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A modular scaffold for cellularly-retained fluorogenic probes for sensitive cell-resolved bioactivity imaging.

Philipp Mauker1,2, Lucas Dessen-Weissenhorn2, Carmen Zecha2

  • 1Faculty of Chemistry and Food Chemistry, Dresden University of Technology Dresden 01069 Germany oliver.thorn-seshold@tu-dresden.de.

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Researchers developed Trappable Green (TraG), a novel fluorogenic probe platform for live-cell imaging. TraG enables accurate quantification of biochemical processes by ensuring bright fluorescent products remain within cells, enhancing sensitivity and resolution.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Chemical Biology

Background:

  • Live cell probes require membrane permeability for intracellular access, but often lead to fluorescent product leakage, limiting sensitivity and resolution.
  • Existing methods for retaining fluorescent products, such as alkylation or precipitation, can disrupt native cellular biology.
  • Quantifying low-turnover biochemical processes in live cells is challenging due to signal loss and limited sensitivity.

Purpose of the Study:

  • To design a general platform for high-quality fluorogenic activity probes that release cell-retained, bright fluorescent products within live cells.
  • To overcome the limitations of current probes, specifically rapid signal loss and disruption of native biology.
  • To develop a modular and adaptable probe system for quantifying diverse biochemical processes.

Main Methods:

  • Developed a rhodol-based fluorogenic probe platform, Trappable Green (TraG), by optimizing charge and polarity for cell retention.
  • Screened various charge- and polarity-based approaches to transition from permeable to cell-retained states.
  • Demonstrated the modularity of the TraG scaffold by creating probes for glutathione (GSH), thioredoxin reductase (TrxR), and hydrogen peroxide (H2O2).

Main Results:

  • The TraG platform effectively balances rapid probe entry with robust retention of fluorescent products across multiple cell lines.
  • TraG probes enable bright, cell-resolved imaging and sensitive quantification of biochemical activities.
  • Successfully developed and validated TraG-based probes for specific targets like GSH, TrxR, and H2O2.

Conclusions:

  • The TraG scaffold offers a simple, rugged, and versatile solution for creating cell-retained enzyme activity probes.
  • This new probe design enables more accurate cell-resolved imaging and higher-sensitivity detection of low-turnover processes.
  • TraG provides a superior alternative to existing methods, avoiding the drawbacks of alkylation or precipitation-based strategies.