusion protein with green fluorescence protein (GFP), energy is transferred from the excited Lumi4-Tb complex to GPF via LRET. As a result of the long-lived excited state of the Lumi4-Tb complex, the fluorescence lifetime of GFP is extended to ~0.8 ms. This enhancement of the lifetime of GFP from the nanosecond to near millisecond range allows for time-resolved microscopy imaging with high signal to noise, as the auto-fluorescence of the cells can be removed by applying a delay on the detector. By simultaneously expressing eDHFR and GFP as fusion proteins with other proteins of interest, the native interaction of these other proteins with each other can be monitored using LRET. It was also demonstrated that the Lumi4-Tb complex is resistant to photobleaching (under these imaging conditions) and is stable in the cells. The authors conclude that this novel technique is fifty-fold more sensitive than conventional steady-state FRET imaging. (H. E. Rajapakse, N. Gahlaut, S. Mohandessi, D. Yu, J. R. Turner, L. W. Miller, PNAS 2010, 107, 13582.).