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Current Chemical Genomics May 2009HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal...
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.
PubMed: 20161833
DOI: 10.2174/1875397300903010022 -
ACS Chemical Neuroscience Apr 2022Alzheimer's disease (AD) is the most common neurodegenerative disorder. Pathologically, the disease is characterized by the deposition of amyloid beta (Aβ) plaques and...
Alzheimer's disease (AD) is the most common neurodegenerative disorder. Pathologically, the disease is characterized by the deposition of amyloid beta (Aβ) plaques and the presence of neurofibrillary tangles. These drive microglia neuroinflammation and consequent neurodegeneration. While the means to affect Aβ plaque accumulation pharmacologically was achieved, how it affects disease outcomes remains uncertain. Cerium oxide (CeO) reduces Aβ plaques, oxidative stress, inflammation, and AD signs and symptoms. In particular, CeO nanoparticles (CeONPs) induce free-radical-scavenging and cell protective intracellular signaling. This can ameliorate the pathobiology of an AD-affected brain. To investigate whether CeONPs affect microglia neurotoxic responses, a novel formulation of europium-doped CeONPs (EuCeONPs) was synthesized. We then tested EuCeONPs for its ability to generate cellular immune homeostasis in AD models. EuCeONPs attenuated microglia BV2 inflammatory activities after Aβ exposure by increasing the cells' phagocytic and Aβ degradation activities. These were associated with increases in the expression of the CD36 scavenger receptor. EuCeONPs facilitated Aβ endolysosomal trafficking and abrogated microglial inflammatory responses. We posit that EuCeONPs may be developed as an AD immunomodulator.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cerium; Europium; Homeostasis; Mice; Mice, Transgenic; Microglia; Nanoparticles; Plaque, Amyloid
PubMed: 35312284
DOI: 10.1021/acschemneuro.1c00847 -
MAbs 2021Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of... (Comparative Study)
Comparative Study
Protein aggregation is a spontaneous process affected by multiple external and internal properties, such as buffer composition and storage temperature. Aggregation of protein-based drugs can endanger patient safety due, for example, to increased immunogenicity. Aggregation can also inactivate protein drugs and prevent target engagement, and thus regulatory requirements are strict regarding drug stability monitoring during manufacturing and storage. Many of the current technologies for aggregation monitoring are time- and material-consuming and require specific instruments and expertise. These types of assays are not only expensive, but also unsuitable for larger sample panels. Here we report a label-free time-resolved luminescence-based method using an external Eu-conjugated probe for the simple and fast detection of protein stability and aggregation. We focused on monitoring the properties of IgG, which is a common format for biological drugs. The Protein-Probe assay enables IgG aggregation detection with a simple single-well mix-and-measure assay performed at room temperature. Further information can be obtained in a thermal ramping, where IgG thermal stability is monitored. We showed that with the Protein-Probe, trastuzumab aggregation was detected already after 18 hours of storage at 60°C, 4 to 8 days earlier compared to SYPRO Orange- and UV250-based assays, respectively. The ultra-high sensitivity of less than 0.1% IgG aggregates enables the Protein-Probe to reduce assay time and material consumption compared to existing techniques.
Topics: Antineoplastic Agents, Immunological; Drug Compounding; Europium; High-Throughput Screening Assays; Hot Temperature; Immunoglobulin G; Luminescent Agents; Luminescent Measurements; Organometallic Compounds; Protein Aggregates; Protein Binding; Protein Denaturation; Protein Stability; Time Factors; Trastuzumab
PubMed: 34455913
DOI: 10.1080/19420862.2021.1955810 -
Molecules (Basel, Switzerland) Sep 2022Theranostics (bifunction of therapeutics and diagnostics) has attracted increasing attention due to its efficiency that can reduce the physical and financial burden on... (Review)
Review
Theranostics (bifunction of therapeutics and diagnostics) has attracted increasing attention due to its efficiency that can reduce the physical and financial burden on patients. One of the promising materials for theranostics is calcium phosphate (CP) and it is biocompatible and can be functionalized not only with drug molecules but also with rare earth ions to show photoluminescence that is necessary for the diagnostic purpose. Such the CP-based hybrids are formed in vivo by interacting between functional groups of organic molecules and inorganic ions. It is of great importance to elucidate the interaction of CP with the photofunctional species and the drug molecules to clarify the relationship between the existing state and function. Well-designed photofunctional CPs will contribute to biomedical fields as highly-functional ormultifunctional theranostic materials at the nanoscales. In this review, we describe the hybridization between CPs and heterogeneous species, mainly focusing on europium(III) ion and methylene blue molecule as the representative photofunctional species for theranostics applications.
Topics: Calcium Phosphates; Europium; Humans; Ions; Methylene Blue; Precision Medicine; Theranostic Nanomedicine
PubMed: 36144659
DOI: 10.3390/molecules27185916 -
Chemistry, An Asian Journal Aug 2022Inorganic and organic phosphates-including orthophosphate, nucleotides, and DNA-are some of the most fundamental anions in cellular biology, regulating numerous... (Review)
Review
Inorganic and organic phosphates-including orthophosphate, nucleotides, and DNA-are some of the most fundamental anions in cellular biology, regulating numerous processes of both medical and environmental significance. The characteristic long lifetimes of emitting lanthanides, including the brighter europium(III) and terbium(III), make them ideally suited for the development of molecular probes for the detection of phosphates directly in complex aqueous media. Moreover, given their high oxophilicity and the exquisite sensitivity of their quantum yields to their hydration number, those luminescent lanthanides are perfect for the detection of phosphates. Herein we discuss the principles that have guided the recent developments of molecular probes selective for inorganic or organic phosphates and how these lanthanide complexes facilitate the study of numerous biological processes.
Topics: Europium; Lanthanoid Series Elements; Luminescence; Luminescent Agents; Luminescent Measurements; Molecular Probes; Organophosphates; Phosphates
PubMed: 35750633
DOI: 10.1002/asia.202200495 -
Journal of Hazardous Materials Feb 2019The final repository for short-lived, low and intermediate level radioactive waste in Sweden is built to act as a passive repository. Already within a few years after...
The final repository for short-lived, low and intermediate level radioactive waste in Sweden is built to act as a passive repository. Already within a few years after closure water will penetrate the repository and conditions of high alkalinity (pH 10.5-13.5) and low temperature (< 7 °C) will prevail. The mobility of radionuclides in the repository is dependent on the radionuclides distribution between solid and liquid phases. In the present work the incorporation of strontium (II) and europium (III) in α-calcium isosaccharinate (ISA) under alkaline conditions (pH ∼10) at 5 °C and 50 °C have been studied. The results show that strontium and europium are incorporated into α-Ca(ISA) when crystallized both at 5 °C and 50 °C. Europium is incorporated to a greater extent than strontium. The highest incorporation of europium and strontium at 5 °C rendered the phase compositions CaEu(ISA) (2.4% of Eu(ISA) by mass) and CaSr(ISA) (2.2% of Sr(ISA) by mass). XPS spectra show that both trivalent and divalent Eu coexist in the Eu incorporated samples. Strontium ions were found to retard the elongated growth of the Ca(ISA) crystals. The incorporation of Sr and Eu into the solid phase of Ca(ISA) is expected to contribute to a decreased mobility of these ions in the repository.
PubMed: 30384240
DOI: 10.1016/j.jhazmat.2018.10.001 -
International Journal of Molecular... Nov 2022In this work, we synthesized a polydimethylsiloxane membrane containing two emitter groups chemically attached to the membrane structure. For this, we attached the...
In this work, we synthesized a polydimethylsiloxane membrane containing two emitter groups chemically attached to the membrane structure. For this, we attached the anthracene group and the [Eu(bzac)] complex as blue and red emitters, respectively, in the matrix via hydrosilylation reactions. The synthesized membrane can be used as a bifunctional temperature and oxygen ratiometric optical probe by analyzing the effects that temperature changes and oxygen levels produce on the ratio of anthracene and europium(III) emission components. As a temperature probe, the system is operational in the 203-323 K range, with an observed maximum relative sensitivity of 2.06% K at 290 K and temperature uncertainties below 0.1 K over all the operational range. As an oxygen probe, we evaluated the ratiometric response at 25, 30, 35, and 40 °C. These results show an interesting approach to obtaining bifunctional ratiometric optical probes and also suggest the presence of an anthracene → europium(III) energy transfer, even though there is no chemical bonding between species.
Topics: Europium; Oxygen; Luminescence; Anthracenes
PubMed: 36498852
DOI: 10.3390/ijms232314526 -
Nano Letters May 2021Cerenkov imaging provides an opportunity to expand the application of approved radiotracers and therapeutic agents by utilizing them for optical approaches, which opens...
Cerenkov imaging provides an opportunity to expand the application of approved radiotracers and therapeutic agents by utilizing them for optical approaches, which opens new avenues for nuclear imaging. The dominating Cerenkov radiation is in the UV/blue region, where it is readily absorbed by human tissue, reducing its utility . To solve this problem, we propose a strategy to shift Cerenkov light to the more penetrative red-light region through the use of a fluorescent down-conversion technique, based upon europium oxide nanoparticles. We synthesized square-shape ultrasmall EuO nanoparticles, functionalized with polyethylene glycol and chelate-free radiolabeled for intravenous injection into mice to visualize the lymph node and tumor. By adding trimethylamine N-oxide during the synthesis, we significantly increased the brightness of the particle and synthesized the (to-date) smallest radiolabeled europium-based nanoparticle. These features allow for the exploration of EuO nanoparticles as a preclinical cancer diagnosis platform with multimodal imaging capability.
Topics: Animals; Europium; Mice; Multimodal Imaging; Nanoparticles
PubMed: 33950695
DOI: 10.1021/acs.nanolett.1c00049 -
Materials Today. Bio Apr 2023The biological functions of rare-earth elements (REEs) have become a focus of intense research. Recent studies have demonstrated that ion doping or alloying of some REEs... (Review)
Review
The biological functions of rare-earth elements (REEs) have become a focus of intense research. Recent studies have demonstrated that ion doping or alloying of some REEs can optimize the properties of traditional biomaterials. Europium (Eu), which is an REE with low toxicity and good biocompatibility, has promising applications in biomedicine. This article systematically reviews the osteogenic, angiogenic, neuritogenic, antibacterial, and anti-tumor properties of Eu-containing biomaterials, thereby paving the way for biomedical applications of Eu. Data collection for this review was completed in October 2022, and 30 relevant articles were finally included. Most articles indicated that doping of Eu ions or Eu-compound nanoparticles in biomaterials can improve their osteogenic, angiogenic, neuritogenic, antibacterial, and anti-tumor properties. The angiogenic, antibacterial, and potential neuritogenic effects of Eu(OH) nanoparticles have also been demonstrated.
PubMed: 36910271
DOI: 10.1016/j.mtbio.2023.100595 -
Materials (Basel, Switzerland) May 2021In this paper, useful models that enable time-efficient computational analyses of the performance of luminescent solar concentrators (LSCs) are developed and thoroughly...
In this paper, useful models that enable time-efficient computational analyses of the performance of luminescent solar concentrators (LSCs) are developed and thoroughly described. These LSCs are based on polymer optical fibers codoped with organic dyes and/or europium chelates. The interest in such dopants lies in the availability of new dyes with higher quantum yields and in the photostability and suitable absorption and emission bands of europium chelates. Time-efficiency without compromising accuracy is especially important for the simulation of europium chelates, in which non-radiative energy transfers from the absorbing ligands to the europium ion and vice versa are so fast that the discretization in time, in the absence of some simplifying assumptions, would have to be very fine. Some available experimental results are also included for the sake of comparison.
PubMed: 34069675
DOI: 10.3390/ma14102667