Homogeneous Time-Resolved Fluorescence Quenching Assay (TruPoint) for Nucleic Acid Detection

Abstract

Reactive oxygen species are highly reactive molecules and have been implicated in the pathophysiology of many diseases, including diabetes mellitus, cancer, rheumatoid arthritis, and cardiovascular, renal, inflammatory, infectious, and neurologic diseases (1, 2 ). Cells and biological fluids have an array of protective antioxidant mechanisms, both for preventing the production of free radicals and for repairing oxidative damage (3 ). These antioxidant systems include enzymes, macromolecules, and small molecules, including ascorbic acid, ␣-tocopherol, ␤-carotene, ubiquinol-10, reduced lipoic acid (DHLA), reduced glutathione (GSH), methionine, uric acid, bilirubin, and some amino acids. Antioxidants within cells, cell membranes, and extracellular fluids can be up-regulated and mobilized to neutralize excessive and inappropriate formation of reactive oxygen species, but a deficiency of antioxidant defense may lead to a situation of increased oxidative stress.Assays that measure the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of ability to resist oxidative damage. Several methods (4 -6 ) have been developed to assess the total antioxidant capacity of human serum or plasma because of the difficulty in measuring each antioxidant component separately and the interactions among different antioxidant components in the serum or plasma. However, the measured antioxidant capacity of a sample depends on which technology and which free radical generator or oxidant is used in the measurement. The ferric reducing ability of plasma (FRAP) assay uses an easily reduced oxidant in a redox-linked colorimetric method (6 ). Because the redox potential of the ferrous/ ferric couple is 0.77 V, any substance or antioxidant with a redox potential Ͻ0.77 V will drive the ferric reduction, assuming stability of redox product. According to a study by Cao and Prior (7 ), the FRAP assay does not measure serum proteins and excludes the low-molecular-weight SH-group-containing antioxidants, such as GSH, DHLA, and some amino acids. We developed a novel method for measuring the ubiquinone-9-reducing ability of plasma (URAP). This new method estimates antioxidant systems with redox potentials Յ0.1 V, which will drive the reduction of ubiquinone-9 (Fig. 1A).HPLC analysis was performed with an ESA Model 582 Solvent Delivery Module, AS1000 autosampler (Thermo Separation Products), and a reversed-phase Microsorb-MV column [150 ϫ 4.6 mm (i.d.); 5-m bead size] from Rainin. The mobile phase consisted of a mixture of anhydrous sodium acetate (4.2 g; Sigma), 15 mL of glacial acetic acid (Mallinckrodt), 15 mL of 2-propanol (Mallinckrodt), 720 mL of methanol (Fisher), and 250 mL of hexane (Fisher). The flow rate was 1.1 mL/min. The electrochemical detector with ESA Model 5200A CouloChem II has been described previously (8 ). The electrochemical cells were a postcolumn guard cell and analytical cell containing dual electrodes in series. The potential of the postcolumn guard cell was set at ϩ0...

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

“…The luminescence yields obtained were high, although the decay time of 40 was surprisingly short. It is worth noting that although the decay times are shorter and luminescence yields much lower in the case of samarium and dysprosium chelates than in the case of europium and terbium chelates, they are valuable when multiparametric assays are developed (4,6,28).…”

“…The development of stable and luminescent lanthanide(III) chelates has enabled the use of homogeneous assay technologies based on time resolution (). Thus, time-resolved fluorescence quenching assays based on energy transfer from a lanthanide(III) chelate to a nonfluorescent quencher have been applied in various assays of hydrolyzing enzymes ( − ) as well as for nucleic acid detection ( , ). The different photochemical properties of europium, terbium, dysprosium, and samarium chelates enable development of even multiparametric homogeneous assays ( , ).…”

Homogeneous Assay Based on Anti-Stokes' Shift Time-Resolved Fluorescence Resonance Energy-Transfer Measurement

“…The result is shown in Figure . Without any further optimization of assay conditions or optical filters, the detection limit (average + 3stdev of 12 blanks) of 0.8 pM DNA target was obtained, which compared favorably to ∼1 nM sensitivity obtained with a respective TR-FQA using the same donor 6 Dilution curve for homogeneous dF508 DNA assay using Alexa Fluor 546 acceptor.…”

Luminescent lanthanide reporters: new concepts for use in bioanalytical applications

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