# Species-selective activation of a β-galactosidase fluorogenic probe

## TL;DR

A new fluorogenic probe activates only human enzymes, not bacterial ones, enabling precise detection in mixed biological samples.

## Contribution

The development of a species-selective β-galactosidase probe with no cross-reactivity to bacterial enzymes.

## Key findings

- PFB-FDG is selectively activated by human β-galactosidase but not bacterial β-galactosidase.
- Molecular docking suggests steric hindrance in bacterial β-gal prevents probe activation.
- The probe works in purified enzyme and live cell experiments without bacterial interference.

## Abstract

Fluorogenic probes capable of distinguishing enzyme activity between species are crucial for precise detection in complex biological environments. Herein, we present 5-(Pentafluorobenzoylamino) Fluorescein di-β-D-galactopyranoside (PFB-FDG), a substrate selectively activated by human β-galactosidase (β-gal) without cross-reactivity with bacterial β-gal. We tested PFB-FDG under various control conditions using purified enzymes and live cells to demonstrate that it does not activate in the presence of bacterial β-gal. Moreover, molecular docking studies suggest that the selectivity results from steric hindrance within the smaller binding pocket of bacterial β-gal, in contrast to the larger binding pocket of the human enzyme. This species-selective probe represents a major advancement, with its unique properties providing a valuable tool for future studies on human-bacterial interactions.

The online version contains supplementary material available at 10.1038/s41598-025-08443-y.