Detection and Analysis of Reactive Oxygen Species (ROS): Buffer Components Are Not Bystanders

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Reactive oxygen species (ROS) are crucial in biological processes, driving interest in their accurate measurement.
• Existing literature shows significant variability in ROS concentration data, hindering comparability.
• A key factor influencing experimental outcomes, the reactivity of buffer components with ROS, is often overlooked.

Purpose of the Study:

• To investigate the chemical reactivity of common organic buffers with ROS.
• To determine how buffer-ROS interactions affect the accuracy of ROS concentration measurements.
• To provide guidance on selecting appropriate buffers for reliable ROS detection.

Main Methods:

• Tested the reactivity of HEPES, Tris, MES, citrate, ammonium acetate, and PBS with sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2) probes.
• Assessed the interaction of buffer components with boronate-based probes used for H2O2 and peroxynitrite (ONOO-) detection.
• Quantified the rate of ROS consumption by buffer molecules.

Main Results:

• Most tested organic buffers rapidly consume NaOCl, leading to false negative results for ROS detection.
• Buffer components interfere with boronate-based probes, causing inaccurate H2O2 and ONOO- measurements.
• Concentrations as low as 20 mM of HEPES, MES, ammonium acetate, and Tris can consume 1 mM hypochlorite within 1 second.

Conclusions:

• Commonly used organic buffers are unsuitable for ROS concentration determination due to chemical interference.
• Avoidance of these buffers is recommended to prevent significant errors in experimental findings and interpretations.
• Researchers must consider buffer chemistry to improve the rigor and reliability of ROS detection experiments.