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A Highly Sensitive Biosensor for ATP Using a Chimeric Firefly Luciferase.

Bruce R Branchini1, Tara L Southworth1

  • 1Connecticut College, New London, CT, United States.

Methods in Enzymology
|March 25, 2017
PubMed
Summary

Researchers engineered a novel luciferase, PLG2, with improved stability and activity for detecting adenosine triphosphate (ATP). This new enzyme enables sensitive ATP measurement in various samples, serving as a biosensor for viable cells.

Keywords:
ATPBioluminescenceChimeraImagingLuciferaseLuciola italicaPLG2Reporterluc2

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Firefly luciferases are widely used for detecting adenosine triphosphate (ATP) due to their light-emitting properties.
  • Existing luciferase systems have limitations in terms of stability and specific activity.
  • Applications include enzyme detection and ATP level quantification.

Purpose of the Study:

  • To engineer a novel luciferase with enhanced properties compared to Photinus pyralis luciferase.
  • To develop a robust assay for sensitive ATP detection.
  • To establish ATP measurement as a biosensor for viable cell detection.

Main Methods:

  • Engineering of a novel luciferase (PLG2) through chimeric studies.
  • Development of a single-mixture assay protocol for PLG2.
  • Testing the assay with various biological samples, including human and bacterial cells.

Main Results:

  • PLG2 exhibits enhanced specific activity, thermal stability, and pH stability.
  • The developed assay can detect femtomole levels of ATP.
  • The methodology is effective for ATP measurement in diverse cellular samples.

Conclusions:

  • PLG2 represents a significant advancement in luciferase technology.
  • The developed assay provides a sensitive and stable method for ATP quantification.
  • This approach facilitates the use of ATP measurements as a reliable biosensor for viable cells.