Real-time bacterial detection with an intracellular ROS sensing platform

Area of Science:

• Biomedical Engineering
• Immunology
• Analytical Chemistry

Background:

• Reactive oxygen species (ROS) are crucial in biological processes and implicated in various diseases.
• Existing technologies struggle with accurate detection and identification of specific ROS.
• Understanding ROS dynamics during bacterial infection is vital for immune response studies.

Purpose of the Study:

• To develop a novel electrochemical biosensor for sensitive and rapid detection of intracellular hydrogen peroxide (H2O2).
• To investigate the real-time H2O2 production by macrophage cells upon bacterial challenge.
• To differentiate immune responses to various bacterial serotypes using H2O2 as an indicator.

Main Methods:

• Fabrication of an electrochemical biosensor using single-walled carbon nanotubes on indium tin oxide, functionalized with an osmium-based compound.
• Integration of the biosensor into RAW 264.7 macrophage cells for intracellular measurements.
• Stimulation of immune response using Gram-negative bacteria and measurement of H2O2 via lipopolysaccharide-Toll-like Receptor 4 interaction.
• Validation of sensor specificity using n-acetylcysteine and sodium pyruvate controls.

Main Results:

• The biosensor demonstrated a low limit of detection for H2O2 at 368 nM.
• Intracellular H2O2 increase was detected within 3 seconds of bacterial interaction with macrophages.
• Distinct H2O2 production patterns were observed for different Gram-negative bacterial serotypes.
• The sensor enabled unprecedented temporal detection of H2O2 during bacterial threat response.

Conclusions:

• The developed electrochemical biosensor offers sensitive, rapid, and specific detection of intracellular H2O2.
• This technology provides real-time insights into the dynamic innate immune response to bacterial infections.
• The findings highlight the potential for advanced diagnostics and understanding of host-pathogen interactions.