Functionalization of cantilever tips with nucleotides by the phosphoramidite method

Area of Science:

• Biophysics
• Surface Science
• Nanotechnology

Background:

• Atomic force microscopy (AFM) enables atomic-level surface scanning and force spectroscopy.
• Force spectroscopy requires specific immobilization of molecules on cantilever tips and substrate surfaces.
• Understanding molecular interactions at the nanoscale is crucial for various biological and material science applications.

Purpose of the Study:

• To describe the covalent immobilization of single adenosine and thymidine nucleotides onto an amino-PEGylated AFM cantilever tip.
• To determine the rupture forces of the coordination bond between immobilized nucleotides and a gold surface using force spectroscopy.
• To explore the potential of nucleotide-functionalized tips for studying molecular binding mechanisms.

Main Methods:

• Covalent immobilization of adenosine and thymidine nucleotides on an amino-PEGylated cantilever tip using the phosphoramidite method.
• Recording force-distance curves between functionalized cantilever tips and gold surfaces via atomic force microscopy.
• Analysis of rupture events to determine bond strengths.

Main Results:

• The rupture force of the coordination bond between adenosine's primary amine and gold atoms was measured at 145 pN.
• Force-distance curves for thymidine-functionalized tips showed no rupture events due to the absence of a primary amine.
• Successful immobilization and force measurements demonstrate the utility of the phosphoramidite method for nucleotide functionalization.

Conclusions:

• Single nucleotide immobilization via the phosphoramidite method is feasible for AFM force spectroscopy.
• The measured rupture force for adenosine-gold interaction aligns with existing literature.
• Nucleotide-functionalized AFM tips offer a promising tool for investigating the binding mechanisms of nucleotide-interacting biomolecules, such as polymerases.