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Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy
Published on: March 16, 2020
Antibody linking to atomic force microscope tips via disulfide bond formation
A S M Kamruzzahan1, Andreas Ebner, Linda Wildling
1Institute of Biophysics and Institute of Organic Chemistry, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria.
Bioconjugate Chemistry
|November 16, 2006
Summary
Atomic force microscope (AFM) tips functionalized with poly(ethylene glycol) (PEG) linkers enable precise biosensing. This study reveals protein pre-adsorption significantly accelerates disulfide bond formation for enhanced ligand-receptor interaction analysis.
Area of Science:
- Biophysics
- Nanotechnology
- Surface Chemistry
Background:
- Atomic force microscopy (AFM) tips can be converted into monomolecular biosensors by covalently attaching bioligands.
- Poly(ethylene glycol) (PEG) linkers are used to tether bioligands to AFM tips, allowing for free reorientation and scanning of sample surfaces.
- A standard coupling scheme involves generating amino groups on the AFM tip, reacting them with PEG linkers, and finally forming disulfide bonds with protein thiols.
Purpose of the Study:
- To critically examine the standard coupling scheme for tethering bioligands to AFM tips.
- To investigate the mechanism and kinetics of protein thiol coupling to PEG linkers on AFM tips and silicon nitride chips.
- To reconcile discrepancies between observed and predicted coupling rates.
Main Methods:
- Functionalization of AFM tips and silicon nitride chips with poly(ethylene glycol) (PEG) linkers.
- Covalent attachment of biotinylated immunoglobulin G (IgG) with free thiols to the functionalized surfaces.
- Characterization of ligand-receptor interactions using AFM force spectroscopy (unbinding force analysis).
- Quantification of bound biotin-IgG using ExtrAvidin-peroxidase conjugate.
- Kinetic analysis of disulfide bond formation in bulk solution by monitoring 2-thiopyridone absorbance.
Main Results:
- AFM tips with PEG-tethered biotin-IgG specifically recognized avidin, with unbinding forces indicating simultaneous unbinding of multiple IgG-linked biotin residues.
- The coupling scheme was reproducible on amino-functionalized silicon nitride chips, with binding confirmed to be via disulfide bonds.
- Observed disulfide coupling rates on AFM tips and chips were approximately 10^3-fold faster than predicted from bulk solution experiments.
Conclusions:
- The standard coupling scheme is effective for creating bioligand-functionalized AFM tips for biosensing applications.
- Protein pre-adsorption to AFM tips and silicon nitride surfaces significantly enhances the rate of disulfide bond formation.
- This enrichment mechanism reconciles the discrepancy between bulk solution kinetics and observed surface coupling rates, improving understanding of biosensor fabrication.
