Fluorogenic Probe Assay
Fluorogenic Probe Assay - Related Videos
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2025
This video demonstrates a flow cytometry assay to quantify the ROS levels in 3D intestinal organoids obtained from GFP-expressing transgenic mice. This method provides an in vitro experimental model to investigate the efficacy of test compounds on ROS production.
09:31
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Cited by 1 •
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02:42
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2025
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13:20
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Cited by 18 •
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We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental...
08:11
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Cited by 3 •
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The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.
FAQs
Signal generation depends on a structural change in the probe. Target binding or enzymatic activity can alter the probe so that a quencher is separated or cleaved from a fluorophore. Once quenching decreases, fluorescence rises, linking the measured signal to the underlying molecular event and allowing the reaction or target level to be monitored.
A quencher suppresses fluorescence while the probe remains in its initial state, helping keep background low before the target-dependent reaction occurs. When the quencher is cleaved from the fluorophore, the signal increases substantially. This contrast between the starting and activated states supports sensitive detection and improves measurement of relatively small biochemical changes.
The measured fluorescence reflects whichever event changes the probe structure. In one design, target binding produces the structural change; in another, an enzyme cleaves part of the probe, including a quencher. Interpreting the signal therefore requires relating fluorescence to the intended molecular event, whether the goal is target detection or activity measurement.
Quantification depends on the relationship between fluorescence and the amount of target or extent of reaction. In these assays, fluorescence is described as increasing in proportion to target concentration or reaction progress. The resulting signal can therefore support quantitative biochemical measurements, provided the measured fluorescence is interpreted within the assay’s intended detection context.
A typical workflow places the probe with the target or reaction system, allows binding or enzymatic activity to alter the probe, and measures the resulting fluorescence. The signal can be followed as the reaction proceeds, producing a real-time record rather than only an endpoint observation. Microplate and imaging platforms can support this measurement.
These assays support several measurement types, including enzyme activity, nucleic-acid detection, cellular-process monitoring, and quantitative biochemical reactions. The same signal-generation principle can therefore be applied to both purified reaction systems and biological samples. Selection depends on whether the experiment needs target detection, activity tracking, or observation of changes within cells.
Their specificity and quantitative fluorescence readout make them useful when experiments must detect or compare molecular activity. Compatibility with microplate formats supports drug-screening workflows, while sensitive target measurement can contribute to diagnostic applications. Real-time monitoring also helps researchers follow biochemical reactions and evaluate how experimental conditions affect the measured signal.