Multivalent nanobody engineering for enhanced physisorption and functional display on gold nanoparticles

Area of Science:

• Bioconjugation chemistry
• Nanotechnology
• Immunodiagnostics

Background:

• Single domain antibodies, or nanobodies, offer advantages over conventional antibodies for point-of-care diagnostics like lateral flow assays.
• Gold nanoparticle bioconjugates are crucial labels in lateral flow assays, but their simple physisorption onto gold nanoparticles often leads to aggregation and loss of function.
• Existing methods for nanobody-gold nanoparticle conjugation face challenges with stability and functionality, hindering their application in robust diagnostic devices.

Purpose of the Study:

• To investigate the potential of engineering nanobodies into multivalent structures to enhance their functionality when passively adsorbed onto gold nanoparticles.
• To develop a facile and stable conjugation strategy for nanobodies on gold nanoparticles for use in lateral flow assays.
• To assess the stability and performance of multivalent nanobody-gold nanoparticle bioconjugates under conditions relevant to lateral flow assay manufacturing and storage.

Main Methods:

• Engineering of a trivalent nanobody (VHH3) targeting the S1 protein of SARS-CoV-2.
• Physisorption of monovalent, bivalent, and trivalent VHHV nanobodies onto gold nanoparticles.
• Assessment of nanoparticle stability under various stress conditions including salt stress, blocking, washing, and freeze-drying.
• Evaluation of the sensitivity of the resulting bioconjugates for S1 protein detection using picomolar sensitivity assays.

Main Results:

• Multivalent nanobody structures significantly improved the stability of gold nanoparticle bioconjugates against aggregation and loss of function during processing and storage.
• The trivalent VHHV3 nanobody, when passively adsorbed onto gold nanoparticles, demonstrated superior stability under harsh conditions compared to its monovalent and bivalent counterparts.
• The VHHV3-gold nanoparticle bioconjugates maintained picomolar sensitivity for detecting the S1 protein of SARS-CoV-2, indicating preserved functionality.
• The one-step physisorption of multivalent nanobodies proved to be a robust and effective method for creating stable and functional bioconjugates.

Conclusions:

• Engineering nanobodies into multivalent structures is a highly effective strategy to overcome the limitations of simple physisorption onto gold nanoparticles.
• This approach enhances the stability and functionality of nanobody-gold nanoparticle bioconjugates, making them suitable for demanding applications like lateral flow assays.
• The developed facile conjugation strategy represents a significant advancement for the integration of nanobodies into robust and reliable point-of-care diagnostic tools.