The overall goal of the single-molecule force spectroscopy technique is to determine the adhesion force and nature of interactions between amino acids or peptides with different surfaces. This matter can help answer key questions in the area of surface chemistry and be used for the development of new composites and chordates. The main advantage of this technique is that it provides information on interaction between the desired molecule and surfaces at the single molecule level.
To begin, purchase silicon nitride AFM cantilevers equipped with silicon tips with a nominal cantilever radius of two nanometers. Mount the tip on the holder. Clean each of the AFM cantilevers by dipping them into ethanol for 20 minutes and then dry them at room temperature.
Next, place the tips into a plasma chamber and treat the cantilevers by exposing them to oxygen plasma for five minutes. Prepare a solution containing 15 parts methyltriethoxysilane and one part three amino propyltriethoxysilane and suspend the canitlevers three centimeters above the solution while under an inert atmosphere. It is important that the tips and the holder do not touch the silane mixture.
In addition, the silane is highly moisture sensitive, and therefore these tips need to be carried out in an inert atmosphere. Once sealed, connect the desiccator to a vacuum pump and apply a vacuum for two hours to form a mono-layer of the two types of mixed silane compounds on the surface of the cantilevers. Carefully remove the tips and place them onto a hot plate preheated to 70 degrees Celsius.
Leave the tips to dry for 10 minutes. Cool the tips at room temperature before immersing them in a solution of Fmoc-PEG-NHS for one hour at room temperature. Next, dip the tips into chloroform for five minutes and then into DMF for an additional five minutes.
After incubation in DMF, de-protect the Fmoc groups of the attached PEG molecules by dipping the tips into 20%piperidine solution in DMF for 30 minutes. Again, dip the tips in DMF for four minutes and then in N-Methyl-2-pyrrolidone for an additional four minutes. To couple amino acids onto the tips, immerse the for one and a half hours into an amino acid coupling solution with a total concentration of 30 millimolar and five milliliters of N-Methyl-2-pyrrolidone.
To protect the remaining free or unreactant PEG residues by the acetyl group, dip the tips for 30 minutes in a 0.65 molar mixture of four parts acetic anhydride and one part diisopropylethylamine and N-Methyl-2-pyrrolidone. Then sequentially dip the peptide amino acid functionalized tips for four minutes each into NMP, chloroform, 50%ethanol and water before finally drying the tip in air. Attach the desired test surface to a metallic holder of the AFM with commercially available epoxy.
Then, place the metallic holder into the glass holder of the AFM, which is shaped as a small petri dish. Fill the glass holder with TRIS buffer. And then place the metallic holder on the AFM stage below the tip holder.
Next, mount the AFM tip on the AFM's optic glass. Place the optic glass block in the AFM head. And put the AFM head on the stage.
Next, calibrate the AFM cantilevers with spring constants ranging from 10 to 30 piconewtons per nanometer. A typical force measurement results in a force distance curve like those shown here. The first peek indicates non-specific interactions between the tip and the surface and the second peek refers to the specific adhesion event.
From several hundred force distance curves, it is possible to construct a histogram by plotting the number of adhesion events versus force. Applying a Gaussian fit on these histograms will determine the most probable force. After watching this video, you should have a good understanding of how to chemically modify AFM tips with amino acids or peptides and how to perform single-molecule force spectroscopy using AFM.
