Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Single molecule force spectroscopy enables us to measure physical parameters that describe the mechanical and adhesive properties of polymers. When using AFM-based force spectroscopy to study single molecules, it is essential to have a reliable and efficient protocol for binding these molecules covalently to an AFM cantilever tip. This protocol can be adopted to many different polymers irrespective of the contour length or hydrophobicity.

All steps should be carried out in a fume hood to avoid inhalation of organic vapors. Additionally, solvent-resistant glass, lab coat, and eye protection are required. First use freshly cleaned tweezers to place AFM cantilever chips in the plasma chamber.

Start the plasma chamber surface activation program by selecting start and then yes. Verify that the plasma process is working properly. A plasma process with a high oxygen content shows a light blue color.

While the surface activation program is executing, dissolve silane-PEG-mal in toluene to obtain a concentration of 1.25 milligrams per milliliter. Place three milliliters of the solution in a flat Petri dish. After the plasma process is complete, ventilate the plasma chamber by selecting confirm and then venting.

Proceed immediately to the next step in order to prevent absorption of contaminants. Place the chips in the Petri dish and incubate the chips for three hours at 60 degrees Celsius. Remove the Petri dish from the oven and allow it to cool for at least 10 minutes.

Next, rinse the chips. For PEG or polystyrene binding, rinse the chips with toluene three times. For polynipam binding, chips should be rinsed once with toluene and twice with ethanol.

To reduce the impact of capillary forces on the AFM cantilever, tilt the chips slightly when rinsing. The AFM cantilever chips need to be rinsed properly to remove any excess of physical polymers which may influence the experiment. Rinsing should be performed carefully to prevent any damage to the AFM cantilevers.

Finally, prepare at least two chips to serve as controls which will not undergo covalent polymer attachment. For controls versus PEG and polystyrene chips, rinse twice with ethanol and once with water. For controls versus polynipam chips, rinse twice with water.

To perform covalent attachment of PEG or polystyrene, prepare three milliliters of polymer solution in toluene at a concentration of 1.25 milligrams per milliliter. Add the solution and the chips to a Petri dish and incubate the chips at 60 degrees Celsius for one hour. After incubation with PEG or polystyrene, allow the chips to cool for 10 minutes.

Rinse the chips twice with toluene, twice with ethanol, and once with water. To perform covalent attachment of polynipam, prepare three milliliters of polymer solution in ethanol at a concentration of 1.25 milligrams per milliliter. Add the solution and the chips to a Petri dish and incubate the chips at room temperature for three hours.

After incubation with polynipam, rinse the chips twice with ethanol and twice with water. To store the chips until use in an experiment, place each chip separately in a one milliliter Petri dish filled with water. Keep the Petri dishes at four degrees Celsius.

First, insert the functionalized AFM cantilever chip into a chip holder. Glue the prepared surface into a sample holder that is suitable for measurements in liquid. Use a pipette to immerse the chip in water.

Mount the sample surface into the AFM. Immerse the sample surface in water. Connect the chip holder to the AFM.

Then approach the chip to the sample surface. Use the environmental panel to set the target temperature and switch the mode and the feedback radio buttons to on. Then let the system equilibrate for about 15 minutes.

To take a force extension curve, approach the AFM cantilever tip to the surface and select single force. The resulting curve displays the deflection against the piezo distance with the approach to the surface shown in red and the retraction shown in blue. Expand the portion of the curve that represents the indentation of the AFM cantilever tip into the underlying surface.

To perform a linear fit, set cursors on either the approach or the retract curve and select update INVOLS from the context menu. The resulting value for the inverse optical lever sensitivity value appears in the panel on the upper left. After repeating this procedure at least five times, calculate an average for inverse optical lever sensitivity and enter the average in the panel.

Position the AFM cantilever at a height of approximately 100 micrometers above the surface by selecting move to pre-engage. To get a satisfactory signal-to-noise ratio for the thermal noise spectrum, set the averaging count to at least 10 and choose the highest possible frequency resolution. Next, record the thermal noise spectrum by selecting capture thermal data.

To fit the thermal noise spectrum with a simple harmonic oscillator function, expand the portion of the curve representing the first resonance peak. Then select initialize fit. Finally, refine the fit by using the fit thermal data button.

The respective force constant will appear in the panel. To begin collecting the data, set the parameters for the experiment. Set pulling velocity to one micrometer per second and force trigger to one nanonewton.

Approach the AFM cantilever tip to the surface and select single force to record a single curve and determine whether the parameters need to be adjusted as described in the manuscript. Select F map from the master panel. To obtain a force map with 100 curves, set the number of force points and force lines to 10.

Start recording the force map by selecting do F map. Take force extension curves in a grid-like fashion to avoid any local surface effects and to average different surface areas. After the experiment, repeat the determination of the inverse optical lever sensitivity and the spring constant to check the consistency and the stability of the system.

Single polynipam and PEG polymers were covalently bound to an AFM cantilever tip at one end and physisorbed on the silicon dioxide surface at the other end. To measure temperature-dependent stretching behavior, a clear single-molecule stretching event followed by a final maximum at the end of the respective force extension curve was identified. Then a single master curve was generated for every temperature.

For PEG, a decrease of the stretching force was observed with increasing temperature. For polynipam, the opposite trend was observed. The desorption of polystyrene from a SAM surface in water can be used to determine the desorption force and length.

When the polymer attachment was successful, the force extension curves showed plateaus of constant force. Each plateau was fitted with a sigmoidal curve to determine the desorption force and desorption length. The observed desorption forces corresponded to previously obtained values.

When more than one polymer attached to the AFM cantilever tip, cascades of plateaus were observed in the force extension curves. With two polymers attached, a bimodal distribution was found for the desorption length while the desorption force showed a narrow distribution. A functionalized AFM cantilever tip can be used to quantify the force response of single molecules in a liquid environment and with external stimuli.

The use of clean equipment, solvents, AFM cantilever tips, and repeated rinsing is very important to reach a high level of cleanliness which should be confirmed prior to the described controlled experiments. The presented protocols and procedures paved the way for better understanding of stimuli responsive polymer systems. The results can be directly compared to molecular dynamic simulations.