Fluorescent Probes for Enzymatic Activity Detection

Fluorescent probes have emerged as indispensable tools for visualising and quantifying enzymatic processes in real time. By coupling enzyme‐specific recognition elements with sensitive fluorophores, these molecular sensors translate catalytic turnover into detectable optical signals. Designs range from “turn‐on” probes, which remain dark until enzymatic cleavage liberates a fluorescent moiety, to ratiometric systems that shift emission wavelengths upon reaction, thereby offering built-in correction for environmental fluctuations. Advances in photophysics—such as aggregation‐induced emission (AIE), large Stokes shifts and near‐infrared (NIR) chromophores—have extended imaging depth and reduced background, while responsive self‐assembly strategies enable on‐site amplification of signal. Collectively, these innovations allow high‐resolution mapping of enzyme distribution and activity in living cells, tissues and whole organisms, unlocking new avenues in diagnostics, drug screening and guided therapy.

Research from Nature Portfolio

In one seminal study, a β-galactosidase‐targeting probe was engineered with an intramolecular spirocyclic switch that produces over 1,400-fold fluorescence enhancement upon activation. This membrane-permeable agent visualises peritoneal metastases smaller than 1 mm in multiple ovarian cancer models, facilitating real-time detection with the naked eye or endoscopic guidance. Another work described a near-infrared sensor for NAD(P)H:quinone oxidoreductase 1 (NQO1) that remains quenched until enzymatic reduction liberates a dicyanoisophorone fluorophore. With a 186 nm Stokes shift and emission at 646 nm, this probe differentiates tumours from normal tissues in live mouse models with high signal-to-noise contrast. Complementing these, a pair of red‐emission probes was devised for monoamine oxidase detection, exhibiting large Stokes shifts (>200 nm), excellent photostability and cell imaging capability, thereby enabling sensitive interrogation of MAO activity in living cells and on paper‐based assays.

Fluorescent Probes for Enzymatic Activity Detection publication trend

The graph below shows the total number of articles in fluorescent probes for enzymatic activity detection across all publications each year (not limited to Nature Index journals).

Technical terms

Fluorogenic probe: A molecule that exhibits minimal fluorescence until enzymatic or chemical activation releases a bright emitter.

Turn-on probe: A sensor that increases in fluorescence intensity upon interaction with its target enzyme.

Ratiometric fluorescence: A method utilising two emission wavelengths to provide self-referencing measurements, reducing artefacts from probe concentration or environment.

Aggregation-induced emission (AIE): A phenomenon in which certain fluorophores emit more brightly when aggregated than when dissolved.

Stokes shift: The wavelength difference between a fluorophore’s peak absorption and peak emission, larger values reducing self-quenching and background interference.

Near-infrared (NIR): Light in the 650–900 nm range, which penetrates deeper into biological tissues with lower phototoxicity.

Self-assembly: The spontaneous organisation of molecular units into ordered structures, here exploited to amplify fluorescence signals upon enzymatic trigger.

References

1. Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo. Nature Communications (2015).
2. Characterization of a highly specific NQO1-activated near-infrared fluorescent probe and its application for in vivo tumor imaging. Scientific Reports (2019).
3. Red emission fluorescent probes for visualization of monoamine oxidase in living cells. Scientific Reports (2016).
4. Hierarchical Self‐Assembly Molecular Building Blocks as Intelligent Nanoplatforms for Ovarian Cancer Theranostics. Advanced Science (2024).
5. Rationally designed monoamine oxidase A‐activatable AIE molecular photosensitizer for the specific imaging and cellular therapy of tumors. Aggregate (2022).
6. A general strategy to the intracellular sensing of glycosidases using AIE-based glycoclusters. Chemical Science (2021).