Fluorescence-Based Protease Detection Strategies

Fluorescence-based protease detection exploits the light-emitting properties of molecular probes that change their emission in response to enzymatic cleavage. Central to these strategies are fluorogenic substrates that remain non-fluorescent until a specific protease hydrolyses a peptide linkage, triggering a “turn-on” signal. Common designs incorporate Förster resonance energy transfer (FRET) pairs in which a donor dye’s emission is quenched by an acceptor until proteolysis separates them. Alternative approaches utilise aggregation-induced quenching, in which dye molecules aggregate on a nano-scaffold and regain fluorescence upon enzyme-mediated release. Recent advances harness near-infrared (NIR) fluorophores, aptamer-based molecular switches and quantum dots or metal-doped nanoparticles to extend detection into biological tissues and living cells. These platforms offer high sensitivity, rapid kinetics and compatibility with real-time and in vivo imaging. Applications span biomedical diagnostics, drug screening and environmental monitoring, with practical examples including point-of-care assays for disease biomarkers and non-invasive tumour imaging. Ongoing research focuses on improving multiplexing, enhancing biocompatibility and achieving sub-nanomolar detection limits in complex media.

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Fluorescence-Based Protease Detection Strategies publication trend

The graph below shows the total number of articles in fluorescence-based protease detection strategies across all publications each year (not limited to Nature Index journals).

Technical terms

Fluorogenic probe: A molecule that emits fluorescence only after enzymatic or chemical activation.
Turn-on sensor: A detection scheme in which a fluorophore gains brightness upon interaction with the target enzyme.
Förster resonance energy transfer (FRET): Distance-dependent energy transfer between a donor fluorophore and an acceptor quencher, disrupted by proteolysis.
Aggregation-induced quenching (AIQ): Self-assembly of dye molecules into non-emissive aggregates, reversed by enzymatic cleavage.
Aptamer: A short nucleic acid sequence selected to bind a target molecule and undergo a conformational change upon interaction.
Near-infrared (NIR) fluorescence: Emission in the 650–900 nm range, advantageous for deep-tissue imaging due to low autofluorescence and high penetration.
Quantum dot: A semiconductor nanocrystal that emits size-tunable fluorescence with high brightness and stability.

References

1. NIR-Sensitive Squaraine Dye—Peptide Conjugate for Trypsin Fluorogenic Detection. Biosensors (2024).
2. A Label-Free Fluorescence Aptasensor Based on G-Quadruplex/Thioflavin T Complex for the Detection of Trypsin. Molecules (2022).
3. Peptide modified manganese-doped iron oxide nanoparticles as a sensitive fluorescence nanosensor for non-invasive detection of trypsin activity in vitro and in vivo. RSC Advances (2021).