High performance immunoassay using immobilized enzyme in nanoporous carbon

Area of Science:

• Biochemistry
• Materials Science
• Analytical Chemistry

Background:

• Immunoassays require stable enzyme and antibody immobilization for reliable detection.
• Conventional immobilization methods often lead to reduced enzyme activity and thermal instability.
• Nanoporous materials offer unique properties for biomolecule stabilization.

Purpose of the Study:

• To develop a highly stable immunoassay format using enzyme-immobilized nanoporous carbon.
• To enhance the thermal stability and activity retention of immobilized enzymes and antibodies.
• To achieve sensitive colorimetric detection of human IgG with improved assay performance.

Main Methods:

• Loading horseradish peroxidase (HRP) into mesocellular carbon foam (MSU-F-C).
• Cross-linking HRP with glutaraldehyde (GA) and surface modification with anti-human IgG using EDC/sulfo-NHS chemistry.
• Evaluating thermal stability and enzyme activity retention at 40°C for 5 hours.
• Performing bead-based immunoassays for human IgG detection.

Main Results:

• The MSU-F-C/HRP/anti-human IgG conjugate demonstrated significantly higher thermal stability compared to conventional conjugates.
• Immobilized enzymes retained approximately 80% of initial activity at 40°C after 5 hours, versus a 90% loss for HRP/anti-human IgG.
• Sensitive colorimetric detection of human IgG was achieved with a detection limit of approximately 33 pM.
• Negligible cross-reactivity was observed against rabbit and chicken IgGs.

Conclusions:

• Immobilization of signal-generating enzymes and antibodies in nanoporous carbon creates a highly stable immunoassay platform.
• This approach significantly enhances thermal stability and enzyme activity retention, leading to improved assay performance.
• The developed immunoassay enables sensitive and specific detection of human IgG, with potential applications in diagnostics.