A new, simple method for linking of antibodies to atomic force microscopy tips

Area of Science:

• Biophysics
• Nanotechnology
• Biochemistry

Background:

• Atomic force microscope (AFM) tips functionalized with bioligands act as monomolecular biosensors.
• Flexible polyethylene glycol (PEG) tethers are preferred for optimal bioligand mobility.
• Existing methods require pre-derivatization of thiol-free biomolecules, increasing protein demand.

Purpose of the Study:

• To develop a convenient alternative for AFM tip functionalization with minimal protein consumption.
• To eliminate the need for pre-derivatization of antibodies and other proteins.
• To introduce a novel heterobifunctional cross-linker for efficient biomolecule tethering.

Main Methods:

• Utilized a new heterobifunctional cross-linker with two distinct amino-reactive functions.
• One end of the cross-linker (N-hydroxysuccinimide ester) rapidly reacts with amino groups on AFM tips.
• The other end (benzaldehyde) covalently binds lysine residues on proteins via Schiff base formation.
• Validated the method using biotinylated IgG on AFM tips and avidin on mica, and with human rhinovirus and its receptor fragments.

Main Results:

• Achieved functionalization with minimal protein consumption (5 µg).
• Successfully tethered biotinylated IgG to AFM tips and detected avidin with high reproducibility.
• Quantified approximately 2000 specifically bound IgG molecules per µm² on silicon nitride chips.
• Demonstrated application in studying specific interactions between human rhinovirus and very-low-density lipoprotein receptor fragments using force microscopy.

Conclusions:

• The new cross-linker provides a convenient and efficient method for AFM tip biosensor preparation.
• This approach significantly reduces protein requirements and eliminates pre-derivatization steps.
• The method is suitable for sensitive detection of molecular interactions in biophysical and biological studies.