Onderzoekt Single Molecule Hechting van Atomic Force Spectroscopie

The overall goal of this procedure is to couple a single polymer to an A FM cantilever tip in order to do four spectroscopic surface desorption experiments with one in the same molecules multiple times. This is achieved by first plasma activating the surface of the tip in order to form hydroxyl groups as a second step. The cantilever tip is amino siloized by an up test solution, which forms amino groups on the tip surface.

The third step is to couple polyethylene glycol molecules to the amino groups. They prevent unspecific interactions between the tip and the substrate and serve as linker molecules for the polymers. Finally, polymers are conjugated to the polyethylene glycol molecules to form a probe.

Measurements suggest this procedure indeed makes it possible to use one and the same polymer force probe for a complete experimental set, or likewise, many hundred force distance curves. The main advantages of single molecule experiments over ensemble methods is that rare confirmations and infrequent states can be studied, which would otherwise be hidden in ensemble averaging. Because of their small size, these experiments can directly be compared to molecular dynamic simulations.

The first step is to covalently attach probe molecules to the cantilever tip. Start with fresh silicon nitride. Cantilever chips, employ chips with a spring constant of 10 to 100 pico newtons per nanometer.

Use tweezers to place them on a clean glass slide or Petri dish. Next, put the slide with the chips in a 100 watt plasma chamber after evacuating the chamber and flushing it with oxygen operated at 0.25 millibar and 20 watts for 15 minutes. In the next steps, use a hood to avoid inhalation of organic vapors during the plasma processing.

Prepare for amino siloization of the cantilevers in a glass petri dish, placed 2.5 milliliters of up test solution. In addition, repair a vessel containing acetone. Retrieve the cantilevers immediately after the plasma processing is complete.

Proceed to dip each cantilever in acetone for one second, and then place it in the Optus solution. Incubate the cantilevers for 15 minutes at room temperature before proceeding. As the cantilevers incubate, begin preparation for pegylation so as to minimize the effects of water and air on the components.

Begin with previously prepared eph tubes, one containing m peg, NHS and the other dye. NHS Peg. Determine the weight of the contents of each tube.

Next, work with the tube containing EG NHS and prepare to add chloroform. Pour the chloroform to achieve 25 millimolar final concentration and sufficient solution to cover the cantilevers in subsequent steps. Place the tube on a vortex mixer to ensure the powder dissolves.

Put the tube with the EG NHS aside to work with the diet. NHS peg. Add a chloroform for it to reach a final concentration of 0.25 millimolar.

Use a vortex mixer to ensure the powder is dissolved. Next, mix the solutions to adjust the ratio between the diet NHS peg and the methyl peg NHS molecules. This ratio is typically one to 500.

Obtain a small glass Petri dish with a lid to limit evaporation and fill it with the mixture as the cantilevers. Finish incubation. In APT test solution, have two containers with about 10 milliliters of acetone and one with chloroform ready for rinsing them.

Dip each of them in the acetone twice and once in chloroform and place them in the polyethylene glycol solution. Cover the dish and keep the cantilevers in the solution. For one hour, prepare for the covalent coupling of a probe molecule in this case poly amino acid poly de tyrosine.

Begin with the volume of one molar sodium hydroxide dissolve into this, the probe molecule to a concentration of one milligram per milliliter. For poly D tyrosine, exchange sodium hydroxide for eight to 8.5 pH sodium bore. Eight buffer immediately before functionalization.

As the cantilevers complete their incubation in polyethylene glycol solution, prepare three containers for rinsing them and a container of the poly tyrosine bore. Eight buffer solution for rinsing is five milliliters of chloroform, five milliliters of ethanol, five milliliters of the bore buffer. Use about 300 microliters of the poly tyrosine bore buffer solution.

Retrieve the cantilevers and dip each first in chloroform, then the ethanol, and then the bore eight buffer. Before placing them in the poly tyrosine bore eight buffer solution. Allow the cantilever chips to incubate for one hour after one hour of ready, about five milliliters of bate buffer in one container, and five milliliters of ultrapure water in another for rinsing the cantilevers also have 20 milliliters of ultrapure water to store them.

Get the cantilevers and dip each one in Boite buffer, followed by ultrapure water for rinsing. Then immerse it in ultrapure water for storage. The cantilevers can now be used for data acquisition.

Once the A FM tip is functionalized and a surface has been prepared and mounted, begin steps for a FM data acquisition. First place the functionalized cantilever into an A FM cantilever holder that is suitable for measuring in liquids. Next, connect the cantilever holder to the A FM head.

Proceed by pouring water into the fluid cell to immerse the cantilever and the surface. Then lower the A FM head on the liquid cell. Let the system equilibrate for at least half an hour.

After e equilibration. Position the cantilever far from the surface, then record thermal noise spectra 10 or more spectra are required to obtain a sufficient signal to noise ratio for excluding surface damping effects. Next, carefully approach the surface to make a measurement of the inverse optical lever sensitivity.

This involves pushing the cantilever tip against a hard surface to record the force versus pizo travel distance. Measure the slope during contact and invert it to determine the inverse optical lever sensitivity. Next work, the inverse optical lever sensitivity and the thermal noise spectra Determine the spring constant of the cantilever by fitting a harmonic oscillator to the thermal noise spectrum.

Now set the appropriate parameters for data collection. Record numerous forced distance curves for each experimental condition at the end of data collection. Determine the sensitivity and the spring constant again, to check for consistency and stability of the system.

This example of a forced distance trace in red is from the retraction of poly tyrosine from a methyl self-assembled monolayer impure water. The single step drop to zero force indicates detachment of a single molecule. The sigmoidal fit is shown in black.

The extracted plateau length and plateau force are indicated with arrows. The extension retraction velocity of the cantilever was 0.5 microns per second, and the dwell time on the surface was one second. Different liquid environments for the measurement caused changes in the extracted plateau force.

These histograms reflect the extracted plateau force for measurements in pure water and measurements in water. Ethanol mixtures at least 300 force distance traces were recorded for each solution with one in the same single polymer. Here analogous histograms are shown for extracted plateau length for in pure water and in water ethanol mixtures.

The addition of ethanol weakens the hydrophobic interaction between poly tyrosine and the surface leading to a lower plateau force and a decrease in plateau length. This is summarized in this plate of peak plateau length versus peak plateau force. Once mastered, the functionalization protocol can be done in about four hours after development.

This technique paved away for researchers in the field of physics and chemistry to explore the adhesion of infraction of polymers at interfaces.