Abstract Background: Nanoparticle-based detection technologies have the potential to improve sensitivity in miniature as well conventional biochemical assays. We introduce a technology that relies on use of europium(III) nanoparticles and time-resolved fluorometry limit assays visualize individual molecules microtiter plate format. Methods: Streptavidin was covalently coated 107-nm containing >30 000 europium entrapped with β-diketones. In model assay system, these were used trace...

The monovalent binding affinity of high site density nanoparticle-antibody bioconjugates is shown to exceed the intrinsic original, monoclonal antibody. were prepared by covalent coupling antibodies long-lifetime fluorescent, europium(III) chelate nanoparticles, 107 nm in diameter. Experiments carried out standard microtitration wells determine solid-phase association and dissociation rate constants, nonspecific binding, constants various conventionally labeled constant for a bioconjugate...

The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and enhanced monovalent binding affinity multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay.We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) (107 nm in diameter) were coated with monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). had an average 214 active sites per...

Preparation and characterization of europium(III), terbium(III), samarium(III), dysprosium(III) polystyrene nanoparticle labels with lanthanide-specific fluorescence properties has been presented. Emulsion copolymerization styrene acrylic acid was used to synthesize uniform-sized nanoparticles ∼45 nm in diameter. Europium(III) samarium(III) lanthanides were chelated 2-naphthoyltrifluoroacetone trioctylphosphine oxide dye the spherical particles, whereas terbium(III) chelate complexes...

We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited linear dose-dependent pattern within range 0.01 to 100 ng/ml and was approximately 100-fold more than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or collected mice injected lethal factor edema alone. For spiked sensitivities ELISA 100% (11/11...

Persistent luminescence (PeL) materials are used in everyday glow‐in‐the‐dark applications and they show high potential for, e.g., medical imaging, night‐vision surveillance, enhancement of solar cells. However, the best performing contain rare earths and/or other heavy metal expensive elements such as Ga Ge, increasing production costs. Here, (Li,Na) 8 Al 6 Si O 24 (Cl,S) 2 :Ti, a heavy‐metal‐ rare‐earth‐free low‐cost material is presented. It can give white PeL that stays 7 h above 0.3 mcd...

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange RAS/RAF-RBD interaction assays. mutations are frequent drivers of multiple different human cancers, but the development therapeutic strategies has been challenging. Traditionally, efforts to disrupt function focused on inhibitors, GTP-RAS activators increasing GTPase activity mutant proteins. As amount biological knowledge grows, targeted biochemical assays enabling...

Abstract Hyperactive Ras signalling is found in most cancers. proteins are only active membrane nanoclusters, which therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with binding domain (RBD) of Raf. Here, we establish B-Raf preference Gal1 emerges from divergence Raf RBDs at their proposed Gal1-binding interface. then identify L5UR peptide, disrupts this by low micromolar affinity to B-...

Adsorption of sample protein to Eu(3+) chelate-labeled nanoparticles is the basis developed noncompetitive and homogeneous method for estimation isoelectric point (pI). The lanthanide ion nanoparticle surface-conjugated chelate dissociated at a low pH, therefore decreasing luminescence signal. A nanoparticle-adsorbed prevents dissociation chelate, leading high adsorption efficiency reduced above due decreased electrostatic attraction between negatively charged particle. Four proteins with...

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent in are poorly characterized for their oncogenic potential. Yet insight into mechanism action may point to novel opportunities target Ras. Here, we show that several cancer-associated the switch III region moderately increase activity all isoforms. Mutants biochemically inconspicuous, while clustering nanoscale signaling complexes on plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates...

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and profiles of biotherapeutics can help to screen or study potential drugs find stabilizing destabilizing conditions. Differential scanning calorimetry (DSC) is 'Gold Standard' for assays (TSA), but there also multitude other methodologies, such differential fluorimetry (DSF). The use an external probe increases assay throughput, making it...

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2ALEKEVAQLEHEG-amide and basic X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study kinetics folding a heterodimeric investigate role electrostatic attraction between oppositely charged peptide chains reaction. Each alone did not form at ionic strength (μ) <1 M because repulsion like charges homodimer....

A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The concentration determined by observing magnitude fluorescence altered quenching particles a homogeneous assay format. Under optimal low pH conditions, allowed determination picogram quantities (7.0 μg/L) with an average variation 4.5% 10 min assay. sensitivity was more than...

In this article, we report on the formation and mode-of-operation of an affinity biosensor, where alternate layers biotin/streptavidin/biotinylated-CRP-antigen/anti-CRP antibody are grown printed gold electrodes disposable paper-substrates. We have successfully demonstrated detected consecutive supra-molecular protein assembly using electrical (impedimetric) technique. The process is also supplemented verified conventional surface plasmon resonance (SPR) measurements sensitive...

Derivatives of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine europium(III) (1) bearing one (6) or two (7) additional iminodiacetate coordinating arms have been synthesized. 6 and 7 were significantly more stable than 1 as evidenced by competition experiments with ethylenediaminetetraacetic acid (EDTA) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic (DOTA). While the luminescence quantum yield remained modest, other complexes displayed...

In this article, a single-label separation-free fluorescence technique is presented as potential screening method for cell-based receptor antagonists and agonists.The time-resolved technique, quenching resonance energy transfer (QRET), relies on single-labeled binding partner in combination with soluble quencher. The quencher efficiently suppresses the luminescence of unbound labeled ligand, whereas bound fraction not affected. This approach allows development assays simple cost-effective...

A non-competitive homogeneous, single-label quenching resonance energy transfer (QRET) assay for protein quantification is now presented using lanthanide-chelate labeled nucleic acid aptamers. ssDNA aptamer binding to a growth factor has been successfully used provide luminescence signal protection of the lanthanide label. The QRET technology previously applied competitive formats, but first time direct presented. system based on Eu(III)-chelate from soluble quencher molecule when interacts...

In modern biochemistry, protein stability and ligand interactions are of high interest. These properties often studied with methods requiring labeled biomolecules, as the existing utilizing luminescent external probes suffer from low sensitivity. Currently available label-free technologies, e.g., thermal shift assays, circular dichroism, differential scanning calorimetry, enable studies on unfolding protein–ligand (PLI). Unfortunately, required micromolar concentration increases costs...