A fluorescence-based method to directly quantify antibodies immobilized on gold nanoparticles

Area of Science:

• Bioconjugation Chemistry
• Nanoparticle-based Biosensors
• Surface Chemistry

Background:

• Antibody immobilization on gold nanoparticles is crucial for biosensor sensitivity.
• Accurate quantification of antibody surface coverage is essential for optimizing biosensor performance.
• Conventional methods for quantifying immobilized antibodies can be inaccurate.

Purpose of the Study:

• To develop a direct and accurate fluorescence-based method for quantifying antibodies on gold nanoparticles.
• To compare the accuracy of the new method with conventional indirect quantification techniques.
• To validate the formation of a single antibody layer on gold nanoparticles.

Main Methods:

• Gold nanoparticles were modified with antibodies.
• Antibodies were released from gold nanoparticles using a KI/I2 etchant.
• Released antibodies were quantified using NanoOrange fluorescent dye after desalting.
• Hydrodynamic diameter changes were measured to assess antibody layer formation.

Main Results:

• A direct fluorescence method was established for quantifying antibodies on gold nanoparticles.
• Approximately 309 ± 93 antibodies were found to adsorb onto a 60 nm gold nanoparticle.
• This coverage is consistent with a single antibody layer (monolayer).
• Indirect supernatant analysis overestimated antibody coverage (660 ± 87 antibodies per nanoparticle).

Conclusions:

• The developed fluorescence method offers a more accurate quantification of antibody immobilization on gold nanoparticles.
• This improved quantification is vital for optimizing nanoparticle-based biosensor sensitivity and reliability.
• The findings confirm the formation of a monolayer, preventing multilayer artifacts in biosensor design.